^DATABASE = GeoMiame
!Database_name = Gene Expression Omnibus (GEO)
!Database_institute = NCBI NLM NIH
!Database_web_link = http://www.ncbi.nlm.nih.gov/geo
!Database_email = geo@ncbi.nlm.nih.gov
^SERIES = GSE154311
!Series_title = RNA-seq on CD16Int versus CD16High in severe COVID-19 patients
!Series_geo_accession = GSE154311
!Series_status = Public on Jul 20 2021
!Series_submission_date = Jul 13 2020
!Series_last_update_date = Jul 20 2021
!Series_pubmed_id = 33986193
!Series_summary = Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel viral pathogen that  causes a clinical disease called coronavirus disease 2019 (COVID-19). Approximately 20% of  infected patients experience a severe manifestation of the disease, causing bilateral pneumonia  and acute respiratory distress syndrome. Severe COVID-19 patients also have a pronounced  coagulopathy with approximately 30% of patients experiencing thromboembolic complications.  However, the cellular etiology driving the coagulopathy remains unknown. Here, we explore  whether the prominent neutrophilia seen in severe COVID-19 patients contributes to  inflammation-associated coagulation. We found in severe patients the emergence of a CD16Int  low-density inflammatory band (LDIB) neutrophil population that trends over time with changes  in disease status. These cells demonstrated spontaneous neutrophil extracellular trap (NET)  formation, higher phagocytic capacity, enhanced cytokine production, and associated clinically  with D-dimer, ferritin, and systemic IL-6 and TNF-α levels. Strikingly, LDIB neutrophils are the  major immune cells within the bronchoalveolar lavage (BAL) fluid with increased CXCR3 and  loss of CD44 and CD38 expression. We conclude that the LDIB subset contributes to COVID-  19-associated coagulopathy (CAC) and systemic inflammation and could be used as an adjunct  clinical marker to monitor disease status and progression. Identifying patients who are trending  towards LDIB crisis and implementing early, appropriate treatment could improve all-cause  mortality rates for severe COVID-19 patients.
!Series_overall_design = Three paired CD16Int and CD16High samples from severe COVID-19 patients were compared with each other, as well as with three healthy donor NDN samples.
!Series_type = Expression profiling by high throughput sequencing
!Series_contributor = Samantha,M,Morrissey
!Series_contributor = Anne,E,Geller
!Series_contributor = Xiaoling,,Hu
!Series_contributor = David,,Tieri
!Series_contributor = Elizabeth,A,Cooke
!Series_contributor = Chuanlin,,Ding
!Series_contributor = Matthew,,Woeste
!Series_contributor = Zachary,C,Martin
!Series_contributor = Huang-ge,,Zhang
!Series_contributor = Rodrigo,,Cavallazzi
!Series_contributor = Sean,P,Clifford
!Series_contributor = James,,Chen
!Series_contributor = Lu,,Cai
!Series_contributor = Maiying,,Kong
!Series_contributor = Corey,T,Watson
!Series_contributor = Jiapeng,,Huang
!Series_contributor = Eric,C,Rouchka
!Series_contributor = Jun,,Yan
!Series_sample_id = GSM4668813
!Series_sample_id = GSM4668814
!Series_sample_id = GSM4668815
!Series_sample_id = GSM4668816
!Series_sample_id = GSM4668817
!Series_sample_id = GSM4668818
!Series_sample_id = GSM4668819
!Series_sample_id = GSM4668820
!Series_sample_id = GSM4668821
!Series_contact_name = Eric,Christian,Rouchka
!Series_contact_email = eric.rouchka@louisville.edu
!Series_contact_laboratory = KY INBRE Bioinformatics Core
!Series_contact_department = Biochemistry and Molecular Genetics
!Series_contact_institute = University of Louisville
!Series_contact_address = 522 East Gray Street
!Series_contact_city = Louisville
!Series_contact_state = Kentucky
!Series_contact_zip/postal_code = 40292
!Series_contact_country = USA
!Series_supplementary_file = ftp://ftp.ncbi.nlm.nih.gov/geo/series/GSE154nnn/GSE154311/suppl/GSE154311_htseq_rawCounts.txt.gz
!Series_platform_id = GPL18573
!Series_platform_taxid = 9606
!Series_sample_taxid = 9606
!Series_relation = BioProject: https://www.ncbi.nlm.nih.gov/bioproject/PRJNA645842
!Series_relation = SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRP271567
^PLATFORM = GPL18573
!Platform_title = Illumina NextSeq 500 (Homo sapiens)
!Platform_geo_accession = GPL18573
!Platform_status = Public on Apr 15 2014
!Platform_submission_date = Apr 15 2014
!Platform_last_update_date = Mar 26 2019
!Platform_technology = high-throughput sequencing
!Platform_distribution = virtual
!Platform_organism = Homo sapiens
!Platform_taxid = 9606
!Platform_contact_name = ,,GEO
!Platform_contact_country = USA
!Platform_data_row_count = 0
^SAMPLE = GSM4668813
!Sample_title = Healthy donor, normal density neutrophils (NDN), replicate 1
!Sample_geo_accession = GSM4668813
!Sample_status = Public on Jul 20 2021
!Sample_submission_date = Jul 13 2020
!Sample_last_update_date = Jul 20 2021
!Sample_type = SRA
!Sample_channel_count = 1
!Sample_source_name_ch1 = Healthy donor, normal density neutrophils (NDN), replicate 1
!Sample_organism_ch1 = Homo sapiens
!Sample_taxid_ch1 = 9606
!Sample_characteristics_ch1 = disease: Healthy
!Sample_characteristics_ch1 = neutrophils: NDN
!Sample_characteristics_ch1 = tissue: peripheral blood
!Sample_treatment_protocol_ch1 = CD16High and CD16Int neutrophils were sorted by FACSAria III from three severe COVID-19 patients and NDN were purified from healthy donor peripheral blood.
!Sample_molecule_ch1 = polyA RNA
!Sample_extract_protocol_ch1 = RNA was extracted using Qiagen's RNAeasy Plus Microkit.
!Sample_extract_protocol_ch1 = Library Preparation: Libraries were prepared using the Universal Plus mRNA-Seq with (NuGEN Cat# 0520-24).  a. Poly(A) Selection: 8-30 ng of RNA samples (in a volume of 50 μl) were mixed with  Oligo (dT) Beads and incubated at 65oC for 5 minutes, mixed, then at room  temperature for 10 mins. The supernatant was discarded and the beads were  washed with wash buffer. Captured polyadenylated RNA was eluted using Elution  buffer at 80°C for 2 min. mRNA is further purified in a second bead clean-up.  b. RNA Fragmentation: Beads were mixed with 20 μl Fragmentation Buffer and  incubated for 8 minutes at 94oC. 20 μl of supernatant was removed from the beads  and proceeded immediately to First Strand cDNA Synthesis.  c. First Strand cDNA Synthesis: 5 μl of First Strand Master Mix was added to each  sample and placed on a thermal cycler to run preprogrammed thermal conditions.  d. Second Strand cDNA Synthesis: 50 μl Second Strand Master Mix was added to  each sample, mixed well and incubated in a thermal cycler at 16oC for one hour. e. cDNA Purification: cDNA was purified using Agencourt AMPure XP Beads (1.8  volumes), eluted in 10 μl of H2O, and stored at -20oC.  f. End Repair: End Repair Mix was added to the purified samples from step e and the  samples were incubated in a preprogrammed thermal cycler.  g. Adaptor Ligation: Samples were mixed with 3 μl adaptor mix, then 12 μl Ligation  Mix, and incubated in a thermal cycler at 25oC for 30 minutes.  Sample and Barcode Information: Samples were barcoded with NuGEN Unique  Dual Indexes. Strand Selection: 70 μl of Strand Selection Master Mix was added to 30 μl of each  sample, incubated in a pre-heated thermal cycler at 72oC for 10 minutes. Strand Selection Purification: Strand selected cDNA was purified using  Agencourt AMPure XP Beads (0.8 volumes), eluted in 15 μl of H2O, and stored at -  20oC.  j. Library Amplification: 76.5 μl of Library Amplification Master Mix was added to  13.5 μl of each sample and incubated in a preprogrammed thermal cycler, for 13-  16 cycles, which was pre-optimized with qPCR.  k. Amplified Library Purification: Amplified libraries were purified using 90 μl  Agencourt AMPure XP Beads (1.0 volumes). 25 μl of eluted libraries were collected  and stored at -20oC.  l. Validate Library: The concentration of libraries were measured by Qubit dsDNA  HS Assay Kit (Invitrogen Q32851). Libraries were diluted and normalized to the  optimal range for Agilent Bioanalyzer analysis using the DNA High Sensitivity Kit  (Agilent Technologies, Cat# 5067-4626).  m. Normalize and Pool Libraries: Same amount of libraries were pooled based on  the molar concentration from Bioanalyzer measurements. Library Denaturing and Diluting for MiSeq Nano 300: Pooled library was run on MiSeq  to test quantity and quality, using the MiSeq Reagent Nano Kit V2 300 cycles (Illumina, Cat.  No. MS-103-1001). Library and PhiX control (Illumina, Cat. No. FC-110-3001) were  denatured and diluted using the standard normalization method following manufacturer's  directions, to a final concentration of 6pM. Equal volume of library and PhiX were combined and  sequenced on Illumina MiSeq.  (https://basespace.illumina.com/run/196126950/JY_Xiaoling07072020_test)  3. Library Denaturing and Diluting for Nextseq 500: Library and PhiX were denatured and  diluted using the standard normalization method following manufacturer's directions. The  loading of library pool was 1.3 ml at 1.8 pM, with 1% PhIX spike in.  Sequencing Run: Sequencing was performed on the University of Louisville Brown Cancer  Center Genomics Core Illumina NextSeq 500 using the NextSeq 500/550 75 cycle High  Output Kit v2.5 (20024906).
!Sample_description = JY_07_HD_1
!Sample_data_processing = Base calling: raw .bcl files were converted to fastq files using bcl2fastq v2.20.0.422.
!Sample_data_processing = Demultiplexing: reads were demultiplexed based on the 8-base NuGen sample barcode using a custom Perl script.
!Sample_data_processing = Removal of PCR duplicates: PCR duplicates were removed by collapsing sequences with 100% identity over all 92 bases (76 sequenced bases plus 8 base sample index plus 8 base UMI) using a custom Perl script.
!Sample_data_processing = Quality Assurance/Quality Control: QA/QC was performed using FastQC v0.10.1.  No issues were found, so no further trimming or other QA/QC steps were deemed necessary.
!Sample_data_processing = Alignment: Raw fastq sequences were aligned to the human reference genome (hg38.p12) using STAR v2.6 guided by an ENSEMBL v93 gtf with mitochondrial and ribosomal RNAs removed.
!Sample_data_processing = Count extraction: Raw read counts were obtained from the STAR aligned bam files using HTSeq v0.10.0.
!Sample_data_processing = Genome_build: hg38.p12
!Sample_data_processing = Supplementary_files_format_and_content: htseq_rawCounts.txt
!Sample_platform_id = GPL18573
!Sample_contact_name = Eric,Christian,Rouchka
!Sample_contact_email = eric.rouchka@louisville.edu
!Sample_contact_laboratory = KY INBRE Bioinformatics Core
!Sample_contact_department = Biochemistry and Molecular Genetics
!Sample_contact_institute = University of Louisville
!Sample_contact_address = 522 East Gray Street
!Sample_contact_city = Louisville
!Sample_contact_state = Kentucky
!Sample_contact_zip/postal_code = 40292
!Sample_contact_country = USA
!Sample_instrument_model = Illumina NextSeq 500
!Sample_library_selection = cDNA
!Sample_library_source = transcriptomic
!Sample_library_strategy = RNA-Seq
!Sample_relation = BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN15517450
!Sample_relation = SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX8719043
!Sample_supplementary_file_1 = NONE
!Sample_series_id = GSE154311
!Sample_data_row_count = 0
^SAMPLE = GSM4668814
!Sample_title = Healthy donor, normal density neutrophils (NDN), replicate 2
!Sample_geo_accession = GSM4668814
!Sample_status = Public on Jul 20 2021
!Sample_submission_date = Jul 13 2020
!Sample_last_update_date = Jul 20 2021
!Sample_type = SRA
!Sample_channel_count = 1
!Sample_source_name_ch1 = Healthy donor, normal density neutrophils (NDN), replicate 2
!Sample_organism_ch1 = Homo sapiens
!Sample_taxid_ch1 = 9606
!Sample_characteristics_ch1 = disease: Healthy
!Sample_characteristics_ch1 = neutrophils: NDN
!Sample_characteristics_ch1 = tissue: peripheral blood
!Sample_treatment_protocol_ch1 = CD16High and CD16Int neutrophils were sorted by FACSAria III from three severe COVID-19 patients and NDN were purified from healthy donor peripheral blood.
!Sample_molecule_ch1 = polyA RNA
!Sample_extract_protocol_ch1 = RNA was extracted using Qiagen's RNAeasy Plus Microkit.
!Sample_extract_protocol_ch1 = Library Preparation: Libraries were prepared using the Universal Plus mRNA-Seq with (NuGEN Cat# 0520-24).  a. Poly(A) Selection: 8-30 ng of RNA samples (in a volume of 50 μl) were mixed with  Oligo (dT) Beads and incubated at 65oC for 5 minutes, mixed, then at room  temperature for 10 mins. The supernatant was discarded and the beads were  washed with wash buffer. Captured polyadenylated RNA was eluted using Elution  buffer at 80°C for 2 min. mRNA is further purified in a second bead clean-up.  b. RNA Fragmentation: Beads were mixed with 20 μl Fragmentation Buffer and  incubated for 8 minutes at 94oC. 20 μl of supernatant was removed from the beads  and proceeded immediately to First Strand cDNA Synthesis.  c. First Strand cDNA Synthesis: 5 μl of First Strand Master Mix was added to each  sample and placed on a thermal cycler to run preprogrammed thermal conditions.  d. Second Strand cDNA Synthesis: 50 μl Second Strand Master Mix was added to  each sample, mixed well and incubated in a thermal cycler at 16oC for one hour. e. cDNA Purification: cDNA was purified using Agencourt AMPure XP Beads (1.8  volumes), eluted in 10 μl of H2O, and stored at -20oC.  f. End Repair: End Repair Mix was added to the purified samples from step e and the  samples were incubated in a preprogrammed thermal cycler.  g. Adaptor Ligation: Samples were mixed with 3 μl adaptor mix, then 12 μl Ligation  Mix, and incubated in a thermal cycler at 25oC for 30 minutes.  Sample and Barcode Information: Samples were barcoded with NuGEN Unique  Dual Indexes. Strand Selection: 70 μl of Strand Selection Master Mix was added to 30 μl of each  sample, incubated in a pre-heated thermal cycler at 72oC for 10 minutes. Strand Selection Purification: Strand selected cDNA was purified using  Agencourt AMPure XP Beads (0.8 volumes), eluted in 15 μl of H2O, and stored at -  20oC.  j. Library Amplification: 76.5 μl of Library Amplification Master Mix was added to  13.5 μl of each sample and incubated in a preprogrammed thermal cycler, for 13-  16 cycles, which was pre-optimized with qPCR.  k. Amplified Library Purification: Amplified libraries were purified using 90 μl  Agencourt AMPure XP Beads (1.0 volumes). 25 μl of eluted libraries were collected  and stored at -20oC.  l. Validate Library: The concentration of libraries were measured by Qubit dsDNA  HS Assay Kit (Invitrogen Q32851). Libraries were diluted and normalized to the  optimal range for Agilent Bioanalyzer analysis using the DNA High Sensitivity Kit  (Agilent Technologies, Cat# 5067-4626).  m. Normalize and Pool Libraries: Same amount of libraries were pooled based on  the molar concentration from Bioanalyzer measurements. Library Denaturing and Diluting for MiSeq Nano 300: Pooled library was run on MiSeq  to test quantity and quality, using the MiSeq Reagent Nano Kit V2 300 cycles (Illumina, Cat.  No. MS-103-1001). Library and PhiX control (Illumina, Cat. No. FC-110-3001) were  denatured and diluted using the standard normalization method following manufacturer's  directions, to a final concentration of 6pM. Equal volume of library and PhiX were combined and  sequenced on Illumina MiSeq.  (https://basespace.illumina.com/run/196126950/JY_Xiaoling07072020_test)  3. Library Denaturing and Diluting for Nextseq 500: Library and PhiX were denatured and  diluted using the standard normalization method following manufacturer's directions. The  loading of library pool was 1.3 ml at 1.8 pM, with 1% PhIX spike in.  Sequencing Run: Sequencing was performed on the University of Louisville Brown Cancer  Center Genomics Core Illumina NextSeq 500 using the NextSeq 500/550 75 cycle High  Output Kit v2.5 (20024906).
!Sample_description = JY_09_HD_3
!Sample_data_processing = Base calling: raw .bcl files were converted to fastq files using bcl2fastq v2.20.0.422.
!Sample_data_processing = Demultiplexing: reads were demultiplexed based on the 8-base NuGen sample barcode using a custom Perl script.
!Sample_data_processing = Removal of PCR duplicates: PCR duplicates were removed by collapsing sequences with 100% identity over all 92 bases (76 sequenced bases plus 8 base sample index plus 8 base UMI) using a custom Perl script.
!Sample_data_processing = Quality Assurance/Quality Control: QA/QC was performed using FastQC v0.10.1.  No issues were found, so no further trimming or other QA/QC steps were deemed necessary.
!Sample_data_processing = Alignment: Raw fastq sequences were aligned to the human reference genome (hg38.p12) using STAR v2.6 guided by an ENSEMBL v93 gtf with mitochondrial and ribosomal RNAs removed.
!Sample_data_processing = Count extraction: Raw read counts were obtained from the STAR aligned bam files using HTSeq v0.10.0.
!Sample_data_processing = Genome_build: hg38.p12
!Sample_data_processing = Supplementary_files_format_and_content: htseq_rawCounts.txt
!Sample_platform_id = GPL18573
!Sample_contact_name = Eric,Christian,Rouchka
!Sample_contact_email = eric.rouchka@louisville.edu
!Sample_contact_laboratory = KY INBRE Bioinformatics Core
!Sample_contact_department = Biochemistry and Molecular Genetics
!Sample_contact_institute = University of Louisville
!Sample_contact_address = 522 East Gray Street
!Sample_contact_city = Louisville
!Sample_contact_state = Kentucky
!Sample_contact_zip/postal_code = 40292
!Sample_contact_country = USA
!Sample_instrument_model = Illumina NextSeq 500
!Sample_library_selection = cDNA
!Sample_library_source = transcriptomic
!Sample_library_strategy = RNA-Seq
!Sample_relation = BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN15517449
!Sample_relation = SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX8719044
!Sample_supplementary_file_1 = NONE
!Sample_series_id = GSE154311
!Sample_data_row_count = 0
^SAMPLE = GSM4668815
!Sample_title = Healthy donor, normal density neutrophils (NDN), replicate 3
!Sample_geo_accession = GSM4668815
!Sample_status = Public on Jul 20 2021
!Sample_submission_date = Jul 13 2020
!Sample_last_update_date = Jul 20 2021
!Sample_type = SRA
!Sample_channel_count = 1
!Sample_source_name_ch1 = Healthy donor, normal density neutrophils (NDN), replicate 3
!Sample_organism_ch1 = Homo sapiens
!Sample_taxid_ch1 = 9606
!Sample_characteristics_ch1 = disease: Healthy
!Sample_characteristics_ch1 = neutrophils: NDN
!Sample_characteristics_ch1 = tissue: peripheral blood
!Sample_treatment_protocol_ch1 = CD16High and CD16Int neutrophils were sorted by FACSAria III from three severe COVID-19 patients and NDN were purified from healthy donor peripheral blood.
!Sample_molecule_ch1 = polyA RNA
!Sample_extract_protocol_ch1 = RNA was extracted using Qiagen's RNAeasy Plus Microkit.
!Sample_extract_protocol_ch1 = Library Preparation: Libraries were prepared using the Universal Plus mRNA-Seq with (NuGEN Cat# 0520-24).  a. Poly(A) Selection: 8-30 ng of RNA samples (in a volume of 50 μl) were mixed with  Oligo (dT) Beads and incubated at 65oC for 5 minutes, mixed, then at room  temperature for 10 mins. The supernatant was discarded and the beads were  washed with wash buffer. Captured polyadenylated RNA was eluted using Elution  buffer at 80°C for 2 min. mRNA is further purified in a second bead clean-up.  b. RNA Fragmentation: Beads were mixed with 20 μl Fragmentation Buffer and  incubated for 8 minutes at 94oC. 20 μl of supernatant was removed from the beads  and proceeded immediately to First Strand cDNA Synthesis.  c. First Strand cDNA Synthesis: 5 μl of First Strand Master Mix was added to each  sample and placed on a thermal cycler to run preprogrammed thermal conditions.  d. Second Strand cDNA Synthesis: 50 μl Second Strand Master Mix was added to  each sample, mixed well and incubated in a thermal cycler at 16oC for one hour. e. cDNA Purification: cDNA was purified using Agencourt AMPure XP Beads (1.8  volumes), eluted in 10 μl of H2O, and stored at -20oC.  f. End Repair: End Repair Mix was added to the purified samples from step e and the  samples were incubated in a preprogrammed thermal cycler.  g. Adaptor Ligation: Samples were mixed with 3 μl adaptor mix, then 12 μl Ligation  Mix, and incubated in a thermal cycler at 25oC for 30 minutes.  Sample and Barcode Information: Samples were barcoded with NuGEN Unique  Dual Indexes. Strand Selection: 70 μl of Strand Selection Master Mix was added to 30 μl of each  sample, incubated in a pre-heated thermal cycler at 72oC for 10 minutes. Strand Selection Purification: Strand selected cDNA was purified using  Agencourt AMPure XP Beads (0.8 volumes), eluted in 15 μl of H2O, and stored at -  20oC.  j. Library Amplification: 76.5 μl of Library Amplification Master Mix was added to  13.5 μl of each sample and incubated in a preprogrammed thermal cycler, for 13-  16 cycles, which was pre-optimized with qPCR.  k. Amplified Library Purification: Amplified libraries were purified using 90 μl  Agencourt AMPure XP Beads (1.0 volumes). 25 μl of eluted libraries were collected  and stored at -20oC.  l. Validate Library: The concentration of libraries were measured by Qubit dsDNA  HS Assay Kit (Invitrogen Q32851). Libraries were diluted and normalized to the  optimal range for Agilent Bioanalyzer analysis using the DNA High Sensitivity Kit  (Agilent Technologies, Cat# 5067-4626).  m. Normalize and Pool Libraries: Same amount of libraries were pooled based on  the molar concentration from Bioanalyzer measurements. Library Denaturing and Diluting for MiSeq Nano 300: Pooled library was run on MiSeq  to test quantity and quality, using the MiSeq Reagent Nano Kit V2 300 cycles (Illumina, Cat.  No. MS-103-1001). Library and PhiX control (Illumina, Cat. No. FC-110-3001) were  denatured and diluted using the standard normalization method following manufacturer's  directions, to a final concentration of 6pM. Equal volume of library and PhiX were combined and  sequenced on Illumina MiSeq.  (https://basespace.illumina.com/run/196126950/JY_Xiaoling07072020_test)  3. Library Denaturing and Diluting for Nextseq 500: Library and PhiX were denatured and  diluted using the standard normalization method following manufacturer's directions. The  loading of library pool was 1.3 ml at 1.8 pM, with 1% PhIX spike in.  Sequencing Run: Sequencing was performed on the University of Louisville Brown Cancer  Center Genomics Core Illumina NextSeq 500 using the NextSeq 500/550 75 cycle High  Output Kit v2.5 (20024906).
!Sample_description = JY_10_HD_4
!Sample_data_processing = Base calling: raw .bcl files were converted to fastq files using bcl2fastq v2.20.0.422.
!Sample_data_processing = Demultiplexing: reads were demultiplexed based on the 8-base NuGen sample barcode using a custom Perl script.
!Sample_data_processing = Removal of PCR duplicates: PCR duplicates were removed by collapsing sequences with 100% identity over all 92 bases (76 sequenced bases plus 8 base sample index plus 8 base UMI) using a custom Perl script.
!Sample_data_processing = Quality Assurance/Quality Control: QA/QC was performed using FastQC v0.10.1.  No issues were found, so no further trimming or other QA/QC steps were deemed necessary.
!Sample_data_processing = Alignment: Raw fastq sequences were aligned to the human reference genome (hg38.p12) using STAR v2.6 guided by an ENSEMBL v93 gtf with mitochondrial and ribosomal RNAs removed.
!Sample_data_processing = Count extraction: Raw read counts were obtained from the STAR aligned bam files using HTSeq v0.10.0.
!Sample_data_processing = Genome_build: hg38.p12
!Sample_data_processing = Supplementary_files_format_and_content: htseq_rawCounts.txt
!Sample_platform_id = GPL18573
!Sample_contact_name = Eric,Christian,Rouchka
!Sample_contact_email = eric.rouchka@louisville.edu
!Sample_contact_laboratory = KY INBRE Bioinformatics Core
!Sample_contact_department = Biochemistry and Molecular Genetics
!Sample_contact_institute = University of Louisville
!Sample_contact_address = 522 East Gray Street
!Sample_contact_city = Louisville
!Sample_contact_state = Kentucky
!Sample_contact_zip/postal_code = 40292
!Sample_contact_country = USA
!Sample_instrument_model = Illumina NextSeq 500
!Sample_library_selection = cDNA
!Sample_library_source = transcriptomic
!Sample_library_strategy = RNA-Seq
!Sample_relation = BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN15517448
!Sample_relation = SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX8719045
!Sample_supplementary_file_1 = NONE
!Sample_series_id = GSE154311
!Sample_data_row_count = 0
^SAMPLE = GSM4668816
!Sample_title = CD16High, replicate 1
!Sample_geo_accession = GSM4668816
!Sample_status = Public on Jul 20 2021
!Sample_submission_date = Jul 13 2020
!Sample_last_update_date = Jul 20 2021
!Sample_type = SRA
!Sample_channel_count = 1
!Sample_source_name_ch1 = CD16High, replicate 1
!Sample_organism_ch1 = Homo sapiens
!Sample_taxid_ch1 = 9606
!Sample_characteristics_ch1 = disease: COVID-19
!Sample_characteristics_ch1 = neutrophils: CD16High
!Sample_characteristics_ch1 = tissue: peripheral blood
!Sample_treatment_protocol_ch1 = CD16High and CD16Int neutrophils were sorted by FACSAria III from three severe COVID-19 patients and NDN were purified from healthy donor peripheral blood.
!Sample_molecule_ch1 = polyA RNA
!Sample_extract_protocol_ch1 = RNA was extracted using Qiagen's RNAeasy Plus Microkit.
!Sample_extract_protocol_ch1 = Library Preparation: Libraries were prepared using the Universal Plus mRNA-Seq with (NuGEN Cat# 0520-24).  a. Poly(A) Selection: 8-30 ng of RNA samples (in a volume of 50 μl) were mixed with  Oligo (dT) Beads and incubated at 65oC for 5 minutes, mixed, then at room  temperature for 10 mins. The supernatant was discarded and the beads were  washed with wash buffer. Captured polyadenylated RNA was eluted using Elution  buffer at 80°C for 2 min. mRNA is further purified in a second bead clean-up.  b. RNA Fragmentation: Beads were mixed with 20 μl Fragmentation Buffer and  incubated for 8 minutes at 94oC. 20 μl of supernatant was removed from the beads  and proceeded immediately to First Strand cDNA Synthesis.  c. First Strand cDNA Synthesis: 5 μl of First Strand Master Mix was added to each  sample and placed on a thermal cycler to run preprogrammed thermal conditions.  d. Second Strand cDNA Synthesis: 50 μl Second Strand Master Mix was added to  each sample, mixed well and incubated in a thermal cycler at 16oC for one hour. e. cDNA Purification: cDNA was purified using Agencourt AMPure XP Beads (1.8  volumes), eluted in 10 μl of H2O, and stored at -20oC.  f. End Repair: End Repair Mix was added to the purified samples from step e and the  samples were incubated in a preprogrammed thermal cycler.  g. Adaptor Ligation: Samples were mixed with 3 μl adaptor mix, then 12 μl Ligation  Mix, and incubated in a thermal cycler at 25oC for 30 minutes.  Sample and Barcode Information: Samples were barcoded with NuGEN Unique  Dual Indexes. Strand Selection: 70 μl of Strand Selection Master Mix was added to 30 μl of each  sample, incubated in a pre-heated thermal cycler at 72oC for 10 minutes. Strand Selection Purification: Strand selected cDNA was purified using  Agencourt AMPure XP Beads (0.8 volumes), eluted in 15 μl of H2O, and stored at -  20oC.  j. Library Amplification: 76.5 μl of Library Amplification Master Mix was added to  13.5 μl of each sample and incubated in a preprogrammed thermal cycler, for 13-  16 cycles, which was pre-optimized with qPCR.  k. Amplified Library Purification: Amplified libraries were purified using 90 μl  Agencourt AMPure XP Beads (1.0 volumes). 25 μl of eluted libraries were collected  and stored at -20oC.  l. Validate Library: The concentration of libraries were measured by Qubit dsDNA  HS Assay Kit (Invitrogen Q32851). Libraries were diluted and normalized to the  optimal range for Agilent Bioanalyzer analysis using the DNA High Sensitivity Kit  (Agilent Technologies, Cat# 5067-4626).  m. Normalize and Pool Libraries: Same amount of libraries were pooled based on  the molar concentration from Bioanalyzer measurements. Library Denaturing and Diluting for MiSeq Nano 300: Pooled library was run on MiSeq  to test quantity and quality, using the MiSeq Reagent Nano Kit V2 300 cycles (Illumina, Cat.  No. MS-103-1001). Library and PhiX control (Illumina, Cat. No. FC-110-3001) were  denatured and diluted using the standard normalization method following manufacturer's  directions, to a final concentration of 6pM. Equal volume of library and PhiX were combined and  sequenced on Illumina MiSeq.  (https://basespace.illumina.com/run/196126950/JY_Xiaoling07072020_test)  3. Library Denaturing and Diluting for Nextseq 500: Library and PhiX were denatured and  diluted using the standard normalization method following manufacturer's directions. The  loading of library pool was 1.3 ml at 1.8 pM, with 1% PhIX spike in.  Sequencing Run: Sequencing was performed on the University of Louisville Brown Cancer  Center Genomics Core Illumina NextSeq 500 using the NextSeq 500/550 75 cycle High  Output Kit v2.5 (20024906).
!Sample_description = JY_04_NDN_2
!Sample_data_processing = Base calling: raw .bcl files were converted to fastq files using bcl2fastq v2.20.0.422.
!Sample_data_processing = Demultiplexing: reads were demultiplexed based on the 8-base NuGen sample barcode using a custom Perl script.
!Sample_data_processing = Removal of PCR duplicates: PCR duplicates were removed by collapsing sequences with 100% identity over all 92 bases (76 sequenced bases plus 8 base sample index plus 8 base UMI) using a custom Perl script.
!Sample_data_processing = Quality Assurance/Quality Control: QA/QC was performed using FastQC v0.10.1.  No issues were found, so no further trimming or other QA/QC steps were deemed necessary.
!Sample_data_processing = Alignment: Raw fastq sequences were aligned to the human reference genome (hg38.p12) using STAR v2.6 guided by an ENSEMBL v93 gtf with mitochondrial and ribosomal RNAs removed.
!Sample_data_processing = Count extraction: Raw read counts were obtained from the STAR aligned bam files using HTSeq v0.10.0.
!Sample_data_processing = Genome_build: hg38.p12
!Sample_data_processing = Supplementary_files_format_and_content: htseq_rawCounts.txt
!Sample_platform_id = GPL18573
!Sample_contact_name = Eric,Christian,Rouchka
!Sample_contact_email = eric.rouchka@louisville.edu
!Sample_contact_laboratory = KY INBRE Bioinformatics Core
!Sample_contact_department = Biochemistry and Molecular Genetics
!Sample_contact_institute = University of Louisville
!Sample_contact_address = 522 East Gray Street
!Sample_contact_city = Louisville
!Sample_contact_state = Kentucky
!Sample_contact_zip/postal_code = 40292
!Sample_contact_country = USA
!Sample_instrument_model = Illumina NextSeq 500
!Sample_library_selection = cDNA
!Sample_library_source = transcriptomic
!Sample_library_strategy = RNA-Seq
!Sample_relation = BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN15517447
!Sample_relation = SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX8719046
!Sample_supplementary_file_1 = NONE
!Sample_series_id = GSE154311
!Sample_data_row_count = 0
^SAMPLE = GSM4668817
!Sample_title = CD16High, replicate 2
!Sample_geo_accession = GSM4668817
!Sample_status = Public on Jul 20 2021
!Sample_submission_date = Jul 13 2020
!Sample_last_update_date = Jul 20 2021
!Sample_type = SRA
!Sample_channel_count = 1
!Sample_source_name_ch1 = CD16High, replicate 2
!Sample_organism_ch1 = Homo sapiens
!Sample_taxid_ch1 = 9606
!Sample_characteristics_ch1 = disease: COVID-19
!Sample_characteristics_ch1 = neutrophils: CD16High
!Sample_characteristics_ch1 = tissue: peripheral blood
!Sample_treatment_protocol_ch1 = CD16High and CD16Int neutrophils were sorted by FACSAria III from three severe COVID-19 patients and NDN were purified from healthy donor peripheral blood.
!Sample_molecule_ch1 = polyA RNA
!Sample_extract_protocol_ch1 = RNA was extracted using Qiagen's RNAeasy Plus Microkit.
!Sample_extract_protocol_ch1 = Library Preparation: Libraries were prepared using the Universal Plus mRNA-Seq with (NuGEN Cat# 0520-24).  a. Poly(A) Selection: 8-30 ng of RNA samples (in a volume of 50 μl) were mixed with  Oligo (dT) Beads and incubated at 65oC for 5 minutes, mixed, then at room  temperature for 10 mins. The supernatant was discarded and the beads were  washed with wash buffer. Captured polyadenylated RNA was eluted using Elution  buffer at 80°C for 2 min. mRNA is further purified in a second bead clean-up.  b. RNA Fragmentation: Beads were mixed with 20 μl Fragmentation Buffer and  incubated for 8 minutes at 94oC. 20 μl of supernatant was removed from the beads  and proceeded immediately to First Strand cDNA Synthesis.  c. First Strand cDNA Synthesis: 5 μl of First Strand Master Mix was added to each  sample and placed on a thermal cycler to run preprogrammed thermal conditions.  d. Second Strand cDNA Synthesis: 50 μl Second Strand Master Mix was added to  each sample, mixed well and incubated in a thermal cycler at 16oC for one hour. e. cDNA Purification: cDNA was purified using Agencourt AMPure XP Beads (1.8  volumes), eluted in 10 μl of H2O, and stored at -20oC.  f. End Repair: End Repair Mix was added to the purified samples from step e and the  samples were incubated in a preprogrammed thermal cycler.  g. Adaptor Ligation: Samples were mixed with 3 μl adaptor mix, then 12 μl Ligation  Mix, and incubated in a thermal cycler at 25oC for 30 minutes.  Sample and Barcode Information: Samples were barcoded with NuGEN Unique  Dual Indexes. Strand Selection: 70 μl of Strand Selection Master Mix was added to 30 μl of each  sample, incubated in a pre-heated thermal cycler at 72oC for 10 minutes. Strand Selection Purification: Strand selected cDNA was purified using  Agencourt AMPure XP Beads (0.8 volumes), eluted in 15 μl of H2O, and stored at -  20oC.  j. Library Amplification: 76.5 μl of Library Amplification Master Mix was added to  13.5 μl of each sample and incubated in a preprogrammed thermal cycler, for 13-  16 cycles, which was pre-optimized with qPCR.  k. Amplified Library Purification: Amplified libraries were purified using 90 μl  Agencourt AMPure XP Beads (1.0 volumes). 25 μl of eluted libraries were collected  and stored at -20oC.  l. Validate Library: The concentration of libraries were measured by Qubit dsDNA  HS Assay Kit (Invitrogen Q32851). Libraries were diluted and normalized to the  optimal range for Agilent Bioanalyzer analysis using the DNA High Sensitivity Kit  (Agilent Technologies, Cat# 5067-4626).  m. Normalize and Pool Libraries: Same amount of libraries were pooled based on  the molar concentration from Bioanalyzer measurements. Library Denaturing and Diluting for MiSeq Nano 300: Pooled library was run on MiSeq  to test quantity and quality, using the MiSeq Reagent Nano Kit V2 300 cycles (Illumina, Cat.  No. MS-103-1001). Library and PhiX control (Illumina, Cat. No. FC-110-3001) were  denatured and diluted using the standard normalization method following manufacturer's  directions, to a final concentration of 6pM. Equal volume of library and PhiX were combined and  sequenced on Illumina MiSeq.  (https://basespace.illumina.com/run/196126950/JY_Xiaoling07072020_test)  3. Library Denaturing and Diluting for Nextseq 500: Library and PhiX were denatured and  diluted using the standard normalization method following manufacturer's directions. The  loading of library pool was 1.3 ml at 1.8 pM, with 1% PhIX spike in.  Sequencing Run: Sequencing was performed on the University of Louisville Brown Cancer  Center Genomics Core Illumina NextSeq 500 using the NextSeq 500/550 75 cycle High  Output Kit v2.5 (20024906).
!Sample_description = JY_06_NDN_3
!Sample_data_processing = Base calling: raw .bcl files were converted to fastq files using bcl2fastq v2.20.0.422.
!Sample_data_processing = Demultiplexing: reads were demultiplexed based on the 8-base NuGen sample barcode using a custom Perl script.
!Sample_data_processing = Removal of PCR duplicates: PCR duplicates were removed by collapsing sequences with 100% identity over all 92 bases (76 sequenced bases plus 8 base sample index plus 8 base UMI) using a custom Perl script.
!Sample_data_processing = Quality Assurance/Quality Control: QA/QC was performed using FastQC v0.10.1.  No issues were found, so no further trimming or other QA/QC steps were deemed necessary.
!Sample_data_processing = Alignment: Raw fastq sequences were aligned to the human reference genome (hg38.p12) using STAR v2.6 guided by an ENSEMBL v93 gtf with mitochondrial and ribosomal RNAs removed.
!Sample_data_processing = Count extraction: Raw read counts were obtained from the STAR aligned bam files using HTSeq v0.10.0.
!Sample_data_processing = Genome_build: hg38.p12
!Sample_data_processing = Supplementary_files_format_and_content: htseq_rawCounts.txt
!Sample_platform_id = GPL18573
!Sample_contact_name = Eric,Christian,Rouchka
!Sample_contact_email = eric.rouchka@louisville.edu
!Sample_contact_laboratory = KY INBRE Bioinformatics Core
!Sample_contact_department = Biochemistry and Molecular Genetics
!Sample_contact_institute = University of Louisville
!Sample_contact_address = 522 East Gray Street
!Sample_contact_city = Louisville
!Sample_contact_state = Kentucky
!Sample_contact_zip/postal_code = 40292
!Sample_contact_country = USA
!Sample_instrument_model = Illumina NextSeq 500
!Sample_library_selection = cDNA
!Sample_library_source = transcriptomic
!Sample_library_strategy = RNA-Seq
!Sample_relation = BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN15517446
!Sample_relation = SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX8719047
!Sample_supplementary_file_1 = NONE
!Sample_series_id = GSE154311
!Sample_data_row_count = 0
^SAMPLE = GSM4668818
!Sample_title = CD16High, replicate 3
!Sample_geo_accession = GSM4668818
!Sample_status = Public on Jul 20 2021
!Sample_submission_date = Jul 13 2020
!Sample_last_update_date = Jul 20 2021
!Sample_type = SRA
!Sample_channel_count = 1
!Sample_source_name_ch1 = CD16High, replicate 3
!Sample_organism_ch1 = Homo sapiens
!Sample_taxid_ch1 = 9606
!Sample_characteristics_ch1 = disease: COVID-19
!Sample_characteristics_ch1 = neutrophils: CD16High
!Sample_characteristics_ch1 = tissue: peripheral blood
!Sample_treatment_protocol_ch1 = CD16High and CD16Int neutrophils were sorted by FACSAria III from three severe COVID-19 patients and NDN were purified from healthy donor peripheral blood.
!Sample_molecule_ch1 = polyA RNA
!Sample_extract_protocol_ch1 = RNA was extracted using Qiagen's RNAeasy Plus Microkit.
!Sample_extract_protocol_ch1 = Library Preparation: Libraries were prepared using the Universal Plus mRNA-Seq with (NuGEN Cat# 0520-24).  a. Poly(A) Selection: 8-30 ng of RNA samples (in a volume of 50 μl) were mixed with  Oligo (dT) Beads and incubated at 65oC for 5 minutes, mixed, then at room  temperature for 10 mins. The supernatant was discarded and the beads were  washed with wash buffer. Captured polyadenylated RNA was eluted using Elution  buffer at 80°C for 2 min. mRNA is further purified in a second bead clean-up.  b. RNA Fragmentation: Beads were mixed with 20 μl Fragmentation Buffer and  incubated for 8 minutes at 94oC. 20 μl of supernatant was removed from the beads  and proceeded immediately to First Strand cDNA Synthesis.  c. First Strand cDNA Synthesis: 5 μl of First Strand Master Mix was added to each  sample and placed on a thermal cycler to run preprogrammed thermal conditions.  d. Second Strand cDNA Synthesis: 50 μl Second Strand Master Mix was added to  each sample, mixed well and incubated in a thermal cycler at 16oC for one hour. e. cDNA Purification: cDNA was purified using Agencourt AMPure XP Beads (1.8  volumes), eluted in 10 μl of H2O, and stored at -20oC.  f. End Repair: End Repair Mix was added to the purified samples from step e and the  samples were incubated in a preprogrammed thermal cycler.  g. Adaptor Ligation: Samples were mixed with 3 μl adaptor mix, then 12 μl Ligation  Mix, and incubated in a thermal cycler at 25oC for 30 minutes.  Sample and Barcode Information: Samples were barcoded with NuGEN Unique  Dual Indexes. Strand Selection: 70 μl of Strand Selection Master Mix was added to 30 μl of each  sample, incubated in a pre-heated thermal cycler at 72oC for 10 minutes. Strand Selection Purification: Strand selected cDNA was purified using  Agencourt AMPure XP Beads (0.8 volumes), eluted in 15 μl of H2O, and stored at -  20oC.  j. Library Amplification: 76.5 μl of Library Amplification Master Mix was added to  13.5 μl of each sample and incubated in a preprogrammed thermal cycler, for 13-  16 cycles, which was pre-optimized with qPCR.  k. Amplified Library Purification: Amplified libraries were purified using 90 μl  Agencourt AMPure XP Beads (1.0 volumes). 25 μl of eluted libraries were collected  and stored at -20oC.  l. Validate Library: The concentration of libraries were measured by Qubit dsDNA  HS Assay Kit (Invitrogen Q32851). Libraries were diluted and normalized to the  optimal range for Agilent Bioanalyzer analysis using the DNA High Sensitivity Kit  (Agilent Technologies, Cat# 5067-4626).  m. Normalize and Pool Libraries: Same amount of libraries were pooled based on  the molar concentration from Bioanalyzer measurements. Library Denaturing and Diluting for MiSeq Nano 300: Pooled library was run on MiSeq  to test quantity and quality, using the MiSeq Reagent Nano Kit V2 300 cycles (Illumina, Cat.  No. MS-103-1001). Library and PhiX control (Illumina, Cat. No. FC-110-3001) were  denatured and diluted using the standard normalization method following manufacturer's  directions, to a final concentration of 6pM. Equal volume of library and PhiX were combined and  sequenced on Illumina MiSeq.  (https://basespace.illumina.com/run/196126950/JY_Xiaoling07072020_test)  3. Library Denaturing and Diluting for Nextseq 500: Library and PhiX were denatured and  diluted using the standard normalization method following manufacturer's directions. The  loading of library pool was 1.3 ml at 1.8 pM, with 1% PhIX spike in.  Sequencing Run: Sequencing was performed on the University of Louisville Brown Cancer  Center Genomics Core Illumina NextSeq 500 using the NextSeq 500/550 75 cycle High  Output Kit v2.5 (20024906).
!Sample_description = JY_13_NDN_4
!Sample_data_processing = Base calling: raw .bcl files were converted to fastq files using bcl2fastq v2.20.0.422.
!Sample_data_processing = Demultiplexing: reads were demultiplexed based on the 8-base NuGen sample barcode using a custom Perl script.
!Sample_data_processing = Removal of PCR duplicates: PCR duplicates were removed by collapsing sequences with 100% identity over all 92 bases (76 sequenced bases plus 8 base sample index plus 8 base UMI) using a custom Perl script.
!Sample_data_processing = Quality Assurance/Quality Control: QA/QC was performed using FastQC v0.10.1.  No issues were found, so no further trimming or other QA/QC steps were deemed necessary.
!Sample_data_processing = Alignment: Raw fastq sequences were aligned to the human reference genome (hg38.p12) using STAR v2.6 guided by an ENSEMBL v93 gtf with mitochondrial and ribosomal RNAs removed.
!Sample_data_processing = Count extraction: Raw read counts were obtained from the STAR aligned bam files using HTSeq v0.10.0.
!Sample_data_processing = Genome_build: hg38.p12
!Sample_data_processing = Supplementary_files_format_and_content: htseq_rawCounts.txt
!Sample_platform_id = GPL18573
!Sample_contact_name = Eric,Christian,Rouchka
!Sample_contact_email = eric.rouchka@louisville.edu
!Sample_contact_laboratory = KY INBRE Bioinformatics Core
!Sample_contact_department = Biochemistry and Molecular Genetics
!Sample_contact_institute = University of Louisville
!Sample_contact_address = 522 East Gray Street
!Sample_contact_city = Louisville
!Sample_contact_state = Kentucky
!Sample_contact_zip/postal_code = 40292
!Sample_contact_country = USA
!Sample_instrument_model = Illumina NextSeq 500
!Sample_library_selection = cDNA
!Sample_library_source = transcriptomic
!Sample_library_strategy = RNA-Seq
!Sample_relation = BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN15517445
!Sample_relation = SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX8719048
!Sample_supplementary_file_1 = NONE
!Sample_series_id = GSE154311
!Sample_data_row_count = 0
^SAMPLE = GSM4668819
!Sample_title = CD16Int, replicate 1
!Sample_geo_accession = GSM4668819
!Sample_status = Public on Jul 20 2021
!Sample_submission_date = Jul 13 2020
!Sample_last_update_date = Jul 20 2021
!Sample_type = SRA
!Sample_channel_count = 1
!Sample_source_name_ch1 = CD16Int, replicate 1
!Sample_organism_ch1 = Homo sapiens
!Sample_taxid_ch1 = 9606
!Sample_characteristics_ch1 = disease: COVID-19
!Sample_characteristics_ch1 = neutrophils: CD16Int
!Sample_characteristics_ch1 = tissue: peripheral blood
!Sample_treatment_protocol_ch1 = CD16High and CD16Int neutrophils were sorted by FACSAria III from three severe COVID-19 patients and NDN were purified from healthy donor peripheral blood.
!Sample_molecule_ch1 = polyA RNA
!Sample_extract_protocol_ch1 = RNA was extracted using Qiagen's RNAeasy Plus Microkit.
!Sample_extract_protocol_ch1 = Library Preparation: Libraries were prepared using the Universal Plus mRNA-Seq with (NuGEN Cat# 0520-24).  a. Poly(A) Selection: 8-30 ng of RNA samples (in a volume of 50 μl) were mixed with  Oligo (dT) Beads and incubated at 65oC for 5 minutes, mixed, then at room  temperature for 10 mins. The supernatant was discarded and the beads were  washed with wash buffer. Captured polyadenylated RNA was eluted using Elution  buffer at 80°C for 2 min. mRNA is further purified in a second bead clean-up.  b. RNA Fragmentation: Beads were mixed with 20 μl Fragmentation Buffer and  incubated for 8 minutes at 94oC. 20 μl of supernatant was removed from the beads  and proceeded immediately to First Strand cDNA Synthesis.  c. First Strand cDNA Synthesis: 5 μl of First Strand Master Mix was added to each  sample and placed on a thermal cycler to run preprogrammed thermal conditions.  d. Second Strand cDNA Synthesis: 50 μl Second Strand Master Mix was added to  each sample, mixed well and incubated in a thermal cycler at 16oC for one hour. e. cDNA Purification: cDNA was purified using Agencourt AMPure XP Beads (1.8  volumes), eluted in 10 μl of H2O, and stored at -20oC.  f. End Repair: End Repair Mix was added to the purified samples from step e and the  samples were incubated in a preprogrammed thermal cycler.  g. Adaptor Ligation: Samples were mixed with 3 μl adaptor mix, then 12 μl Ligation  Mix, and incubated in a thermal cycler at 25oC for 30 minutes.  Sample and Barcode Information: Samples were barcoded with NuGEN Unique  Dual Indexes. Strand Selection: 70 μl of Strand Selection Master Mix was added to 30 μl of each  sample, incubated in a pre-heated thermal cycler at 72oC for 10 minutes. Strand Selection Purification: Strand selected cDNA was purified using  Agencourt AMPure XP Beads (0.8 volumes), eluted in 15 μl of H2O, and stored at -  20oC.  j. Library Amplification: 76.5 μl of Library Amplification Master Mix was added to  13.5 μl of each sample and incubated in a preprogrammed thermal cycler, for 13-  16 cycles, which was pre-optimized with qPCR.  k. Amplified Library Purification: Amplified libraries were purified using 90 μl  Agencourt AMPure XP Beads (1.0 volumes). 25 μl of eluted libraries were collected  and stored at -20oC.  l. Validate Library: The concentration of libraries were measured by Qubit dsDNA  HS Assay Kit (Invitrogen Q32851). Libraries were diluted and normalized to the  optimal range for Agilent Bioanalyzer analysis using the DNA High Sensitivity Kit  (Agilent Technologies, Cat# 5067-4626).  m. Normalize and Pool Libraries: Same amount of libraries were pooled based on  the molar concentration from Bioanalyzer measurements. Library Denaturing and Diluting for MiSeq Nano 300: Pooled library was run on MiSeq  to test quantity and quality, using the MiSeq Reagent Nano Kit V2 300 cycles (Illumina, Cat.  No. MS-103-1001). Library and PhiX control (Illumina, Cat. No. FC-110-3001) were  denatured and diluted using the standard normalization method following manufacturer's  directions, to a final concentration of 6pM. Equal volume of library and PhiX were combined and  sequenced on Illumina MiSeq.  (https://basespace.illumina.com/run/196126950/JY_Xiaoling07072020_test)  3. Library Denaturing and Diluting for Nextseq 500: Library and PhiX were denatured and  diluted using the standard normalization method following manufacturer's directions. The  loading of library pool was 1.3 ml at 1.8 pM, with 1% PhIX spike in.  Sequencing Run: Sequencing was performed on the University of Louisville Brown Cancer  Center Genomics Core Illumina NextSeq 500 using the NextSeq 500/550 75 cycle High  Output Kit v2.5 (20024906).
!Sample_description = JY_03_LDN_2
!Sample_data_processing = Base calling: raw .bcl files were converted to fastq files using bcl2fastq v2.20.0.422.
!Sample_data_processing = Demultiplexing: reads were demultiplexed based on the 8-base NuGen sample barcode using a custom Perl script.
!Sample_data_processing = Removal of PCR duplicates: PCR duplicates were removed by collapsing sequences with 100% identity over all 92 bases (76 sequenced bases plus 8 base sample index plus 8 base UMI) using a custom Perl script.
!Sample_data_processing = Quality Assurance/Quality Control: QA/QC was performed using FastQC v0.10.1.  No issues were found, so no further trimming or other QA/QC steps were deemed necessary.
!Sample_data_processing = Alignment: Raw fastq sequences were aligned to the human reference genome (hg38.p12) using STAR v2.6 guided by an ENSEMBL v93 gtf with mitochondrial and ribosomal RNAs removed.
!Sample_data_processing = Count extraction: Raw read counts were obtained from the STAR aligned bam files using HTSeq v0.10.0.
!Sample_data_processing = Genome_build: hg38.p12
!Sample_data_processing = Supplementary_files_format_and_content: htseq_rawCounts.txt
!Sample_platform_id = GPL18573
!Sample_contact_name = Eric,Christian,Rouchka
!Sample_contact_email = eric.rouchka@louisville.edu
!Sample_contact_laboratory = KY INBRE Bioinformatics Core
!Sample_contact_department = Biochemistry and Molecular Genetics
!Sample_contact_institute = University of Louisville
!Sample_contact_address = 522 East Gray Street
!Sample_contact_city = Louisville
!Sample_contact_state = Kentucky
!Sample_contact_zip/postal_code = 40292
!Sample_contact_country = USA
!Sample_instrument_model = Illumina NextSeq 500
!Sample_library_selection = cDNA
!Sample_library_source = transcriptomic
!Sample_library_strategy = RNA-Seq
!Sample_relation = BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN15517444
!Sample_relation = SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX8719049
!Sample_supplementary_file_1 = NONE
!Sample_series_id = GSE154311
!Sample_data_row_count = 0
^SAMPLE = GSM4668820
!Sample_title = CD16Int, replicate 2
!Sample_geo_accession = GSM4668820
!Sample_status = Public on Jul 20 2021
!Sample_submission_date = Jul 13 2020
!Sample_last_update_date = Jul 20 2021
!Sample_type = SRA
!Sample_channel_count = 1
!Sample_source_name_ch1 = CD16Int, replicate 2
!Sample_organism_ch1 = Homo sapiens
!Sample_taxid_ch1 = 9606
!Sample_characteristics_ch1 = disease: COVID-19
!Sample_characteristics_ch1 = neutrophils: CD16Int
!Sample_characteristics_ch1 = tissue: peripheral blood
!Sample_treatment_protocol_ch1 = CD16High and CD16Int neutrophils were sorted by FACSAria III from three severe COVID-19 patients and NDN were purified from healthy donor peripheral blood.
!Sample_molecule_ch1 = polyA RNA
!Sample_extract_protocol_ch1 = RNA was extracted using Qiagen's RNAeasy Plus Microkit.
!Sample_extract_protocol_ch1 = Library Preparation: Libraries were prepared using the Universal Plus mRNA-Seq with (NuGEN Cat# 0520-24).  a. Poly(A) Selection: 8-30 ng of RNA samples (in a volume of 50 μl) were mixed with  Oligo (dT) Beads and incubated at 65oC for 5 minutes, mixed, then at room  temperature for 10 mins. The supernatant was discarded and the beads were  washed with wash buffer. Captured polyadenylated RNA was eluted using Elution  buffer at 80°C for 2 min. mRNA is further purified in a second bead clean-up.  b. RNA Fragmentation: Beads were mixed with 20 μl Fragmentation Buffer and  incubated for 8 minutes at 94oC. 20 μl of supernatant was removed from the beads  and proceeded immediately to First Strand cDNA Synthesis.  c. First Strand cDNA Synthesis: 5 μl of First Strand Master Mix was added to each  sample and placed on a thermal cycler to run preprogrammed thermal conditions.  d. Second Strand cDNA Synthesis: 50 μl Second Strand Master Mix was added to  each sample, mixed well and incubated in a thermal cycler at 16oC for one hour. e. cDNA Purification: cDNA was purified using Agencourt AMPure XP Beads (1.8  volumes), eluted in 10 μl of H2O, and stored at -20oC.  f. End Repair: End Repair Mix was added to the purified samples from step e and the  samples were incubated in a preprogrammed thermal cycler.  g. Adaptor Ligation: Samples were mixed with 3 μl adaptor mix, then 12 μl Ligation  Mix, and incubated in a thermal cycler at 25oC for 30 minutes.  Sample and Barcode Information: Samples were barcoded with NuGEN Unique  Dual Indexes. Strand Selection: 70 μl of Strand Selection Master Mix was added to 30 μl of each  sample, incubated in a pre-heated thermal cycler at 72oC for 10 minutes. Strand Selection Purification: Strand selected cDNA was purified using  Agencourt AMPure XP Beads (0.8 volumes), eluted in 15 μl of H2O, and stored at -  20oC.  j. Library Amplification: 76.5 μl of Library Amplification Master Mix was added to  13.5 μl of each sample and incubated in a preprogrammed thermal cycler, for 13-  16 cycles, which was pre-optimized with qPCR.  k. Amplified Library Purification: Amplified libraries were purified using 90 μl  Agencourt AMPure XP Beads (1.0 volumes). 25 μl of eluted libraries were collected  and stored at -20oC.  l. Validate Library: The concentration of libraries were measured by Qubit dsDNA  HS Assay Kit (Invitrogen Q32851). Libraries were diluted and normalized to the  optimal range for Agilent Bioanalyzer analysis using the DNA High Sensitivity Kit  (Agilent Technologies, Cat# 5067-4626).  m. Normalize and Pool Libraries: Same amount of libraries were pooled based on  the molar concentration from Bioanalyzer measurements. Library Denaturing and Diluting for MiSeq Nano 300: Pooled library was run on MiSeq  to test quantity and quality, using the MiSeq Reagent Nano Kit V2 300 cycles (Illumina, Cat.  No. MS-103-1001). Library and PhiX control (Illumina, Cat. No. FC-110-3001) were  denatured and diluted using the standard normalization method following manufacturer's  directions, to a final concentration of 6pM. Equal volume of library and PhiX were combined and  sequenced on Illumina MiSeq.  (https://basespace.illumina.com/run/196126950/JY_Xiaoling07072020_test)  3. Library Denaturing and Diluting for Nextseq 500: Library and PhiX were denatured and  diluted using the standard normalization method following manufacturer's directions. The  loading of library pool was 1.3 ml at 1.8 pM, with 1% PhIX spike in.  Sequencing Run: Sequencing was performed on the University of Louisville Brown Cancer  Center Genomics Core Illumina NextSeq 500 using the NextSeq 500/550 75 cycle High  Output Kit v2.5 (20024906).
!Sample_description = JY_05_LDN_3
!Sample_data_processing = Base calling: raw .bcl files were converted to fastq files using bcl2fastq v2.20.0.422.
!Sample_data_processing = Demultiplexing: reads were demultiplexed based on the 8-base NuGen sample barcode using a custom Perl script.
!Sample_data_processing = Removal of PCR duplicates: PCR duplicates were removed by collapsing sequences with 100% identity over all 92 bases (76 sequenced bases plus 8 base sample index plus 8 base UMI) using a custom Perl script.
!Sample_data_processing = Quality Assurance/Quality Control: QA/QC was performed using FastQC v0.10.1.  No issues were found, so no further trimming or other QA/QC steps were deemed necessary.
!Sample_data_processing = Alignment: Raw fastq sequences were aligned to the human reference genome (hg38.p12) using STAR v2.6 guided by an ENSEMBL v93 gtf with mitochondrial and ribosomal RNAs removed.
!Sample_data_processing = Count extraction: Raw read counts were obtained from the STAR aligned bam files using HTSeq v0.10.0.
!Sample_data_processing = Genome_build: hg38.p12
!Sample_data_processing = Supplementary_files_format_and_content: htseq_rawCounts.txt
!Sample_platform_id = GPL18573
!Sample_contact_name = Eric,Christian,Rouchka
!Sample_contact_email = eric.rouchka@louisville.edu
!Sample_contact_laboratory = KY INBRE Bioinformatics Core
!Sample_contact_department = Biochemistry and Molecular Genetics
!Sample_contact_institute = University of Louisville
!Sample_contact_address = 522 East Gray Street
!Sample_contact_city = Louisville
!Sample_contact_state = Kentucky
!Sample_contact_zip/postal_code = 40292
!Sample_contact_country = USA
!Sample_instrument_model = Illumina NextSeq 500
!Sample_library_selection = cDNA
!Sample_library_source = transcriptomic
!Sample_library_strategy = RNA-Seq
!Sample_relation = BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN15517443
!Sample_relation = SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX8719050
!Sample_supplementary_file_1 = NONE
!Sample_series_id = GSE154311
!Sample_data_row_count = 0
^SAMPLE = GSM4668821
!Sample_title = CD16Int, replicate 3
!Sample_geo_accession = GSM4668821
!Sample_status = Public on Jul 20 2021
!Sample_submission_date = Jul 13 2020
!Sample_last_update_date = Jul 20 2021
!Sample_type = SRA
!Sample_channel_count = 1
!Sample_source_name_ch1 = CD16Int, replicate 3
!Sample_organism_ch1 = Homo sapiens
!Sample_taxid_ch1 = 9606
!Sample_characteristics_ch1 = disease: COVID-19
!Sample_characteristics_ch1 = neutrophils: CD16Int
!Sample_characteristics_ch1 = tissue: peripheral blood
!Sample_treatment_protocol_ch1 = CD16High and CD16Int neutrophils were sorted by FACSAria III from three severe COVID-19 patients and NDN were purified from healthy donor peripheral blood.
!Sample_molecule_ch1 = polyA RNA
!Sample_extract_protocol_ch1 = RNA was extracted using Qiagen's RNAeasy Plus Microkit.
!Sample_extract_protocol_ch1 = Library Preparation: Libraries were prepared using the Universal Plus mRNA-Seq with (NuGEN Cat# 0520-24).  a. Poly(A) Selection: 8-30 ng of RNA samples (in a volume of 50 μl) were mixed with  Oligo (dT) Beads and incubated at 65oC for 5 minutes, mixed, then at room  temperature for 10 mins. The supernatant was discarded and the beads were  washed with wash buffer. Captured polyadenylated RNA was eluted using Elution  buffer at 80°C for 2 min. mRNA is further purified in a second bead clean-up.  b. RNA Fragmentation: Beads were mixed with 20 μl Fragmentation Buffer and  incubated for 8 minutes at 94oC. 20 μl of supernatant was removed from the beads  and proceeded immediately to First Strand cDNA Synthesis.  c. First Strand cDNA Synthesis: 5 μl of First Strand Master Mix was added to each  sample and placed on a thermal cycler to run preprogrammed thermal conditions.  d. Second Strand cDNA Synthesis: 50 μl Second Strand Master Mix was added to  each sample, mixed well and incubated in a thermal cycler at 16oC for one hour. e. cDNA Purification: cDNA was purified using Agencourt AMPure XP Beads (1.8  volumes), eluted in 10 μl of H2O, and stored at -20oC.  f. End Repair: End Repair Mix was added to the purified samples from step e and the  samples were incubated in a preprogrammed thermal cycler.  g. Adaptor Ligation: Samples were mixed with 3 μl adaptor mix, then 12 μl Ligation  Mix, and incubated in a thermal cycler at 25oC for 30 minutes.  Sample and Barcode Information: Samples were barcoded with NuGEN Unique  Dual Indexes. Strand Selection: 70 μl of Strand Selection Master Mix was added to 30 μl of each  sample, incubated in a pre-heated thermal cycler at 72oC for 10 minutes. Strand Selection Purification: Strand selected cDNA was purified using  Agencourt AMPure XP Beads (0.8 volumes), eluted in 15 μl of H2O, and stored at -  20oC.  j. Library Amplification: 76.5 μl of Library Amplification Master Mix was added to  13.5 μl of each sample and incubated in a preprogrammed thermal cycler, for 13-  16 cycles, which was pre-optimized with qPCR.  k. Amplified Library Purification: Amplified libraries were purified using 90 μl  Agencourt AMPure XP Beads (1.0 volumes). 25 μl of eluted libraries were collected  and stored at -20oC.  l. Validate Library: The concentration of libraries were measured by Qubit dsDNA  HS Assay Kit (Invitrogen Q32851). Libraries were diluted and normalized to the  optimal range for Agilent Bioanalyzer analysis using the DNA High Sensitivity Kit  (Agilent Technologies, Cat# 5067-4626).  m. Normalize and Pool Libraries: Same amount of libraries were pooled based on  the molar concentration from Bioanalyzer measurements. Library Denaturing and Diluting for MiSeq Nano 300: Pooled library was run on MiSeq  to test quantity and quality, using the MiSeq Reagent Nano Kit V2 300 cycles (Illumina, Cat.  No. MS-103-1001). Library and PhiX control (Illumina, Cat. No. FC-110-3001) were  denatured and diluted using the standard normalization method following manufacturer's  directions, to a final concentration of 6pM. Equal volume of library and PhiX were combined and  sequenced on Illumina MiSeq.  (https://basespace.illumina.com/run/196126950/JY_Xiaoling07072020_test)  3. Library Denaturing and Diluting for Nextseq 500: Library and PhiX were denatured and  diluted using the standard normalization method following manufacturer's directions. The  loading of library pool was 1.3 ml at 1.8 pM, with 1% PhIX spike in.  Sequencing Run: Sequencing was performed on the University of Louisville Brown Cancer  Center Genomics Core Illumina NextSeq 500 using the NextSeq 500/550 75 cycle High  Output Kit v2.5 (20024906).
!Sample_description = JY_12_LDN_4
!Sample_data_processing = Base calling: raw .bcl files were converted to fastq files using bcl2fastq v2.20.0.422.
!Sample_data_processing = Demultiplexing: reads were demultiplexed based on the 8-base NuGen sample barcode using a custom Perl script.
!Sample_data_processing = Removal of PCR duplicates: PCR duplicates were removed by collapsing sequences with 100% identity over all 92 bases (76 sequenced bases plus 8 base sample index plus 8 base UMI) using a custom Perl script.
!Sample_data_processing = Quality Assurance/Quality Control: QA/QC was performed using FastQC v0.10.1.  No issues were found, so no further trimming or other QA/QC steps were deemed necessary.
!Sample_data_processing = Alignment: Raw fastq sequences were aligned to the human reference genome (hg38.p12) using STAR v2.6 guided by an ENSEMBL v93 gtf with mitochondrial and ribosomal RNAs removed.
!Sample_data_processing = Count extraction: Raw read counts were obtained from the STAR aligned bam files using HTSeq v0.10.0.
!Sample_data_processing = Genome_build: hg38.p12
!Sample_data_processing = Supplementary_files_format_and_content: htseq_rawCounts.txt
!Sample_platform_id = GPL18573
!Sample_contact_name = Eric,Christian,Rouchka
!Sample_contact_email = eric.rouchka@louisville.edu
!Sample_contact_laboratory = KY INBRE Bioinformatics Core
!Sample_contact_department = Biochemistry and Molecular Genetics
!Sample_contact_institute = University of Louisville
!Sample_contact_address = 522 East Gray Street
!Sample_contact_city = Louisville
!Sample_contact_state = Kentucky
!Sample_contact_zip/postal_code = 40292
!Sample_contact_country = USA
!Sample_instrument_model = Illumina NextSeq 500
!Sample_library_selection = cDNA
!Sample_library_source = transcriptomic
!Sample_library_strategy = RNA-Seq
!Sample_relation = BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN15517442
!Sample_relation = SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX8719051
!Sample_supplementary_file_1 = NONE
!Sample_series_id = GSE154311
!Sample_data_row_count = 0