^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 = GSE178883
!Series_title = Hydrogen gas inhibits the formation of neutrophil extracellular traps
!Series_geo_accession = GSE178883
!Series_status = Public on Feb 23 2022
!Series_submission_date = Jun 25 2021
!Series_last_update_date = Feb 23 2022
!Series_summary = Neutrophil extracellular traps (NETs) contribute to inflammatory pathogenesis, especially in infectious and cardiovascular diseases. H2 gas acts as an antioxidant and has been clinically and experimentally proven to ameliorate inflammation. Therefore, we investigated whether H2 gas could inhibit NET formation associated with excessive neutrophil activation. Phorbol-12-myristate-13-acetate (PMA)-stimulated human neutrophils exposed to H2 exhibited reduced citrullination of histones, membrane disruption by chromatin complexes, and release of NET components over controls. Mechanistically, H2 suppressed the phosphorylation of Ser-139 residue in H2AX, a marker of DNA damage, thereby reducing the expression of CXC-chemokine receptor 4 (CXCR4) in PMA-stimulated neutrophils. Along with the upregulation of CXCR4, the intracellular content of myeloperoxidase (MPO) and the production of MPO-derived hypochlorous acid (HOCl) was increased; however, these effects were markedly suppressed in H2-exposed cultures. Thus, H2 neutralized HOCl produced by the oxidative burst, inhibited DNA damage, and subsequently inhibited NET formation. We further confirmed that inhalation of H2 inhibited the formation and release of NET components in the blood and bronchoalveolar lavage of a lipopolysaccharide-induced sepsis model in mice and aged minipigs. Therefore, H2 therapy has the potential to be a new therapeutic agent for inflammatory diseases involving NETs associated with excessive neutrophil activation.
!Series_overall_design = 9 samples; Cell cultures of neutrophils in RPMI 1640 medium containing 15 mM HEPES were exposed to hydrogen gas using a hydrogen-filling device with a built-in hydrogen-absorbing alloy. The control medium was left untreated. Aliquots containing 1×10^7 isolated human neutrophils were stimulated with 100 nM phorbol 12-myristate 13-acetate (PMA) in the control or hydrogen gas-exposed medium at 37 °C for 1 h.
!Series_type = Expression profiling by high throughput sequencing
!Series_contributor = Kohsuke,,Shirakawa
!Series_contributor = Motoaki,,Sano
!Series_sample_id = GSM5399898
!Series_sample_id = GSM5399899
!Series_sample_id = GSM5399900
!Series_sample_id = GSM5399901
!Series_sample_id = GSM5399902
!Series_sample_id = GSM5399903
!Series_sample_id = GSM5399904
!Series_sample_id = GSM5399905
!Series_sample_id = GSM5399906
!Series_contact_name = Kohsuke,,Shirakawa
!Series_contact_email = shirakawa19840905.z6@keio.jp
!Series_contact_department = Cardiology
!Series_contact_institute = Keio University School of Medicine
!Series_contact_address = 35 Shinanomachi, Shinjuku-ku
!Series_contact_city = Tokyo
!Series_contact_zip/postal_code = 1608582
!Series_contact_country = Japan
!Series_supplementary_file = ftp://ftp.ncbi.nlm.nih.gov/geo/series/GSE178nnn/GSE178883/suppl/GSE178883_RawCount_Matrix_ann.txt.gz
!Series_platform_id = GPL24676
!Series_platform_taxid = 9606
!Series_sample_taxid = 9606
!Series_relation = BioProject: https://www.ncbi.nlm.nih.gov/bioproject/PRJNA741395
!Series_relation = SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRP325603
^PLATFORM = GPL24676
!Platform_title = Illumina NovaSeq 6000 (Homo sapiens)
!Platform_geo_accession = GPL24676
!Platform_status = Public on Mar 02 2018
!Platform_submission_date = Mar 02 2018
!Platform_last_update_date = Nov 05 2018
!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 = GSM5399898
!Sample_title = NeuStiCont3187
!Sample_geo_accession = GSM5399898
!Sample_status = Public on Feb 23 2022
!Sample_submission_date = Jun 25 2021
!Sample_last_update_date = Feb 23 2022
!Sample_type = SRA
!Sample_channel_count = 1
!Sample_source_name_ch1 = human neutrophil
!Sample_organism_ch1 = Homo sapiens
!Sample_taxid_ch1 = 9606
!Sample_characteristics_ch1 = cell type: circulating neutrophil
!Sample_characteristics_ch1 = treatment: stimulated with 100 nM PMA in control medium
!Sample_molecule_ch1 = total RNA
!Sample_extract_protocol_ch1 = Human neutrophils were isolated from the peripheral blood of healthy volunteers, with written informed consent, using Lympholyte®-poly (CEDARLANE Laboratories, Burlington, Canada) according to the manufacturer’s instructions, after approval from the Ethics Review Committee of Keio University (#20200183). Polynuclear cells containing neutrophils were collected, washed, and resuspended in 5 mL of red blood cell (RBC) lysis buffer (BioLegend, San Diego, CA, USA), then washed twice in Hank’s buffered salt solution (HBSS) without Ca2+/Mg2+ (Wako Pure Chemicals, Osaka, Japan), and finally resuspended in the Roswell Park Memorial Institute (RPMI) 1640 medium (Wako). Cell cultures of neutrophils in RPMI 1640 medium (Wako) containing 15 mM HEPES were exposed to hydrogen gas using a hydrogen-filling device with a built-in hydrogen-absorbing alloy (Doctors Man Co, Ltd., Kanagawa, Japan). The control medium was left untreated. Aliquots containing 1×10^7 isolated human neutrophils were stimulated with 100 nM phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich, St. Louis, MO, USA) in the control or hydrogen gas-exposed medium at 37 °C for 1 h in 15-mL Falcon tubes (Fisher Scientific, Waltham, MA, USA).
!Sample_extract_protocol_ch1 = Libraries were prepared for sequencing using library creation kits (NEBNext Ultra Directional RNA Library Prep Kit, Illumina, San Diego, CA, USA)
!Sample_data_processing = Samples were sequenced using Novaseq with paired-end 2 x 150-bp cycle
!Sample_data_processing = Quality check for sequence reads using FastQC
!Sample_data_processing = Pre-processing of sequence reads using Trimmomatic version 0.38 with LEADING:20 TRAILING:20 SLIDINGWINDOW:4:15 MINLEN:36 as command option
!Sample_data_processing = Mapping of sequence reads using HISAT2 version 2.1.0
!Sample_data_processing = Raw read counts were determined using featureCounts version 1.6.3
!Sample_data_processing = Differentially expressed genes were identified using DESeq2 version 1.22.0
!Sample_data_processing = Genome_build: hg38
!Sample_data_processing = Supplementary_files_format_and_content: Fastq reads were generated using bcl2fastq Conversion Software version 1.8.4; First line begins with a '@' character and is followed by a sequence identifier. Second line represent raw nucleotide sequence letters. The last line encodes the quality values for the sequence.
!Sample_platform_id = GPL24676
!Sample_contact_name = Kohsuke,,Shirakawa
!Sample_contact_email = shirakawa19840905.z6@keio.jp
!Sample_contact_department = Cardiology
!Sample_contact_institute = Keio University School of Medicine
!Sample_contact_address = 35 Shinanomachi, Shinjuku-ku
!Sample_contact_city = Tokyo
!Sample_contact_zip/postal_code = 1608582
!Sample_contact_country = Japan
!Sample_instrument_model = Illumina NovaSeq 6000
!Sample_library_selection = cDNA
!Sample_library_source = transcriptomic
!Sample_library_strategy = RNA-Seq
!Sample_relation = BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN19872187
!Sample_relation = SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX11225411
!Sample_supplementary_file_1 = NONE
!Sample_series_id = GSE178883
!Sample_data_row_count = 0
^SAMPLE = GSM5399899
!Sample_title = NeuStiCont3218
!Sample_geo_accession = GSM5399899
!Sample_status = Public on Feb 23 2022
!Sample_submission_date = Jun 25 2021
!Sample_last_update_date = Feb 23 2022
!Sample_type = SRA
!Sample_channel_count = 1
!Sample_source_name_ch1 = human neutrophil
!Sample_organism_ch1 = Homo sapiens
!Sample_taxid_ch1 = 9606
!Sample_characteristics_ch1 = cell type: circulating neutrophil
!Sample_characteristics_ch1 = treatment: stimulated with 100 nM PMA in control medium
!Sample_molecule_ch1 = total RNA
!Sample_extract_protocol_ch1 = Human neutrophils were isolated from the peripheral blood of healthy volunteers, with written informed consent, using Lympholyte®-poly (CEDARLANE Laboratories, Burlington, Canada) according to the manufacturer’s instructions, after approval from the Ethics Review Committee of Keio University (#20200183). Polynuclear cells containing neutrophils were collected, washed, and resuspended in 5 mL of red blood cell (RBC) lysis buffer (BioLegend, San Diego, CA, USA), then washed twice in Hank’s buffered salt solution (HBSS) without Ca2+/Mg2+ (Wako Pure Chemicals, Osaka, Japan), and finally resuspended in the Roswell Park Memorial Institute (RPMI) 1640 medium (Wako). Cell cultures of neutrophils in RPMI 1640 medium (Wako) containing 15 mM HEPES were exposed to hydrogen gas using a hydrogen-filling device with a built-in hydrogen-absorbing alloy (Doctors Man Co, Ltd., Kanagawa, Japan). The control medium was left untreated. Aliquots containing 1×10^7 isolated human neutrophils were stimulated with 100 nM phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich, St. Louis, MO, USA) in the control or hydrogen gas-exposed medium at 37 °C for 1 h in 15-mL Falcon tubes (Fisher Scientific, Waltham, MA, USA).
!Sample_extract_protocol_ch1 = Libraries were prepared for sequencing using library creation kits (NEBNext Ultra Directional RNA Library Prep Kit, Illumina, San Diego, CA, USA)
!Sample_data_processing = Samples were sequenced using Novaseq with paired-end 2 x 150-bp cycle
!Sample_data_processing = Quality check for sequence reads using FastQC
!Sample_data_processing = Pre-processing of sequence reads using Trimmomatic version 0.38 with LEADING:20 TRAILING:20 SLIDINGWINDOW:4:15 MINLEN:36 as command option
!Sample_data_processing = Mapping of sequence reads using HISAT2 version 2.1.0
!Sample_data_processing = Raw read counts were determined using featureCounts version 1.6.3
!Sample_data_processing = Differentially expressed genes were identified using DESeq2 version 1.22.0
!Sample_data_processing = Genome_build: hg38
!Sample_data_processing = Supplementary_files_format_and_content: Fastq reads were generated using bcl2fastq Conversion Software version 1.8.4; First line begins with a '@' character and is followed by a sequence identifier. Second line represent raw nucleotide sequence letters. The last line encodes the quality values for the sequence.
!Sample_platform_id = GPL24676
!Sample_contact_name = Kohsuke,,Shirakawa
!Sample_contact_email = shirakawa19840905.z6@keio.jp
!Sample_contact_department = Cardiology
!Sample_contact_institute = Keio University School of Medicine
!Sample_contact_address = 35 Shinanomachi, Shinjuku-ku
!Sample_contact_city = Tokyo
!Sample_contact_zip/postal_code = 1608582
!Sample_contact_country = Japan
!Sample_instrument_model = Illumina NovaSeq 6000
!Sample_library_selection = cDNA
!Sample_library_source = transcriptomic
!Sample_library_strategy = RNA-Seq
!Sample_relation = BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN19872186
!Sample_relation = SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX11225412
!Sample_supplementary_file_1 = NONE
!Sample_series_id = GSE178883
!Sample_data_row_count = 0
^SAMPLE = GSM5399900
!Sample_title = NeuStiCont3225
!Sample_geo_accession = GSM5399900
!Sample_status = Public on Feb 23 2022
!Sample_submission_date = Jun 25 2021
!Sample_last_update_date = Feb 23 2022
!Sample_type = SRA
!Sample_channel_count = 1
!Sample_source_name_ch1 = human neutrophil
!Sample_organism_ch1 = Homo sapiens
!Sample_taxid_ch1 = 9606
!Sample_characteristics_ch1 = cell type: circulating neutrophil
!Sample_characteristics_ch1 = treatment: stimulated with 100 nM PMA in control medium
!Sample_molecule_ch1 = total RNA
!Sample_extract_protocol_ch1 = Human neutrophils were isolated from the peripheral blood of healthy volunteers, with written informed consent, using Lympholyte®-poly (CEDARLANE Laboratories, Burlington, Canada) according to the manufacturer’s instructions, after approval from the Ethics Review Committee of Keio University (#20200183). Polynuclear cells containing neutrophils were collected, washed, and resuspended in 5 mL of red blood cell (RBC) lysis buffer (BioLegend, San Diego, CA, USA), then washed twice in Hank’s buffered salt solution (HBSS) without Ca2+/Mg2+ (Wako Pure Chemicals, Osaka, Japan), and finally resuspended in the Roswell Park Memorial Institute (RPMI) 1640 medium (Wako). Cell cultures of neutrophils in RPMI 1640 medium (Wako) containing 15 mM HEPES were exposed to hydrogen gas using a hydrogen-filling device with a built-in hydrogen-absorbing alloy (Doctors Man Co, Ltd., Kanagawa, Japan). The control medium was left untreated. Aliquots containing 1×10^7 isolated human neutrophils were stimulated with 100 nM phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich, St. Louis, MO, USA) in the control or hydrogen gas-exposed medium at 37 °C for 1 h in 15-mL Falcon tubes (Fisher Scientific, Waltham, MA, USA).
!Sample_extract_protocol_ch1 = Libraries were prepared for sequencing using library creation kits (NEBNext Ultra Directional RNA Library Prep Kit, Illumina, San Diego, CA, USA)
!Sample_data_processing = Samples were sequenced using Novaseq with paired-end 2 x 150-bp cycle
!Sample_data_processing = Quality check for sequence reads using FastQC
!Sample_data_processing = Pre-processing of sequence reads using Trimmomatic version 0.38 with LEADING:20 TRAILING:20 SLIDINGWINDOW:4:15 MINLEN:36 as command option
!Sample_data_processing = Mapping of sequence reads using HISAT2 version 2.1.0
!Sample_data_processing = Raw read counts were determined using featureCounts version 1.6.3
!Sample_data_processing = Differentially expressed genes were identified using DESeq2 version 1.22.0
!Sample_data_processing = Genome_build: hg38
!Sample_data_processing = Supplementary_files_format_and_content: Fastq reads were generated using bcl2fastq Conversion Software version 1.8.4; First line begins with a '@' character and is followed by a sequence identifier. Second line represent raw nucleotide sequence letters. The last line encodes the quality values for the sequence.
!Sample_platform_id = GPL24676
!Sample_contact_name = Kohsuke,,Shirakawa
!Sample_contact_email = shirakawa19840905.z6@keio.jp
!Sample_contact_department = Cardiology
!Sample_contact_institute = Keio University School of Medicine
!Sample_contact_address = 35 Shinanomachi, Shinjuku-ku
!Sample_contact_city = Tokyo
!Sample_contact_zip/postal_code = 1608582
!Sample_contact_country = Japan
!Sample_instrument_model = Illumina NovaSeq 6000
!Sample_library_selection = cDNA
!Sample_library_source = transcriptomic
!Sample_library_strategy = RNA-Seq
!Sample_relation = BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN19872185
!Sample_relation = SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX11225413
!Sample_supplementary_file_1 = NONE
!Sample_series_id = GSE178883
!Sample_data_row_count = 0
^SAMPLE = GSM5399901
!Sample_title = NeuStiHydo3187
!Sample_geo_accession = GSM5399901
!Sample_status = Public on Feb 23 2022
!Sample_submission_date = Jun 25 2021
!Sample_last_update_date = Feb 23 2022
!Sample_type = SRA
!Sample_channel_count = 1
!Sample_source_name_ch1 = human neutrophil
!Sample_organism_ch1 = Homo sapiens
!Sample_taxid_ch1 = 9606
!Sample_characteristics_ch1 = cell type: circulating neutrophil
!Sample_characteristics_ch1 = treatment: stimulated with 100 nM PMA in hydrogen gas-exposed medium
!Sample_molecule_ch1 = total RNA
!Sample_extract_protocol_ch1 = Human neutrophils were isolated from the peripheral blood of healthy volunteers, with written informed consent, using Lympholyte®-poly (CEDARLANE Laboratories, Burlington, Canada) according to the manufacturer’s instructions, after approval from the Ethics Review Committee of Keio University (#20200183). Polynuclear cells containing neutrophils were collected, washed, and resuspended in 5 mL of red blood cell (RBC) lysis buffer (BioLegend, San Diego, CA, USA), then washed twice in Hank’s buffered salt solution (HBSS) without Ca2+/Mg2+ (Wako Pure Chemicals, Osaka, Japan), and finally resuspended in the Roswell Park Memorial Institute (RPMI) 1640 medium (Wako). Cell cultures of neutrophils in RPMI 1640 medium (Wako) containing 15 mM HEPES were exposed to hydrogen gas using a hydrogen-filling device with a built-in hydrogen-absorbing alloy (Doctors Man Co, Ltd., Kanagawa, Japan). The control medium was left untreated. Aliquots containing 1×10^7 isolated human neutrophils were stimulated with 100 nM phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich, St. Louis, MO, USA) in the control or hydrogen gas-exposed medium at 37 °C for 1 h in 15-mL Falcon tubes (Fisher Scientific, Waltham, MA, USA).
!Sample_extract_protocol_ch1 = Libraries were prepared for sequencing using library creation kits (NEBNext Ultra Directional RNA Library Prep Kit, Illumina, San Diego, CA, USA)
!Sample_data_processing = Samples were sequenced using Novaseq with paired-end 2 x 150-bp cycle
!Sample_data_processing = Quality check for sequence reads using FastQC
!Sample_data_processing = Pre-processing of sequence reads using Trimmomatic version 0.38 with LEADING:20 TRAILING:20 SLIDINGWINDOW:4:15 MINLEN:36 as command option
!Sample_data_processing = Mapping of sequence reads using HISAT2 version 2.1.0
!Sample_data_processing = Raw read counts were determined using featureCounts version 1.6.3
!Sample_data_processing = Differentially expressed genes were identified using DESeq2 version 1.22.0
!Sample_data_processing = Genome_build: hg38
!Sample_data_processing = Supplementary_files_format_and_content: Fastq reads were generated using bcl2fastq Conversion Software version 1.8.4; First line begins with a '@' character and is followed by a sequence identifier. Second line represent raw nucleotide sequence letters. The last line encodes the quality values for the sequence.
!Sample_platform_id = GPL24676
!Sample_contact_name = Kohsuke,,Shirakawa
!Sample_contact_email = shirakawa19840905.z6@keio.jp
!Sample_contact_department = Cardiology
!Sample_contact_institute = Keio University School of Medicine
!Sample_contact_address = 35 Shinanomachi, Shinjuku-ku
!Sample_contact_city = Tokyo
!Sample_contact_zip/postal_code = 1608582
!Sample_contact_country = Japan
!Sample_instrument_model = Illumina NovaSeq 6000
!Sample_library_selection = cDNA
!Sample_library_source = transcriptomic
!Sample_library_strategy = RNA-Seq
!Sample_relation = BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN19872184
!Sample_relation = SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX11225414
!Sample_supplementary_file_1 = NONE
!Sample_series_id = GSE178883
!Sample_data_row_count = 0
^SAMPLE = GSM5399902
!Sample_title = NeuStiHydo3218
!Sample_geo_accession = GSM5399902
!Sample_status = Public on Feb 23 2022
!Sample_submission_date = Jun 25 2021
!Sample_last_update_date = Feb 23 2022
!Sample_type = SRA
!Sample_channel_count = 1
!Sample_source_name_ch1 = human neutrophil
!Sample_organism_ch1 = Homo sapiens
!Sample_taxid_ch1 = 9606
!Sample_characteristics_ch1 = cell type: circulating neutrophil
!Sample_characteristics_ch1 = treatment: stimulated with 100 nM PMA in hydrogen gas-exposed medium
!Sample_molecule_ch1 = total RNA
!Sample_extract_protocol_ch1 = Human neutrophils were isolated from the peripheral blood of healthy volunteers, with written informed consent, using Lympholyte®-poly (CEDARLANE Laboratories, Burlington, Canada) according to the manufacturer’s instructions, after approval from the Ethics Review Committee of Keio University (#20200183). Polynuclear cells containing neutrophils were collected, washed, and resuspended in 5 mL of red blood cell (RBC) lysis buffer (BioLegend, San Diego, CA, USA), then washed twice in Hank’s buffered salt solution (HBSS) without Ca2+/Mg2+ (Wako Pure Chemicals, Osaka, Japan), and finally resuspended in the Roswell Park Memorial Institute (RPMI) 1640 medium (Wako). Cell cultures of neutrophils in RPMI 1640 medium (Wako) containing 15 mM HEPES were exposed to hydrogen gas using a hydrogen-filling device with a built-in hydrogen-absorbing alloy (Doctors Man Co, Ltd., Kanagawa, Japan). The control medium was left untreated. Aliquots containing 1×10^7 isolated human neutrophils were stimulated with 100 nM phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich, St. Louis, MO, USA) in the control or hydrogen gas-exposed medium at 37 °C for 1 h in 15-mL Falcon tubes (Fisher Scientific, Waltham, MA, USA).
!Sample_extract_protocol_ch1 = Libraries were prepared for sequencing using library creation kits (NEBNext Ultra Directional RNA Library Prep Kit, Illumina, San Diego, CA, USA)
!Sample_data_processing = Samples were sequenced using Novaseq with paired-end 2 x 150-bp cycle
!Sample_data_processing = Quality check for sequence reads using FastQC
!Sample_data_processing = Pre-processing of sequence reads using Trimmomatic version 0.38 with LEADING:20 TRAILING:20 SLIDINGWINDOW:4:15 MINLEN:36 as command option
!Sample_data_processing = Mapping of sequence reads using HISAT2 version 2.1.0
!Sample_data_processing = Raw read counts were determined using featureCounts version 1.6.3
!Sample_data_processing = Differentially expressed genes were identified using DESeq2 version 1.22.0
!Sample_data_processing = Genome_build: hg38
!Sample_data_processing = Supplementary_files_format_and_content: Fastq reads were generated using bcl2fastq Conversion Software version 1.8.4; First line begins with a '@' character and is followed by a sequence identifier. Second line represent raw nucleotide sequence letters. The last line encodes the quality values for the sequence.
!Sample_platform_id = GPL24676
!Sample_contact_name = Kohsuke,,Shirakawa
!Sample_contact_email = shirakawa19840905.z6@keio.jp
!Sample_contact_department = Cardiology
!Sample_contact_institute = Keio University School of Medicine
!Sample_contact_address = 35 Shinanomachi, Shinjuku-ku
!Sample_contact_city = Tokyo
!Sample_contact_zip/postal_code = 1608582
!Sample_contact_country = Japan
!Sample_instrument_model = Illumina NovaSeq 6000
!Sample_library_selection = cDNA
!Sample_library_source = transcriptomic
!Sample_library_strategy = RNA-Seq
!Sample_relation = BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN19872183
!Sample_relation = SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX11225415
!Sample_supplementary_file_1 = NONE
!Sample_series_id = GSE178883
!Sample_data_row_count = 0
^SAMPLE = GSM5399903
!Sample_title = NeuStiHydo3225
!Sample_geo_accession = GSM5399903
!Sample_status = Public on Feb 23 2022
!Sample_submission_date = Jun 25 2021
!Sample_last_update_date = Feb 23 2022
!Sample_type = SRA
!Sample_channel_count = 1
!Sample_source_name_ch1 = human neutrophil
!Sample_organism_ch1 = Homo sapiens
!Sample_taxid_ch1 = 9606
!Sample_characteristics_ch1 = cell type: circulating neutrophil
!Sample_characteristics_ch1 = treatment: stimulated with 100 nM PMA in hydrogen gas-exposed medium
!Sample_molecule_ch1 = total RNA
!Sample_extract_protocol_ch1 = Human neutrophils were isolated from the peripheral blood of healthy volunteers, with written informed consent, using Lympholyte®-poly (CEDARLANE Laboratories, Burlington, Canada) according to the manufacturer’s instructions, after approval from the Ethics Review Committee of Keio University (#20200183). Polynuclear cells containing neutrophils were collected, washed, and resuspended in 5 mL of red blood cell (RBC) lysis buffer (BioLegend, San Diego, CA, USA), then washed twice in Hank’s buffered salt solution (HBSS) without Ca2+/Mg2+ (Wako Pure Chemicals, Osaka, Japan), and finally resuspended in the Roswell Park Memorial Institute (RPMI) 1640 medium (Wako). Cell cultures of neutrophils in RPMI 1640 medium (Wako) containing 15 mM HEPES were exposed to hydrogen gas using a hydrogen-filling device with a built-in hydrogen-absorbing alloy (Doctors Man Co, Ltd., Kanagawa, Japan). The control medium was left untreated. Aliquots containing 1×10^7 isolated human neutrophils were stimulated with 100 nM phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich, St. Louis, MO, USA) in the control or hydrogen gas-exposed medium at 37 °C for 1 h in 15-mL Falcon tubes (Fisher Scientific, Waltham, MA, USA).
!Sample_extract_protocol_ch1 = Libraries were prepared for sequencing using library creation kits (NEBNext Ultra Directional RNA Library Prep Kit, Illumina, San Diego, CA, USA)
!Sample_data_processing = Samples were sequenced using Novaseq with paired-end 2 x 150-bp cycle
!Sample_data_processing = Quality check for sequence reads using FastQC
!Sample_data_processing = Pre-processing of sequence reads using Trimmomatic version 0.38 with LEADING:20 TRAILING:20 SLIDINGWINDOW:4:15 MINLEN:36 as command option
!Sample_data_processing = Mapping of sequence reads using HISAT2 version 2.1.0
!Sample_data_processing = Raw read counts were determined using featureCounts version 1.6.3
!Sample_data_processing = Differentially expressed genes were identified using DESeq2 version 1.22.0
!Sample_data_processing = Genome_build: hg38
!Sample_data_processing = Supplementary_files_format_and_content: Fastq reads were generated using bcl2fastq Conversion Software version 1.8.4; First line begins with a '@' character and is followed by a sequence identifier. Second line represent raw nucleotide sequence letters. The last line encodes the quality values for the sequence.
!Sample_platform_id = GPL24676
!Sample_contact_name = Kohsuke,,Shirakawa
!Sample_contact_email = shirakawa19840905.z6@keio.jp
!Sample_contact_department = Cardiology
!Sample_contact_institute = Keio University School of Medicine
!Sample_contact_address = 35 Shinanomachi, Shinjuku-ku
!Sample_contact_city = Tokyo
!Sample_contact_zip/postal_code = 1608582
!Sample_contact_country = Japan
!Sample_instrument_model = Illumina NovaSeq 6000
!Sample_library_selection = cDNA
!Sample_library_source = transcriptomic
!Sample_library_strategy = RNA-Seq
!Sample_relation = BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN19872182
!Sample_relation = SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX11225416
!Sample_supplementary_file_1 = NONE
!Sample_series_id = GSE178883
!Sample_data_row_count = 0
^SAMPLE = GSM5399904
!Sample_title = NeuStino3187
!Sample_geo_accession = GSM5399904
!Sample_status = Public on Feb 23 2022
!Sample_submission_date = Jun 25 2021
!Sample_last_update_date = Feb 23 2022
!Sample_type = SRA
!Sample_channel_count = 1
!Sample_source_name_ch1 = human neutrophil
!Sample_organism_ch1 = Homo sapiens
!Sample_taxid_ch1 = 9606
!Sample_characteristics_ch1 = cell type: circulating neutrophil
!Sample_characteristics_ch1 = treatment: control medium
!Sample_molecule_ch1 = total RNA
!Sample_extract_protocol_ch1 = Human neutrophils were isolated from the peripheral blood of healthy volunteers, with written informed consent, using Lympholyte®-poly (CEDARLANE Laboratories, Burlington, Canada) according to the manufacturer’s instructions, after approval from the Ethics Review Committee of Keio University (#20200183). Polynuclear cells containing neutrophils were collected, washed, and resuspended in 5 mL of red blood cell (RBC) lysis buffer (BioLegend, San Diego, CA, USA), then washed twice in Hank’s buffered salt solution (HBSS) without Ca2+/Mg2+ (Wako Pure Chemicals, Osaka, Japan), and finally resuspended in the Roswell Park Memorial Institute (RPMI) 1640 medium (Wako). Cell cultures of neutrophils in RPMI 1640 medium (Wako) containing 15 mM HEPES were exposed to hydrogen gas using a hydrogen-filling device with a built-in hydrogen-absorbing alloy (Doctors Man Co, Ltd., Kanagawa, Japan). The control medium was left untreated. Aliquots containing 1×10^7 isolated human neutrophils were stimulated with 100 nM phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich, St. Louis, MO, USA) in the control or hydrogen gas-exposed medium at 37 °C for 1 h in 15-mL Falcon tubes (Fisher Scientific, Waltham, MA, USA).
!Sample_extract_protocol_ch1 = Libraries were prepared for sequencing using library creation kits (NEBNext Ultra Directional RNA Library Prep Kit, Illumina, San Diego, CA, USA)
!Sample_data_processing = Samples were sequenced using Novaseq with paired-end 2 x 150-bp cycle
!Sample_data_processing = Quality check for sequence reads using FastQC
!Sample_data_processing = Pre-processing of sequence reads using Trimmomatic version 0.38 with LEADING:20 TRAILING:20 SLIDINGWINDOW:4:15 MINLEN:36 as command option
!Sample_data_processing = Mapping of sequence reads using HISAT2 version 2.1.0
!Sample_data_processing = Raw read counts were determined using featureCounts version 1.6.3
!Sample_data_processing = Differentially expressed genes were identified using DESeq2 version 1.22.0
!Sample_data_processing = Genome_build: hg38
!Sample_data_processing = Supplementary_files_format_and_content: Fastq reads were generated using bcl2fastq Conversion Software version 1.8.4; First line begins with a '@' character and is followed by a sequence identifier. Second line represent raw nucleotide sequence letters. The last line encodes the quality values for the sequence.
!Sample_platform_id = GPL24676
!Sample_contact_name = Kohsuke,,Shirakawa
!Sample_contact_email = shirakawa19840905.z6@keio.jp
!Sample_contact_department = Cardiology
!Sample_contact_institute = Keio University School of Medicine
!Sample_contact_address = 35 Shinanomachi, Shinjuku-ku
!Sample_contact_city = Tokyo
!Sample_contact_zip/postal_code = 1608582
!Sample_contact_country = Japan
!Sample_instrument_model = Illumina NovaSeq 6000
!Sample_library_selection = cDNA
!Sample_library_source = transcriptomic
!Sample_library_strategy = RNA-Seq
!Sample_relation = BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN19872181
!Sample_relation = SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX11225417
!Sample_supplementary_file_1 = NONE
!Sample_series_id = GSE178883
!Sample_data_row_count = 0
^SAMPLE = GSM5399905
!Sample_title = NeuStino3218
!Sample_geo_accession = GSM5399905
!Sample_status = Public on Feb 23 2022
!Sample_submission_date = Jun 25 2021
!Sample_last_update_date = Feb 23 2022
!Sample_type = SRA
!Sample_channel_count = 1
!Sample_source_name_ch1 = human neutrophil
!Sample_organism_ch1 = Homo sapiens
!Sample_taxid_ch1 = 9606
!Sample_characteristics_ch1 = cell type: circulating neutrophil
!Sample_characteristics_ch1 = treatment: control medium
!Sample_molecule_ch1 = total RNA
!Sample_extract_protocol_ch1 = Human neutrophils were isolated from the peripheral blood of healthy volunteers, with written informed consent, using Lympholyte®-poly (CEDARLANE Laboratories, Burlington, Canada) according to the manufacturer’s instructions, after approval from the Ethics Review Committee of Keio University (#20200183). Polynuclear cells containing neutrophils were collected, washed, and resuspended in 5 mL of red blood cell (RBC) lysis buffer (BioLegend, San Diego, CA, USA), then washed twice in Hank’s buffered salt solution (HBSS) without Ca2+/Mg2+ (Wako Pure Chemicals, Osaka, Japan), and finally resuspended in the Roswell Park Memorial Institute (RPMI) 1640 medium (Wako). Cell cultures of neutrophils in RPMI 1640 medium (Wako) containing 15 mM HEPES were exposed to hydrogen gas using a hydrogen-filling device with a built-in hydrogen-absorbing alloy (Doctors Man Co, Ltd., Kanagawa, Japan). The control medium was left untreated. Aliquots containing 1×10^7 isolated human neutrophils were stimulated with 100 nM phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich, St. Louis, MO, USA) in the control or hydrogen gas-exposed medium at 37 °C for 1 h in 15-mL Falcon tubes (Fisher Scientific, Waltham, MA, USA).
!Sample_extract_protocol_ch1 = Libraries were prepared for sequencing using library creation kits (NEBNext Ultra Directional RNA Library Prep Kit, Illumina, San Diego, CA, USA)
!Sample_data_processing = Samples were sequenced using Novaseq with paired-end 2 x 150-bp cycle
!Sample_data_processing = Quality check for sequence reads using FastQC
!Sample_data_processing = Pre-processing of sequence reads using Trimmomatic version 0.38 with LEADING:20 TRAILING:20 SLIDINGWINDOW:4:15 MINLEN:36 as command option
!Sample_data_processing = Mapping of sequence reads using HISAT2 version 2.1.0
!Sample_data_processing = Raw read counts were determined using featureCounts version 1.6.3
!Sample_data_processing = Differentially expressed genes were identified using DESeq2 version 1.22.0
!Sample_data_processing = Genome_build: hg38
!Sample_data_processing = Supplementary_files_format_and_content: Fastq reads were generated using bcl2fastq Conversion Software version 1.8.4; First line begins with a '@' character and is followed by a sequence identifier. Second line represent raw nucleotide sequence letters. The last line encodes the quality values for the sequence.
!Sample_platform_id = GPL24676
!Sample_contact_name = Kohsuke,,Shirakawa
!Sample_contact_email = shirakawa19840905.z6@keio.jp
!Sample_contact_department = Cardiology
!Sample_contact_institute = Keio University School of Medicine
!Sample_contact_address = 35 Shinanomachi, Shinjuku-ku
!Sample_contact_city = Tokyo
!Sample_contact_zip/postal_code = 1608582
!Sample_contact_country = Japan
!Sample_instrument_model = Illumina NovaSeq 6000
!Sample_library_selection = cDNA
!Sample_library_source = transcriptomic
!Sample_library_strategy = RNA-Seq
!Sample_relation = BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN19872180
!Sample_relation = SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX11225418
!Sample_supplementary_file_1 = NONE
!Sample_series_id = GSE178883
!Sample_data_row_count = 0
^SAMPLE = GSM5399906
!Sample_title = NeuStino3225
!Sample_geo_accession = GSM5399906
!Sample_status = Public on Feb 23 2022
!Sample_submission_date = Jun 25 2021
!Sample_last_update_date = Feb 23 2022
!Sample_type = SRA
!Sample_channel_count = 1
!Sample_source_name_ch1 = human neutrophil
!Sample_organism_ch1 = Homo sapiens
!Sample_taxid_ch1 = 9606
!Sample_characteristics_ch1 = cell type: circulating neutrophil
!Sample_characteristics_ch1 = treatment: control medium
!Sample_molecule_ch1 = total RNA
!Sample_extract_protocol_ch1 = Human neutrophils were isolated from the peripheral blood of healthy volunteers, with written informed consent, using Lympholyte®-poly (CEDARLANE Laboratories, Burlington, Canada) according to the manufacturer’s instructions, after approval from the Ethics Review Committee of Keio University (#20200183). Polynuclear cells containing neutrophils were collected, washed, and resuspended in 5 mL of red blood cell (RBC) lysis buffer (BioLegend, San Diego, CA, USA), then washed twice in Hank’s buffered salt solution (HBSS) without Ca2+/Mg2+ (Wako Pure Chemicals, Osaka, Japan), and finally resuspended in the Roswell Park Memorial Institute (RPMI) 1640 medium (Wako). Cell cultures of neutrophils in RPMI 1640 medium (Wako) containing 15 mM HEPES were exposed to hydrogen gas using a hydrogen-filling device with a built-in hydrogen-absorbing alloy (Doctors Man Co, Ltd., Kanagawa, Japan). The control medium was left untreated. Aliquots containing 1×10^7 isolated human neutrophils were stimulated with 100 nM phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich, St. Louis, MO, USA) in the control or hydrogen gas-exposed medium at 37 °C for 1 h in 15-mL Falcon tubes (Fisher Scientific, Waltham, MA, USA).
!Sample_extract_protocol_ch1 = Libraries were prepared for sequencing using library creation kits (NEBNext Ultra Directional RNA Library Prep Kit, Illumina, San Diego, CA, USA)
!Sample_data_processing = Samples were sequenced using Novaseq with paired-end 2 x 150-bp cycle
!Sample_data_processing = Quality check for sequence reads using FastQC
!Sample_data_processing = Pre-processing of sequence reads using Trimmomatic version 0.38 with LEADING:20 TRAILING:20 SLIDINGWINDOW:4:15 MINLEN:36 as command option
!Sample_data_processing = Mapping of sequence reads using HISAT2 version 2.1.0
!Sample_data_processing = Raw read counts were determined using featureCounts version 1.6.3
!Sample_data_processing = Differentially expressed genes were identified using DESeq2 version 1.22.0
!Sample_data_processing = Genome_build: hg38
!Sample_data_processing = Supplementary_files_format_and_content: Fastq reads were generated using bcl2fastq Conversion Software version 1.8.4; First line begins with a '@' character and is followed by a sequence identifier. Second line represent raw nucleotide sequence letters. The last line encodes the quality values for the sequence.
!Sample_platform_id = GPL24676
!Sample_contact_name = Kohsuke,,Shirakawa
!Sample_contact_email = shirakawa19840905.z6@keio.jp
!Sample_contact_department = Cardiology
!Sample_contact_institute = Keio University School of Medicine
!Sample_contact_address = 35 Shinanomachi, Shinjuku-ku
!Sample_contact_city = Tokyo
!Sample_contact_zip/postal_code = 1608582
!Sample_contact_country = Japan
!Sample_instrument_model = Illumina NovaSeq 6000
!Sample_library_selection = cDNA
!Sample_library_source = transcriptomic
!Sample_library_strategy = RNA-Seq
!Sample_relation = BioSample: https://www.ncbi.nlm.nih.gov/biosample/SAMN19872179
!Sample_relation = SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRX11225419
!Sample_supplementary_file_1 = NONE
!Sample_series_id = GSE178883
!Sample_data_row_count = 0