{"database": "metadata", "table": "run_metadata", "rows": [[41695, "SRR5125207", "SRX2439963", "SRS1874550", "SRP095522", "PRJNA358490", "Transcriptomic  analysis  reveals  inflammasome dependent  mechanism  for early neutrophil response to Mycobacterium Marinum", "GSE92740", "Transcriptome Analysis", "The mechanisms that govern the neutrophil response to Mycobacterium tuberculosis remain poorly understood.  In this study we utilize an innovative binary approach in zebrafish to perform in depth profiling of the initial response of neutrophils to Mycobacterium marinum  by in vivo biotinylation of neutrophil nuclei and subsequent rapid isolation directly from the in vivo context. RNA seq and differential expression analysis of the neutrophil nuclear transcriptome reveals a significant upregulation in both damage sensing and effector components of the inflammasome  including caspase b  wu:fb15h11 NLRC3 ortholog and il1\u00df. Crispr/Cas9 mediated knockdown of caspase b  which mediates its effects by the proteolytic processing of il1\u00df  results in increased bacterial burden and less infiltration of macrophages to sites of mycobacterium infection  thus impairing granuloma development. We also show that a number of immediate early response genes IEGs are responsible for orchestrating the initial response to mycobacterial infection in neutrophils. Further perturbation of the IEGs revealed egr3 as a key transcription factor regulating il1\u00df transcription. Overall design: We report the design of a binary approach in zebrafish that allows for profiling of the initial response of neutrophils to Mycobacterium Marinum by in vivo biotinylation of neutrophil nuclei and subsequent rapid isolation directly from the in vivo context allowing for RNA seq analysis of the active transcriptome.", null, "pubmed:28747644", null, "mpo RNAseqnuclear +Mm1 3", "GSM2436723", null, "tissue:mpo RNAseqnuclear +Mm1|strain:Tgmpo:BirA 2A Citrine; bactin:Avi Cerulean Rangap128|development:3 dpf method:InVivoBiotinylatedNuclei|treatment:M. marinum 32 512 cell stage|amplification:SMART seqTMv4|strand:reverse", "mpo RNAseqnuclear +Mm1 3", "Reads were mapped using STAR v.2.4.2a to danRer10 and read counts were obtained using subreads FeatureCounts Genome build: danRer10 Supplementary files format and content: read counts", "mpo RNAseqnuclear +Mm1", "For M. marinum infection  embryos were injected at the 32 512 cell stage with M. marinum", "For nuclei isolation  zebrafish embryos were anaesthetised with 0.01% Tricaine 0.01% and aggregates of mycobacterium dissected based on tdTomato fluorescence. Embryos were washed in hypotonic buffer H 20 mM HEPES pH 7.9  15 mM MgCl\u00ad2  10 mM KCl  1 mM DTT  and 1 X Complete protease inhibitor and subsequently resuspended in 500 \u03bcL of buffer H. Embryos were transferred to a Dounce homogenizer and dissociated by 10 strokes with the loose fitting pestal A and incubated on ice for 5 minutes. Further dissociation was carried out by 10 strokes with tight fitting pestal B  every 5 minutes for 15 minutes. Nuclei were collected by centrifugation 2000 g  4 \u00b0C and resuspended in 1 mL buffer NDP 10 mM HEPES pH 7.9  40 mM NaCl  90 mM KCl  0.5 mM EDTA  0.5 mM spermidine  0.15 mM spermine  1 mM dithiothreitol and 1 X Complete protease inhibitor. For nuclei purification  nuclei were incubated with 5 mg of M 280 streptavidin coated dynabeads with rotation for 30 minutes at 4 \u00b0C. A flow based system based on the previously published protocol by was used to capture the nuclei bound on the streptavidin beads. A 10 mL seriological pipette VWR  cat # 8913 898 attached to a 1 mL micropipette tip Rainin reach pipet tip  cat # RT L1000S  both pre treated with NPB/BSA for 30 minutes  and added to a MiniMACS separator magnet OctoMACS Separator  Miltenyl Biotec  cat # 130 042 109. A two way stopcock Biorad cat # 732 8102 was connected to the end of the 1mL micropipette tip via a piece of Tygon tubing Fisher Scientific  cat # 8220 and the flow rate set to 0.75 mL/min. The nuclei beads suspension was diluted by addition of 9 mLs of nuclei pulldown buffer with 0.01 % Triton X 100 NPBt and added to the slow flow setup. The tip was subsequently removed from the stand and the nuclei beads released from the tip by slowly pipetting 1 mL of NPBt in and out of the tip. The solution was then diluted again to 10 mL with NPBt and added again to the slow flow setup. Nuclei beads were eluted in 1 mL of NPBt as described above and the NPBt removed using a magnetic stand DynaMag TM 2 magnet  Invitrogen  cat # 12321D. Nuclei beads were then processed for RNA extraction. RNA extraction and DNAse treatment were carried out using the RNAqueous\u00ae Micro Kit Life Technologies  cat # AM1931. RNA integrity was checked with a RNA pico chip Agilent Technologies  cat # 5067 1513 using the Agilent 2100 Bioanalyzer. cDNA was synthesized and amplified from 100 pg \u2013 1 ng of input RNA using SMART seqTMv4 ultra low input kit for RNA Clontech laboratories  cat #\u2019s 634888  634889  634890  634891  634892  634893  and 634894. Sequencing libraries were prepared using the Nextera XT DNA library preparation kit and sequencing performed on a NextSeq platform using a NextSeq\u2122 500 High Output Kit\u00a0for the generation of 80 base pair paired end reads 150 cycles Illumina  cat # FC 404 1002.", null, "strain:Tgmpo:BirA 2A Citrine;bactin:Avi Cerulean Rangap128|development:3 dpf method:InVivoBiotinylatedNuclei|treatment:M. marinum 32 512 cell stage|amplification:SMART seqTMv4|strand:reverse", "GSM2436723", "GSM2436723: mpo RNAseqnuclear +Mm1 3; Danio rerio; RNA Seq", "GSM2436723", null, "1", "For nuclei isolation  zebrafish embryos were anaesthetised with 0.01% Tricaine 0.01% and aggregates of mycobacterium dissected based on tdTomato fluorescence. Embryos were washed in hypotonic buffer H 20 mM HEPES pH 7.9  15 mM MgCl\u00ad2  10 mM KCl  1 mM DTT  and 1 X Complete protease inhibitor and subsequently resuspended in 500 \u03bcL of buffer H. Embryos were transferred to a Dounce homogenizer and dissociated by 10 strokes with the loose fitting pestal A and incubated on ice for 5 minutes. Further dissociation was carried out by 10 strokes with tight fitting pestal B  every 5 minutes for 15 minutes. Nuclei were collected by centrifugation 2000 g  4 \u00b0C and resuspended in 1 mL buffer NDP 10 mM HEPES pH 7.9  40 mM NaCl  90 mM KCl  0.5 mM EDTA  0.5 mM spermidine  0.15 mM spermine  1 mM dithiothreitol and 1 X Complete protease inhibitor. For nuclei purification  nuclei were incubated with 5 mg of M 280 streptavidin coated dynabeads with rotation for 30 minutes at 4 \u00b0C. A flow based system based on the previously published protocol by was used to capture the nuclei bound on the streptavidin beads. A 10 mL seriological pipette VWR  cat # 8913 898 attached to a 1 mL micropipette tip Rainin reach pipet tip  cat # RT L1000S  both pre treated with NPB/BSA for 30 minutes  and added to a MiniMACS separator magnet OctoMACS Separator  Miltenyl Biotec  cat # 130 042 109. A two way stopcock Biorad cat # 732 8102 was connected to the end of the 1mL micropipette tip via a piece of Tygon tubing Fisher Scientific  cat # 8220 and the flow rate set to 0.75 mL/min. The nuclei beads suspension was diluted by addition of 9 mLs of nuclei pulldown buffer with 0.01 % Triton X 100 NPBt and added to the slow flow setup. The tip was subsequently removed from the stand and the nuclei beads released from the tip by slowly pipetting 1 mL of NPBt in and out of the tip. The solution was then diluted again to 10 mL with NPBt and added again to the slow flow setup. Nuclei beads were eluted in 1 mL of NPBt as described above and the NPBt removed using a magnetic stand DynaMag TM 2 magnet  Invitrogen  cat # 12321D. Nuclei beads were then processed for RNA extraction. RNA extraction and DNase treatment were carried out using the RNAqueous\u00ae Micro Kit Life Technologies  cat # AM1931. RNA integrity was checked with a RNA pico chip Agilent Technologies  cat # 5067 1513 using the Agilent 2100 Bioanalyzer. cDNA was synthesized and amplified from 100 pg \u2013 1 ng of input RNA using SMART seqTMv4 ultra low input kit for RNA Clontech laboratories  cat #\u2019s 634888  634889  634890  634891  634892  634893  and 634894. Sequencing libraries were prepared using the Nextera XT DNA library preparation kit and sequencing performed on a NextSeq platform using a NextSeq\u2122 500 High Output Kit\u00a0for the generation of 80 base pair paired end reads 150 cycles Illumina  cat # FC 404 1002.", "GEO Accession:GSM2436723", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP095522", null, null, "mpo_RNAseqnuclear_+Mm1_3_R1.fastq.gz mpo_RNAseqnuclear_+Mm1_3_R2.fastq.gz", "fastq fastq", 3577998648.0, 22545816.0, "GSM2436723 r1", "0:79.35 1:79.35", "A:1011625838;C:729918557;G:775924617;T:1059494973;N:1034663", 79, 79, null, null, 1011625838, 729918557, 775924617, 1059494973, 1034663, "SRX2439963", "SRS1874550", "SRA507700", "GEO", "Sauka-Spengler lab, University of Oxford, MRC Weatherall Institute of Molecular Medicine", 2, 0.87174, 0.87231, 0.17198, 0.16358, 0.83575, 0.83816, 0.39726, 0.47698, 80, 80, "B", "B", "biological fallback assumption", "illumina", "nextseq", "unknown", "cdna_unspecified", "nextera", "sc", "single_cell_plate", "smartseq", null, "United Kingdom", "2016-12-22", "Multi-stage", "Multi-stage", "Embryo Imprecise", "All anatomical structures"]], "columns": ["rowid", "run.accession", "experiment.accession", "sample.accession", "study.accession", "bioproject", "study.title", "study.alias", "study.type", "study.abstract", "study.attributes", "study.PMIDs", "sample.description", "sample.title", "sample.alias", "sample.centername", "sample.attributes", "GEOsample.title", "GEOsample.dataprocessing", "GEOsample.source", "GEOsample.treatmentprotocol", "GEOsample.extractprotocol", "GEOsample.growthprotocol", "GEOsample.characteristics", "GEOsample.accession", "experiment.title", "experiment.alias", "experiment.library_name", "experiment.design_description", "experiment.library_construction_protocol", "experiment.attributes", "experiment.library_strategy", "experiment.library_source", "experiment.library_selection", "experiment.library_layout", "experiment.platform", "experiment.instrument_model", "experiment.spot_descriptor", "experiment.study_ref", "run.title", "run.attributes", "run.filename", "run.semantic_name", "run.total_bases", "run.total_spots", "run.alias", "run.read_lengths", "run.base_counts", "run.r1_length", "run.r2_length", "run.r3_length", "run.r4_length", "run.Acount", "run.Ccount", "run.Gcount", "run.Tcount", "run.Ncount", "run.experiment", "run.pool_member", "submission.accession", "submission.srasource", "submission.bioprojectsource", "seqdetective.n_mates", "seqdetective.mapping_rate.mate1", "seqdetective.mapping_rate.mate2", "seqdetective.nofeature_rate.mate1", "seqdetective.nofeature_rate.mate2", "seqdetective.sparsity.mate1", "seqdetective.sparsity.mate2", "seqdetective.pos_strand_rate.mate1", "seqdetective.pos_strand_rate.mate2", "seqdetective.readlen.mate1", "seqdetective.readlen.mate2", "seqdetective.judgement.mate1", "seqdetective.judgement.mate2", "seqdetective.judgement.reason", "platform_family", "instrument_generation", "read_bias", "selection_class", "prep_kit", "sc_or_bulk", "tech_class", "technology", "tech_variant", "submission.bioprojectsource.country", "earliest_date", "devstage_curation", "devstage_curation_coarse", "tissue_curation", "tissue_curation_coarse"], "primary_keys": ["rowid"], "primary_key_values": ["41695"], "units": {}, "query_ms": 9.367030004796106}