run_metadata: 41697

This data as json

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
41697	SRR5125204	SRX2439961	SRS1874548	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 il1ß. Crispr/Cas9 mediated knockdown of caspase b which mediates its effects by the proteolytic processing of il1ß 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 il1ß 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.		pubmed:28747644		mpo RNAseqnuclear +Mm1 1	GSM2436721		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 1	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 MgCl2 10 mM KCl 1 mM DTT and 1 X Complete protease inhibitor and subsequently resuspended in 500 μL 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 °C 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 °C. 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® 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 – 1 ng of input RNA using SMART seqTMv4 ultra low input kit for RNA Clontech laboratories cat #’s 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™ 500 High Output Kit for the generation of 80 base pair paired end reads 150 cycles Illumina cat # FC 404 1002.		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	GSM2436721	GSM2436721: mpo RNAseqnuclear +Mm1 1; Danio rerio; RNA Seq	GSM2436721		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 MgCl2 10 mM KCl 1 mM DTT and 1 X Complete protease inhibitor and subsequently resuspended in 500 μL 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 °C 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 °C. 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® 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 – 1 ng of input RNA using SMART seqTMv4 ultra low input kit for RNA Clontech laboratories cat #’s 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™ 500 High Output Kit for the generation of 80 base pair paired end reads 150 cycles Illumina cat # FC 404 1002.	GEO Accession:GSM2436721	RNA-Seq	TRANSCRIPTOMIC	cDNA	PAIRED	ILLUMINA	NextSeq 500		SRP095522			mpo_RNAseqnuclear_+Mm1_1_run1_R1.fastq.gz mpo_RNAseqnuclear_+Mm1_1_run1_R2.fastq.gz	fastq fastq	1726239158.0	10963548.0	GSM2436721 r1	0:78.71 1:78.74	A:513744700;C:341699560;G:360634107;T:508787617;N:1373174	78	78			513744700	341699560	360634107	508787617	1373174	SRX2439961	SRS1874548	SRA507700	GEO	Sauka-Spengler lab, University of Oxford, MRC Weatherall Institute of Molecular Medicine	2	0.84846	0.85305	0.24598	0.24259	0.79813	0.80316	0.41378	0.40578	80	80	B	B	biological fallback assumption	illumina	nextseq	unknown	cdna_unspecified	nextera	sc	single_cell_plate	smartseq		United Kingdom	2016-12-22	Multi-stage	Multi-stage	Embryo Imprecise	All anatomical structures