run_metadata: 41910

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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
41910	SRR5338227	SRX2635541	SRS2044320	SRP101781	PRJNA378947	Generation of a binary transgenic zebrafish model to study myeloid gene regulation in response to pre neoplastic melanocytes	GSE96534	Transcriptome Analysis	A complex network of inflammation succeeds somatic cell transformation and malignant disease. Immune cells and their associated molecules are responsible for detecting and eliminating cancer cells as they establish themselves as the precursors of a tumour. By the time a patient has a detectable solid tumour cancer cells have escaped the initial immune response mechanisms. To date no model exists to allow us to study the underlying mechanisms that govern the initial phase of the immune response as cells are transformed to become the precursors of cancer. Here we describe the development of an innovative double binary animal model designed in zebrafish for exploring regulatory programming of the myeloid cells as they respond to oncogenic transformed melanocytes. This modular system harnesses the power of zebrafish genetics. For studies of melanocyte transformation we generated a hormone inducible binary system allowing for temporal control of different Ras oncogene NRasK61Q HRasG12V KRasG12V expression in melanocytes allowing us to truly study melanoma initiation. This binary model was then coupled to a model for regulatory profiling of the active transcriptome of macrophages and neutrophils which is based on the in vivo biotinylation of nuclei and their subsequent isolation by streptavidin affinity purification. For the first time regulatory profiling of neutrophils as they respond to the earliest precursors of melanoma revealed a number of factors upregulated in neutrophils that may promote progression to melanoma including fgf1 fgf6 cathepsin H cathepsin L galectin 1 and galectin 3. Overall design: We report the design of a double binary approach in zebrafish to study the neutrophil response to transformed melanocytes. By coupling a novel inducible model for melanocyte transformation to a model for the in vivo biotinylation of neutrophil nuclei we can isolate the neutrophil nuclei directly from the in vivo context allowing for RNA seq analysis of the active transcriptome.		pubmed:29666124		mpo RNAseqnuclear mifcntrl 2	GSM2535108		tissue:mpo RNAseqnuclear mif head 2\|strain:Tgmpo:BirA Citrine; bactin:Avi Cerulean Rangapox128 X Tgkita:LexPR Cerulean; PcrysB:ECFP LexOP:mCherry NRasox130\|development:5 dpf method:In Vivo Biotinylated Nuclei\|treatment:n1\|amplification:SMART seqTMv4\|strand:reverse\|assay:RNA seq	mpo RNAseqnuclear mifcntrl 2	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: counts	mpo RNAseqnuclear mif head 2	Incrossed larvae were treated with 1 μM from 24 hpf	For nuclei isolation zebrafish embryos were anaesthetised with 0.01% Tricaine and embryos dissected to separate the head from the trunk and tail. Heads 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 Citrine;bactin:Avi Cerulean Rangapox128 X Tgkita:LexPR Cerulean;PcrysB:ECFP LexOP:mCherry NRasox130\|development:5 dpf method:In Vivo Biotinylated Nuclei\|treatment:n1\|amplification:SMART seqTMv4\|strand:reverse\|assay:RNA seq	GSM2535108	GSM2535108: mpo RNAseqnuclear mifcntrl 2; Danio rerio; RNA Seq	GSM2535108		1	For nuclei isolation zebrafish embryos were anaesthetised with 0.01% Tricaine and embryos dissected to separate the head from the trunk and tail. Heads 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:GSM2535108	RNA-Seq	TRANSCRIPTOMIC	cDNA	PAIRED	ILLUMINA	NextSeq 500		SRP101781			shead2Aligned.out.sort.bam	bam	8216508851.0	51890331.0	GSM2535108 r1	0:79.18 1:79.17	A:2297720467;C:1839611761;G:1894426911;T:2184654439;N:95273	79	79			2297720467	1839611761	1894426911	2184654439	95273	SRX2635541	SRS2044320	SRA544939	GEO	Sauka-Spengler lab, University of Oxford, MRC Weatherall Institute of Molecular Medicine	2	0.91404	0.91129	0.16863	0.17306	0.76108	0.76347	0.47228	0.47663	80	80	B	B	biological fallback assumption	illumina	nextseq	unknown	cdna_unspecified	nextera	sc	single_cell_plate	smartseq		United Kingdom	2017-03-13	Multi-stage	Multi-stage	Head	Nervous System