{"database": "metadata", "table": "run_metadata", "rows": [[41908, "SRR5338229", "SRX2635543", "SRS2044322", "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.", null, "pubmed:29666124", null, "mpo RNAseqnuclear +mifNRas+ 2", "GSM2535110", null, "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:1 \u03bcM mifeprist1 added to embryo water from 24 hpf|amplification:SMART seqTMv4|strand:reverse|assay:RNA seq", "mpo RNAseqnuclear +mifNRas+ 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 \u03bcM 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 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 Citrine;bactin:Avi Cerulean Rangapox128 X Tgkita:LexPR Cerulean;PcrysB:ECFP LexOP:mCherry NRasox130|development:5 dpf method:In Vivo Biotinylated Nuclei|treatment:1 \u03bcM mifeprist1 added to embryo water from 24 hpf|amplification:SMART seqTMv4|strand:reverse|assay:RNA seq", "GSM2535110", "GSM2535110: mpo RNAseqnuclear +mifNRas+ 2; Danio rerio; RNA Seq", "GSM2535110", null, "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 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 #'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\u2122 500 High Output Kit\u00a0for the generation of 80 base pair paired end reads 150 cycles Illumina  cat # FC 404 1002.", "GEO Accession:GSM2535110", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP101781", null, null, "shead_mif2Aligned.out.sort.bam", "bam", 7581928543.0, 47699195.0, "GSM2535110 r1", "0:79.48 1:79.48", "A:2083595704;C:1751859031;G:1833743542;T:1912626493;N:103773", 79, 79, null, null, 2083595704, 1751859031, 1833743542, 1912626493, 103773, "SRX2635543", "SRS2044322", "SRA544939", "GEO", "Sauka-Spengler lab, University of Oxford, MRC Weatherall Institute of Molecular Medicine", 2, 0.93437, 0.93246, 0.08098, 0.08538, 0.78589, 0.79125, 0.39046, 0.38599, 80, 80, "B", "B", "biological fallback assumption", "illumina", "nextseq", "unknown", "cdna_unspecified", "nextera", "sc", "single_cell_plate", "smartseq", null, "United Kingdom", "2017-03-13", "Multi-stage", "Multi-stage", "Head", "Nervous System"]], "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": ["41908"], "units": {}, "query_ms": 9.71052200475242}