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90 rows where devstage_curation = "Multi-stage" and tissue_curation = "Head"

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technology 2

experiment.library_layout 2

devstage_curation_coarse 2

tissue_curation_coarse 1

tissue_curation 1

  • Head · 90 ✖

experiment.platform 1

experiment.library_strategy 1

experiment.library_source 1

experiment.library_selection 1

devstage_curation 1

  • Multi-stage · 90 ✖
Link 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
41908 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.   pubmed:29666124   mpo RNAseqnuclear +mifNRas+ 2 GSM2535110   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 μM 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 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 D…   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 μM 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   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 D… GEO Accession:GSM2535110 RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA NextSeq 500   SRP101781     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     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   United Kingdom 2017-03-13 Multi-stage Multi-stage Head Nervous System
41909 41909 SRR5338228 SRX2635542 SRS2044321 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 +mifNRas+ 1 GSM2535109   tissue:mpo RNAseqnuclear +mif head 1|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 μM mifeprist1 added to embryo water from 24 hpf|amplification:SMART seqTMv4|strand:reverse|assay:RNA seq mpo RNAseqnuclear +mifNRas+ 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: counts mpo RNAseqnuclear +mif head 1 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 D…   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 μM mifeprist1 added to embryo water from 24 hpf|amplification:SMART seqTMv4|strand:reverse|assay:RNA seq GSM2535109 GSM2535109: mpo RNAseqnuclear +mifNRas+ 1; Danio rerio; RNA Seq GSM2535109   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 D… GEO Accession:GSM2535109 RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA NextSeq 500   SRP101781     shead_mif1Aligned.out.sort.bam bam 7904423201.0 49746704.0 GSM2535109 r1 0:79.45 1:79.45 A:2153404550;C:1843316968;G:1926669197;T:1980935189;N:97297 79 79     2153404550 1843316968 1926669197 1980935189 97297 SRX2635542 SRS2044321 SRA544939 GEO Sauka-Spengler lab, University of Oxford, MRC Weatherall Institute of Molecular Medicine 2 0.9341 0.931 0.07739 0.07981 0.79971 0.80568 0.38474 0.38892 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
41910 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 D…   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 D… 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
41911 41911 SRR5338226 SRX2635540 SRS2044319 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 1 GSM2535107   tissue:mpo RNAseqnuclear mif head 1|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 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: counts mpo RNAseqnuclear mif head 1 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 D…   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 GSM2535107 GSM2535107: mpo RNAseqnuclear mifcntrl 1; Danio rerio; RNA Seq GSM2535107   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 D… GEO Accession:GSM2535107 RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA NextSeq 500   SRP101781     shead1Aligned.out.sort.bam bam 6223616133.0 39173742.0 GSM2535107 r1 0:79.44 1:79.43 A:1628842481;C:1512759691;G:1559820323;T:1522127590;N:66048 79 79     1628842481 1512759691 1559820323 1522127590 66048 SRX2635540 SRS2044319 SRA544939 GEO Sauka-Spengler lab, University of Oxford, MRC Weatherall Institute of Molecular Medicine 2 0.91702 0.91416 0.08978 0.09081 0.80825 0.81225 0.47575 0.46407 78 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
50953 50953 SRR8383314 SRX5193232 SRS4199232 SRP175015 PRJNA512489 Selective roles of vertebrate PCF11 in premature and full length transcript termination zebrafish three prime mRNA seq GSE124555 Other The pervasive nature of RNA polymerase II Pol II transcription requires efficient termination. A key player in this process is the cleavage and polyadenylation CPA factor PCF11 which directly binds to the Pol II C terminal domain and dismantles elongating Pol II from DNA in vitro. We demonstrate that PCF11 mediated termination is essential for vertebrate development. A range of genomic analyses including: mNET seq three prime mRNA seq chromatin RNA seq and ChIP seq reveals that PCF11 enhances transcription termination and stimulates early polyadenylation genome wide. PCF11 binds preferentially between closely spaced genes where it prevents transcriptional interference and downstream gene silencing. Notably PCF11 is sub stoichiometric to the CPA complex. Low levels of PCF11 are maintained by an auto regulatory mechanism involving premature termination of its own transcript and are important for normal development. Both in human cell culture and during zebrafish development PCF11 selectively attenuates the expression of other transcriptional regulators by premature CPA and termination. Overall design: three prime mRNA seq in individual zebrafish embryo heads. Two types of mutants: zPCF11 null and zPCF11 with deletion of PAS1. Wild type wt +/+ heterozygous het +/ and homozygous mutant hom / embryos were analyzed. Wild type and heterozygous animals were phenotypically indistinguishable. parent bioproject:PRJNA507553 pubmed:30819644   3 primemRNAseq zPCF11deltaPAS1 hom e6 GSM3536476   tissue:single embryo head 32hpf|molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11deltaPAS1 / 3 primemRNAseq zPCF11deltaPAS1 hom e6 3’ mRNA seq data were mapped according to the guidelines of QuantSeq library kit manufacturer Lexogen. Unaligned bam files from HiSeq2500 were converted to FASTQ files with bam2fastx TopHat 2 component Kim et al. 2013. post overview with FastQC reads were trimmed with BBtools sourceforge.net/projects/bbmap/ script bbduk using following settings: k=13 ktrim=r useshortkmers=t mink=5 qtrim=r trimq=10 minlength=20 . post trimming control with FastQC curated reads were mapped with STAR2.5b Dobin et al. 2013 aligner. STAR index was generated with the GRCz10/danRer10 was used as the reference zebrafish genome together with the corresponding ENSEMBL annotation. Aligned 3’ mRNA seq reads were filtered to remove false positives due to internal priming of the QuantSeq assay on genome encoded polyA stretches. To do this first a crude genomic mask was generated that contained all loci harbouring 6 or more consecutive A bases as well as any 10 nucleotide windows containing more than 6 A bases. For genes expressed from the reverse strand an analogous T rich mask was generated. Those crude masks were then corrected to allow for detection of genuine PAS falling in A/T rich regions by strand specifically unmasking 20 nucleotide intervals centred at annotated 3’ gene ends. 3’ mRNA seq reads falling into those refined strand specific masks were then removed and the filtered reads reduced to the most distal nucleotide 3’ end nucleotide. Genome build: GRCz10/danRer10 Supplementary files format and content: bigWig files: genome coverage files mean from replicates single embryo head 32hpf   Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq 3’mRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. Zebrafish Danio rerio were raised according to standard protocols 28°C water temperature 14/10 hr light/dark cycle. TLAB fish generated by crossing zebrafish AB and the natural variant TL Tupfel Long fin stocks served as wild type zebrafish for all experiments. zPCF11 null and zPCF11 ∆PAS1 mutant zebrafish were generated by Cas9 mediated mutagenesis Gagnon et al. 2014 molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11deltaPAS1 / GSM3536476 GSM3536476: 3 primemRNAseq zPCF11deltaPAS1 hom e6; Danio rerio; RNA Seq GSM3536476   1 Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq three primemRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. GEO Accession:GSM3536476 RNA-Seq TRANSCRIPTOMIC cDNA SINGLE ILLUMINA Illumina HiSeq 2500   SRP175015     bam4__sel_GCTCGAATC.bam.fastq_trimmed.fastq.gz fastq 170117572.0 1936836.0 GSM3536476 r1 0:87.83 A:57043415;C:29867475;G:33401453;T:49804679;N:550 87       57043415 29867475 33401453 49804679 550 SRX5193232 SRS4199232 SRA828489 GEO Sir William Dunn School of Pathology, University of Oxford 1 0.82781   0.11752   0.7791   0.51275   89   B   usable mapping rate illumina hiseq_era 3prime poly_a lexogen bulk unknown unknown   United Kingdom 2019-01-02 Multi-stage Embryo Head Nervous System
50954 50954 SRR8383315 SRX5193232 SRS4199232 SRP175015 PRJNA512489 Selective roles of vertebrate PCF11 in premature and full length transcript termination zebrafish three prime mRNA seq GSE124555 Other The pervasive nature of RNA polymerase II Pol II transcription requires efficient termination. A key player in this process is the cleavage and polyadenylation CPA factor PCF11 which directly binds to the Pol II C terminal domain and dismantles elongating Pol II from DNA in vitro. We demonstrate that PCF11 mediated termination is essential for vertebrate development. A range of genomic analyses including: mNET seq three prime mRNA seq chromatin RNA seq and ChIP seq reveals that PCF11 enhances transcription termination and stimulates early polyadenylation genome wide. PCF11 binds preferentially between closely spaced genes where it prevents transcriptional interference and downstream gene silencing. Notably PCF11 is sub stoichiometric to the CPA complex. Low levels of PCF11 are maintained by an auto regulatory mechanism involving premature termination of its own transcript and are important for normal development. Both in human cell culture and during zebrafish development PCF11 selectively attenuates the expression of other transcriptional regulators by premature CPA and termination. Overall design: three prime mRNA seq in individual zebrafish embryo heads. Two types of mutants: zPCF11 null and zPCF11 with deletion of PAS1. Wild type wt +/+ heterozygous het +/ and homozygous mutant hom / embryos were analyzed. Wild type and heterozygous animals were phenotypically indistinguishable. parent bioproject:PRJNA507553 pubmed:30819644   3 primemRNAseq zPCF11deltaPAS1 hom e6 GSM3536476   tissue:single embryo head 32hpf|molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11deltaPAS1 / 3 primemRNAseq zPCF11deltaPAS1 hom e6 3’ mRNA seq data were mapped according to the guidelines of QuantSeq library kit manufacturer Lexogen. Unaligned bam files from HiSeq2500 were converted to FASTQ files with bam2fastx TopHat 2 component Kim et al. 2013. post overview with FastQC reads were trimmed with BBtools sourceforge.net/projects/bbmap/ script bbduk using following settings: k=13 ktrim=r useshortkmers=t mink=5 qtrim=r trimq=10 minlength=20 . post trimming control with FastQC curated reads were mapped with STAR2.5b Dobin et al. 2013 aligner. STAR index was generated with the GRCz10/danRer10 was used as the reference zebrafish genome together with the corresponding ENSEMBL annotation. Aligned 3’ mRNA seq reads were filtered to remove false positives due to internal priming of the QuantSeq assay on genome encoded polyA stretches. To do this first a crude genomic mask was generated that contained all loci harbouring 6 or more consecutive A bases as well as any 10 nucleotide windows containing more than 6 A bases. For genes expressed from the reverse strand an analogous T rich mask was generated. Those crude masks were then corrected to allow for detection of genuine PAS falling in A/T rich regions by strand specifically unmasking 20 nucleotide intervals centred at annotated 3’ gene ends. 3’ mRNA seq reads falling into those refined strand specific masks were then removed and the filtered reads reduced to the most distal nucleotide 3’ end nucleotide. Genome build: GRCz10/danRer10 Supplementary files format and content: bigWig files: genome coverage files mean from replicates single embryo head 32hpf   Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq 3’mRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. Zebrafish Danio rerio were raised according to standard protocols 28°C water temperature 14/10 hr light/dark cycle. TLAB fish generated by crossing zebrafish AB and the natural variant TL Tupfel Long fin stocks served as wild type zebrafish for all experiments. zPCF11 null and zPCF11 ∆PAS1 mutant zebrafish were generated by Cas9 mediated mutagenesis Gagnon et al. 2014 molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11deltaPAS1 / GSM3536476 GSM3536476: 3 primemRNAseq zPCF11deltaPAS1 hom e6; Danio rerio; RNA Seq GSM3536476   1 Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq three primemRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. GEO Accession:GSM3536476 RNA-Seq TRANSCRIPTOMIC cDNA SINGLE ILLUMINA Illumina HiSeq 2500   SRP175015     bam5__sel_GCTCGAATC.bam.fastq_trimmed.fastq.gz fastq 175173536.0 1995191.0 GSM3536476 r2 0:87.80 A:58844991;C:30708254;G:34327735;T:51292146;N:410 87       58844991 30708254 34327735 51292146 410 SRX5193232 SRS4199232 SRA828489 GEO Sir William Dunn School of Pathology, University of Oxford 1 0.82604   0.11764   0.78029   0.51756   89   B   usable mapping rate illumina hiseq_era 3prime poly_a lexogen bulk unknown unknown   United Kingdom 2019-01-02 Multi-stage Embryo Head Nervous System
50955 50955 SRR8383312 SRX5193231 SRS4199231 SRP175015 PRJNA512489 Selective roles of vertebrate PCF11 in premature and full length transcript termination zebrafish three prime mRNA seq GSE124555 Other The pervasive nature of RNA polymerase II Pol II transcription requires efficient termination. A key player in this process is the cleavage and polyadenylation CPA factor PCF11 which directly binds to the Pol II C terminal domain and dismantles elongating Pol II from DNA in vitro. We demonstrate that PCF11 mediated termination is essential for vertebrate development. A range of genomic analyses including: mNET seq three prime mRNA seq chromatin RNA seq and ChIP seq reveals that PCF11 enhances transcription termination and stimulates early polyadenylation genome wide. PCF11 binds preferentially between closely spaced genes where it prevents transcriptional interference and downstream gene silencing. Notably PCF11 is sub stoichiometric to the CPA complex. Low levels of PCF11 are maintained by an auto regulatory mechanism involving premature termination of its own transcript and are important for normal development. Both in human cell culture and during zebrafish development PCF11 selectively attenuates the expression of other transcriptional regulators by premature CPA and termination. Overall design: three prime mRNA seq in individual zebrafish embryo heads. Two types of mutants: zPCF11 null and zPCF11 with deletion of PAS1. Wild type wt +/+ heterozygous het +/ and homozygous mutant hom / embryos were analyzed. Wild type and heterozygous animals were phenotypically indistinguishable. parent bioproject:PRJNA507553 pubmed:30819644   3 primemRNAseq zPCF11deltaPAS1 hom e5 GSM3536475   tissue:single embryo head 32hpf|molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11deltaPAS1 / 3 primemRNAseq zPCF11deltaPAS1 hom e5 3’ mRNA seq data were mapped according to the guidelines of QuantSeq library kit manufacturer Lexogen. Unaligned bam files from HiSeq2500 were converted to FASTQ files with bam2fastx TopHat 2 component Kim et al. 2013. post overview with FastQC reads were trimmed with BBtools sourceforge.net/projects/bbmap/ script bbduk using following settings: k=13 ktrim=r useshortkmers=t mink=5 qtrim=r trimq=10 minlength=20 . post trimming control with FastQC curated reads were mapped with STAR2.5b Dobin et al. 2013 aligner. STAR index was generated with the GRCz10/danRer10 was used as the reference zebrafish genome together with the corresponding ENSEMBL annotation. Aligned 3’ mRNA seq reads were filtered to remove false positives due to internal priming of the QuantSeq assay on genome encoded polyA stretches. To do this first a crude genomic mask was generated that contained all loci harbouring 6 or more consecutive A bases as well as any 10 nucleotide windows containing more than 6 A bases. For genes expressed from the reverse strand an analogous T rich mask was generated. Those crude masks were then corrected to allow for detection of genuine PAS falling in A/T rich regions by strand specifically unmasking 20 nucleotide intervals centred at annotated 3’ gene ends. 3’ mRNA seq reads falling into those refined strand specific masks were then removed and the filtered reads reduced to the most distal nucleotide 3’ end nucleotide. Genome build: GRCz10/danRer10 Supplementary files format and content: bigWig files: genome coverage files mean from replicates single embryo head 32hpf   Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq 3’mRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. Zebrafish Danio rerio were raised according to standard protocols 28°C water temperature 14/10 hr light/dark cycle. TLAB fish generated by crossing zebrafish AB and the natural variant TL Tupfel Long fin stocks served as wild type zebrafish for all experiments. zPCF11 null and zPCF11 ∆PAS1 mutant zebrafish were generated by Cas9 mediated mutagenesis Gagnon et al. 2014 molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11deltaPAS1 / GSM3536475 GSM3536475: 3 primemRNAseq zPCF11deltaPAS1 hom e5; Danio rerio; RNA Seq GSM3536475   1 Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq three primemRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. GEO Accession:GSM3536475 RNA-Seq TRANSCRIPTOMIC cDNA SINGLE ILLUMINA Illumina HiSeq 2500   SRP175015     bam4__sel_CAAGCAATC.bam.fastq_trimmed.fastq.gz fastq 606360323.0 6905543.0 GSM3536475 r1 0:87.81 A:198106425;C:109559199;G:122893662;T:175799225;N:1812 87       198106425 109559199 122893662 175799225 1812 SRX5193231 SRS4199231 SRA828489 GEO Sir William Dunn School of Pathology, University of Oxford 1 0.8427   0.1325   0.78879   0.51921   89   B   usable mapping rate illumina hiseq_era 3prime poly_a lexogen bulk unknown unknown   United Kingdom 2019-01-02 Multi-stage Embryo Head Nervous System
50956 50956 SRR8383313 SRX5193231 SRS4199231 SRP175015 PRJNA512489 Selective roles of vertebrate PCF11 in premature and full length transcript termination zebrafish three prime mRNA seq GSE124555 Other The pervasive nature of RNA polymerase II Pol II transcription requires efficient termination. A key player in this process is the cleavage and polyadenylation CPA factor PCF11 which directly binds to the Pol II C terminal domain and dismantles elongating Pol II from DNA in vitro. We demonstrate that PCF11 mediated termination is essential for vertebrate development. A range of genomic analyses including: mNET seq three prime mRNA seq chromatin RNA seq and ChIP seq reveals that PCF11 enhances transcription termination and stimulates early polyadenylation genome wide. PCF11 binds preferentially between closely spaced genes where it prevents transcriptional interference and downstream gene silencing. Notably PCF11 is sub stoichiometric to the CPA complex. Low levels of PCF11 are maintained by an auto regulatory mechanism involving premature termination of its own transcript and are important for normal development. Both in human cell culture and during zebrafish development PCF11 selectively attenuates the expression of other transcriptional regulators by premature CPA and termination. Overall design: three prime mRNA seq in individual zebrafish embryo heads. Two types of mutants: zPCF11 null and zPCF11 with deletion of PAS1. Wild type wt +/+ heterozygous het +/ and homozygous mutant hom / embryos were analyzed. Wild type and heterozygous animals were phenotypically indistinguishable. parent bioproject:PRJNA507553 pubmed:30819644   3 primemRNAseq zPCF11deltaPAS1 hom e5 GSM3536475   tissue:single embryo head 32hpf|molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11deltaPAS1 / 3 primemRNAseq zPCF11deltaPAS1 hom e5 3’ mRNA seq data were mapped according to the guidelines of QuantSeq library kit manufacturer Lexogen. Unaligned bam files from HiSeq2500 were converted to FASTQ files with bam2fastx TopHat 2 component Kim et al. 2013. post overview with FastQC reads were trimmed with BBtools sourceforge.net/projects/bbmap/ script bbduk using following settings: k=13 ktrim=r useshortkmers=t mink=5 qtrim=r trimq=10 minlength=20 . post trimming control with FastQC curated reads were mapped with STAR2.5b Dobin et al. 2013 aligner. STAR index was generated with the GRCz10/danRer10 was used as the reference zebrafish genome together with the corresponding ENSEMBL annotation. Aligned 3’ mRNA seq reads were filtered to remove false positives due to internal priming of the QuantSeq assay on genome encoded polyA stretches. To do this first a crude genomic mask was generated that contained all loci harbouring 6 or more consecutive A bases as well as any 10 nucleotide windows containing more than 6 A bases. For genes expressed from the reverse strand an analogous T rich mask was generated. Those crude masks were then corrected to allow for detection of genuine PAS falling in A/T rich regions by strand specifically unmasking 20 nucleotide intervals centred at annotated 3’ gene ends. 3’ mRNA seq reads falling into those refined strand specific masks were then removed and the filtered reads reduced to the most distal nucleotide 3’ end nucleotide. Genome build: GRCz10/danRer10 Supplementary files format and content: bigWig files: genome coverage files mean from replicates single embryo head 32hpf   Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq 3’mRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. Zebrafish Danio rerio were raised according to standard protocols 28°C water temperature 14/10 hr light/dark cycle. TLAB fish generated by crossing zebrafish AB and the natural variant TL Tupfel Long fin stocks served as wild type zebrafish for all experiments. zPCF11 null and zPCF11 ∆PAS1 mutant zebrafish were generated by Cas9 mediated mutagenesis Gagnon et al. 2014 molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11deltaPAS1 / GSM3536475 GSM3536475: 3 primemRNAseq zPCF11deltaPAS1 hom e5; Danio rerio; RNA Seq GSM3536475   1 Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq three primemRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. GEO Accession:GSM3536475 RNA-Seq TRANSCRIPTOMIC cDNA SINGLE ILLUMINA Illumina HiSeq 2500   SRP175015     bam5__sel_CAAGCAATC.bam.fastq_trimmed.fastq.gz fastq 624201360.0 7110625.0 GSM3536475 r2 0:87.78 A:204175038;C:112645351;G:126392625;T:180986966;N:1380 87       204175038 112645351 126392625 180986966 1380 SRX5193231 SRS4199231 SRA828489 GEO Sir William Dunn School of Pathology, University of Oxford 1 0.84211   0.13385   0.7895   0.52023   89   B   usable mapping rate illumina hiseq_era 3prime poly_a lexogen bulk unknown unknown   United Kingdom 2019-01-02 Multi-stage Embryo Head Nervous System
50957 50957 SRR8383310 SRX5193230 SRS4199230 SRP175015 PRJNA512489 Selective roles of vertebrate PCF11 in premature and full length transcript termination zebrafish three prime mRNA seq GSE124555 Other The pervasive nature of RNA polymerase II Pol II transcription requires efficient termination. A key player in this process is the cleavage and polyadenylation CPA factor PCF11 which directly binds to the Pol II C terminal domain and dismantles elongating Pol II from DNA in vitro. We demonstrate that PCF11 mediated termination is essential for vertebrate development. A range of genomic analyses including: mNET seq three prime mRNA seq chromatin RNA seq and ChIP seq reveals that PCF11 enhances transcription termination and stimulates early polyadenylation genome wide. PCF11 binds preferentially between closely spaced genes where it prevents transcriptional interference and downstream gene silencing. Notably PCF11 is sub stoichiometric to the CPA complex. Low levels of PCF11 are maintained by an auto regulatory mechanism involving premature termination of its own transcript and are important for normal development. Both in human cell culture and during zebrafish development PCF11 selectively attenuates the expression of other transcriptional regulators by premature CPA and termination. Overall design: three prime mRNA seq in individual zebrafish embryo heads. Two types of mutants: zPCF11 null and zPCF11 with deletion of PAS1. Wild type wt +/+ heterozygous het +/ and homozygous mutant hom / embryos were analyzed. Wild type and heterozygous animals were phenotypically indistinguishable. parent bioproject:PRJNA507553 pubmed:30819644   3 primemRNAseq zPCF11deltaPAS1 hom e4 GSM3536474   tissue:single embryo head 32hpf|molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11deltaPAS1 / 3 primemRNAseq zPCF11deltaPAS1 hom e4 3’ mRNA seq data were mapped according to the guidelines of QuantSeq library kit manufacturer Lexogen. Unaligned bam files from HiSeq2500 were converted to FASTQ files with bam2fastx TopHat 2 component Kim et al. 2013. post overview with FastQC reads were trimmed with BBtools sourceforge.net/projects/bbmap/ script bbduk using following settings: k=13 ktrim=r useshortkmers=t mink=5 qtrim=r trimq=10 minlength=20 . post trimming control with FastQC curated reads were mapped with STAR2.5b Dobin et al. 2013 aligner. STAR index was generated with the GRCz10/danRer10 was used as the reference zebrafish genome together with the corresponding ENSEMBL annotation. Aligned 3’ mRNA seq reads were filtered to remove false positives due to internal priming of the QuantSeq assay on genome encoded polyA stretches. To do this first a crude genomic mask was generated that contained all loci harbouring 6 or more consecutive A bases as well as any 10 nucleotide windows containing more than 6 A bases. For genes expressed from the reverse strand an analogous T rich mask was generated. Those crude masks were then corrected to allow for detection of genuine PAS falling in A/T rich regions by strand specifically unmasking 20 nucleotide intervals centred at annotated 3’ gene ends. 3’ mRNA seq reads falling into those refined strand specific masks were then removed and the filtered reads reduced to the most distal nucleotide 3’ end nucleotide. Genome build: GRCz10/danRer10 Supplementary files format and content: bigWig files: genome coverage files mean from replicates single embryo head 32hpf   Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq 3’mRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. Zebrafish Danio rerio were raised according to standard protocols 28°C water temperature 14/10 hr light/dark cycle. TLAB fish generated by crossing zebrafish AB and the natural variant TL Tupfel Long fin stocks served as wild type zebrafish for all experiments. zPCF11 null and zPCF11 ∆PAS1 mutant zebrafish were generated by Cas9 mediated mutagenesis Gagnon et al. 2014 molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11deltaPAS1 / GSM3536474 GSM3536474: 3 primemRNAseq zPCF11deltaPAS1 hom e4; Danio rerio; RNA Seq GSM3536474   1 Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq three primemRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. GEO Accession:GSM3536474 RNA-Seq TRANSCRIPTOMIC cDNA SINGLE ILLUMINA Illumina HiSeq 2500   SRP175015     bam4__sel_AACGCCATC.bam.fastq_trimmed.fastq.gz fastq 892780335.0 10219904.0 GSM3536474 r1 0:87.36 A:300258267;C:163189097;G:178114941;T:251215317;N:2713 87       300258267 163189097 178114941 251215317 2713 SRX5193230 SRS4199230 SRA828489 GEO Sir William Dunn School of Pathology, University of Oxford 1 0.83419   0.14585   0.80255   0.50109   89   B   usable mapping rate illumina hiseq_era 3prime poly_a lexogen bulk unknown unknown   United Kingdom 2019-01-02 Multi-stage Embryo Head Nervous System
50958 50958 SRR8383311 SRX5193230 SRS4199230 SRP175015 PRJNA512489 Selective roles of vertebrate PCF11 in premature and full length transcript termination zebrafish three prime mRNA seq GSE124555 Other The pervasive nature of RNA polymerase II Pol II transcription requires efficient termination. A key player in this process is the cleavage and polyadenylation CPA factor PCF11 which directly binds to the Pol II C terminal domain and dismantles elongating Pol II from DNA in vitro. We demonstrate that PCF11 mediated termination is essential for vertebrate development. A range of genomic analyses including: mNET seq three prime mRNA seq chromatin RNA seq and ChIP seq reveals that PCF11 enhances transcription termination and stimulates early polyadenylation genome wide. PCF11 binds preferentially between closely spaced genes where it prevents transcriptional interference and downstream gene silencing. Notably PCF11 is sub stoichiometric to the CPA complex. Low levels of PCF11 are maintained by an auto regulatory mechanism involving premature termination of its own transcript and are important for normal development. Both in human cell culture and during zebrafish development PCF11 selectively attenuates the expression of other transcriptional regulators by premature CPA and termination. Overall design: three prime mRNA seq in individual zebrafish embryo heads. Two types of mutants: zPCF11 null and zPCF11 with deletion of PAS1. Wild type wt +/+ heterozygous het +/ and homozygous mutant hom / embryos were analyzed. Wild type and heterozygous animals were phenotypically indistinguishable. parent bioproject:PRJNA507553 pubmed:30819644   3 primemRNAseq zPCF11deltaPAS1 hom e4 GSM3536474   tissue:single embryo head 32hpf|molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11deltaPAS1 / 3 primemRNAseq zPCF11deltaPAS1 hom e4 3’ mRNA seq data were mapped according to the guidelines of QuantSeq library kit manufacturer Lexogen. Unaligned bam files from HiSeq2500 were converted to FASTQ files with bam2fastx TopHat 2 component Kim et al. 2013. post overview with FastQC reads were trimmed with BBtools sourceforge.net/projects/bbmap/ script bbduk using following settings: k=13 ktrim=r useshortkmers=t mink=5 qtrim=r trimq=10 minlength=20 . post trimming control with FastQC curated reads were mapped with STAR2.5b Dobin et al. 2013 aligner. STAR index was generated with the GRCz10/danRer10 was used as the reference zebrafish genome together with the corresponding ENSEMBL annotation. Aligned 3’ mRNA seq reads were filtered to remove false positives due to internal priming of the QuantSeq assay on genome encoded polyA stretches. To do this first a crude genomic mask was generated that contained all loci harbouring 6 or more consecutive A bases as well as any 10 nucleotide windows containing more than 6 A bases. For genes expressed from the reverse strand an analogous T rich mask was generated. Those crude masks were then corrected to allow for detection of genuine PAS falling in A/T rich regions by strand specifically unmasking 20 nucleotide intervals centred at annotated 3’ gene ends. 3’ mRNA seq reads falling into those refined strand specific masks were then removed and the filtered reads reduced to the most distal nucleotide 3’ end nucleotide. Genome build: GRCz10/danRer10 Supplementary files format and content: bigWig files: genome coverage files mean from replicates single embryo head 32hpf   Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq 3’mRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. Zebrafish Danio rerio were raised according to standard protocols 28°C water temperature 14/10 hr light/dark cycle. TLAB fish generated by crossing zebrafish AB and the natural variant TL Tupfel Long fin stocks served as wild type zebrafish for all experiments. zPCF11 null and zPCF11 ∆PAS1 mutant zebrafish were generated by Cas9 mediated mutagenesis Gagnon et al. 2014 molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11deltaPAS1 / GSM3536474 GSM3536474: 3 primemRNAseq zPCF11deltaPAS1 hom e4; Danio rerio; RNA Seq GSM3536474   1 Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq three primemRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. GEO Accession:GSM3536474 RNA-Seq TRANSCRIPTOMIC cDNA SINGLE ILLUMINA Illumina HiSeq 2500   SRP175015     bam5__sel_AACGCCATC.bam.fastq_trimmed.fastq.gz fastq 921074852.0 10547533.0 GSM3536474 r2 0:87.33 A:310174374;C:168106596;G:183538421;T:259253357;N:2104 87       310174374 168106596 183538421 259253357 2104 SRX5193230 SRS4199230 SRA828489 GEO Sir William Dunn School of Pathology, University of Oxford 1 0.83245   0.14487   0.80359   0.50415   89   B   usable mapping rate illumina hiseq_era 3prime poly_a lexogen bulk unknown unknown   United Kingdom 2019-01-02 Multi-stage Embryo Head Nervous System
50959 50959 SRR8383308 SRX5193229 SRS4199229 SRP175015 PRJNA512489 Selective roles of vertebrate PCF11 in premature and full length transcript termination zebrafish three prime mRNA seq GSE124555 Other The pervasive nature of RNA polymerase II Pol II transcription requires efficient termination. A key player in this process is the cleavage and polyadenylation CPA factor PCF11 which directly binds to the Pol II C terminal domain and dismantles elongating Pol II from DNA in vitro. We demonstrate that PCF11 mediated termination is essential for vertebrate development. A range of genomic analyses including: mNET seq three prime mRNA seq chromatin RNA seq and ChIP seq reveals that PCF11 enhances transcription termination and stimulates early polyadenylation genome wide. PCF11 binds preferentially between closely spaced genes where it prevents transcriptional interference and downstream gene silencing. Notably PCF11 is sub stoichiometric to the CPA complex. Low levels of PCF11 are maintained by an auto regulatory mechanism involving premature termination of its own transcript and are important for normal development. Both in human cell culture and during zebrafish development PCF11 selectively attenuates the expression of other transcriptional regulators by premature CPA and termination. Overall design: three prime mRNA seq in individual zebrafish embryo heads. Two types of mutants: zPCF11 null and zPCF11 with deletion of PAS1. Wild type wt +/+ heterozygous het +/ and homozygous mutant hom / embryos were analyzed. Wild type and heterozygous animals were phenotypically indistinguishable. parent bioproject:PRJNA507553 pubmed:30819644   3 primemRNAseq zPCF11deltaPAS1 hom e3 GSM3536473   tissue:single embryo head 32hpf|molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11deltaPAS1 / 3 primemRNAseq zPCF11deltaPAS1 hom e3 3’ mRNA seq data were mapped according to the guidelines of QuantSeq library kit manufacturer Lexogen. Unaligned bam files from HiSeq2500 were converted to FASTQ files with bam2fastx TopHat 2 component Kim et al. 2013. post overview with FastQC reads were trimmed with BBtools sourceforge.net/projects/bbmap/ script bbduk using following settings: k=13 ktrim=r useshortkmers=t mink=5 qtrim=r trimq=10 minlength=20 . post trimming control with FastQC curated reads were mapped with STAR2.5b Dobin et al. 2013 aligner. STAR index was generated with the GRCz10/danRer10 was used as the reference zebrafish genome together with the corresponding ENSEMBL annotation. Aligned 3’ mRNA seq reads were filtered to remove false positives due to internal priming of the QuantSeq assay on genome encoded polyA stretches. To do this first a crude genomic mask was generated that contained all loci harbouring 6 or more consecutive A bases as well as any 10 nucleotide windows containing more than 6 A bases. For genes expressed from the reverse strand an analogous T rich mask was generated. Those crude masks were then corrected to allow for detection of genuine PAS falling in A/T rich regions by strand specifically unmasking 20 nucleotide intervals centred at annotated 3’ gene ends. 3’ mRNA seq reads falling into those refined strand specific masks were then removed and the filtered reads reduced to the most distal nucleotide 3’ end nucleotide. Genome build: GRCz10/danRer10 Supplementary files format and content: bigWig files: genome coverage files mean from replicates single embryo head 32hpf   Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq 3’mRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. Zebrafish Danio rerio were raised according to standard protocols 28°C water temperature 14/10 hr light/dark cycle. TLAB fish generated by crossing zebrafish AB and the natural variant TL Tupfel Long fin stocks served as wild type zebrafish for all experiments. zPCF11 null and zPCF11 ∆PAS1 mutant zebrafish were generated by Cas9 mediated mutagenesis Gagnon et al. 2014 molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11deltaPAS1 / GSM3536473 GSM3536473: 3 primemRNAseq zPCF11deltaPAS1 hom e3; Danio rerio; RNA Seq GSM3536473   1 Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq three primemRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. GEO Accession:GSM3536473 RNA-Seq TRANSCRIPTOMIC cDNA SINGLE ILLUMINA Illumina HiSeq 2500   SRP175015     bam4__sel_TTTATGATC.bam.fastq_trimmed.fastq.gz fastq 1018559174.0 11616470.0 GSM3536473 r1 0:87.68 A:347433965;C:179438057;G:202582370;T:289101606;N:3176 87       347433965 179438057 202582370 289101606 3176 SRX5193229 SRS4199229 SRA828489 GEO Sir William Dunn School of Pathology, University of Oxford 1 0.81678   0.16376   0.79295   0.52685   89   B   usable mapping rate illumina hiseq_era 3prime poly_a lexogen bulk unknown unknown   United Kingdom 2019-01-02 Multi-stage Embryo Head Nervous System
50960 50960 SRR8383309 SRX5193229 SRS4199229 SRP175015 PRJNA512489 Selective roles of vertebrate PCF11 in premature and full length transcript termination zebrafish three prime mRNA seq GSE124555 Other The pervasive nature of RNA polymerase II Pol II transcription requires efficient termination. A key player in this process is the cleavage and polyadenylation CPA factor PCF11 which directly binds to the Pol II C terminal domain and dismantles elongating Pol II from DNA in vitro. We demonstrate that PCF11 mediated termination is essential for vertebrate development. A range of genomic analyses including: mNET seq three prime mRNA seq chromatin RNA seq and ChIP seq reveals that PCF11 enhances transcription termination and stimulates early polyadenylation genome wide. PCF11 binds preferentially between closely spaced genes where it prevents transcriptional interference and downstream gene silencing. Notably PCF11 is sub stoichiometric to the CPA complex. Low levels of PCF11 are maintained by an auto regulatory mechanism involving premature termination of its own transcript and are important for normal development. Both in human cell culture and during zebrafish development PCF11 selectively attenuates the expression of other transcriptional regulators by premature CPA and termination. Overall design: three prime mRNA seq in individual zebrafish embryo heads. Two types of mutants: zPCF11 null and zPCF11 with deletion of PAS1. Wild type wt +/+ heterozygous het +/ and homozygous mutant hom / embryos were analyzed. Wild type and heterozygous animals were phenotypically indistinguishable. parent bioproject:PRJNA507553 pubmed:30819644   3 primemRNAseq zPCF11deltaPAS1 hom e3 GSM3536473   tissue:single embryo head 32hpf|molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11deltaPAS1 / 3 primemRNAseq zPCF11deltaPAS1 hom e3 3’ mRNA seq data were mapped according to the guidelines of QuantSeq library kit manufacturer Lexogen. Unaligned bam files from HiSeq2500 were converted to FASTQ files with bam2fastx TopHat 2 component Kim et al. 2013. post overview with FastQC reads were trimmed with BBtools sourceforge.net/projects/bbmap/ script bbduk using following settings: k=13 ktrim=r useshortkmers=t mink=5 qtrim=r trimq=10 minlength=20 . post trimming control with FastQC curated reads were mapped with STAR2.5b Dobin et al. 2013 aligner. STAR index was generated with the GRCz10/danRer10 was used as the reference zebrafish genome together with the corresponding ENSEMBL annotation. Aligned 3’ mRNA seq reads were filtered to remove false positives due to internal priming of the QuantSeq assay on genome encoded polyA stretches. To do this first a crude genomic mask was generated that contained all loci harbouring 6 or more consecutive A bases as well as any 10 nucleotide windows containing more than 6 A bases. For genes expressed from the reverse strand an analogous T rich mask was generated. Those crude masks were then corrected to allow for detection of genuine PAS falling in A/T rich regions by strand specifically unmasking 20 nucleotide intervals centred at annotated 3’ gene ends. 3’ mRNA seq reads falling into those refined strand specific masks were then removed and the filtered reads reduced to the most distal nucleotide 3’ end nucleotide. Genome build: GRCz10/danRer10 Supplementary files format and content: bigWig files: genome coverage files mean from replicates single embryo head 32hpf   Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq 3’mRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. Zebrafish Danio rerio were raised according to standard protocols 28°C water temperature 14/10 hr light/dark cycle. TLAB fish generated by crossing zebrafish AB and the natural variant TL Tupfel Long fin stocks served as wild type zebrafish for all experiments. zPCF11 null and zPCF11 ∆PAS1 mutant zebrafish were generated by Cas9 mediated mutagenesis Gagnon et al. 2014 molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11deltaPAS1 / GSM3536473 GSM3536473: 3 primemRNAseq zPCF11deltaPAS1 hom e3; Danio rerio; RNA Seq GSM3536473   1 Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq three primemRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. GEO Accession:GSM3536473 RNA-Seq TRANSCRIPTOMIC cDNA SINGLE ILLUMINA Illumina HiSeq 2500   SRP175015     bam5__sel_TTTATGATC.bam.fastq_trimmed.fastq.gz fastq 1049029756.0 11967431.0 GSM3536473 r2 0:87.66 A:358315722;C:184586479;G:208348099;T:297777016;N:2440 87       358315722 184586479 208348099 297777016 2440 SRX5193229 SRS4199229 SRA828489 GEO Sir William Dunn School of Pathology, University of Oxford 1 0.81613   0.16396   0.7931   0.51637   89   B   usable mapping rate illumina hiseq_era 3prime poly_a lexogen bulk unknown unknown   United Kingdom 2019-01-02 Multi-stage Embryo Head Nervous System
50961 50961 SRR8383306 SRX5193228 SRS4199228 SRP175015 PRJNA512489 Selective roles of vertebrate PCF11 in premature and full length transcript termination zebrafish three prime mRNA seq GSE124555 Other The pervasive nature of RNA polymerase II Pol II transcription requires efficient termination. A key player in this process is the cleavage and polyadenylation CPA factor PCF11 which directly binds to the Pol II C terminal domain and dismantles elongating Pol II from DNA in vitro. We demonstrate that PCF11 mediated termination is essential for vertebrate development. A range of genomic analyses including: mNET seq three prime mRNA seq chromatin RNA seq and ChIP seq reveals that PCF11 enhances transcription termination and stimulates early polyadenylation genome wide. PCF11 binds preferentially between closely spaced genes where it prevents transcriptional interference and downstream gene silencing. Notably PCF11 is sub stoichiometric to the CPA complex. Low levels of PCF11 are maintained by an auto regulatory mechanism involving premature termination of its own transcript and are important for normal development. Both in human cell culture and during zebrafish development PCF11 selectively attenuates the expression of other transcriptional regulators by premature CPA and termination. Overall design: three prime mRNA seq in individual zebrafish embryo heads. Two types of mutants: zPCF11 null and zPCF11 with deletion of PAS1. Wild type wt +/+ heterozygous het +/ and homozygous mutant hom / embryos were analyzed. Wild type and heterozygous animals were phenotypically indistinguishable. parent bioproject:PRJNA507553 pubmed:30819644   3 primemRNAseq zPCF11deltaPAS1 hom e2 GSM3536472   tissue:single embryo head 32hpf|molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11deltaPAS1 / 3 primemRNAseq zPCF11deltaPAS1 hom e2 3’ mRNA seq data were mapped according to the guidelines of QuantSeq library kit manufacturer Lexogen. Unaligned bam files from HiSeq2500 were converted to FASTQ files with bam2fastx TopHat 2 component Kim et al. 2013. post overview with FastQC reads were trimmed with BBtools sourceforge.net/projects/bbmap/ script bbduk using following settings: k=13 ktrim=r useshortkmers=t mink=5 qtrim=r trimq=10 minlength=20 . post trimming control with FastQC curated reads were mapped with STAR2.5b Dobin et al. 2013 aligner. STAR index was generated with the GRCz10/danRer10 was used as the reference zebrafish genome together with the corresponding ENSEMBL annotation. Aligned 3’ mRNA seq reads were filtered to remove false positives due to internal priming of the QuantSeq assay on genome encoded polyA stretches. To do this first a crude genomic mask was generated that contained all loci harbouring 6 or more consecutive A bases as well as any 10 nucleotide windows containing more than 6 A bases. For genes expressed from the reverse strand an analogous T rich mask was generated. Those crude masks were then corrected to allow for detection of genuine PAS falling in A/T rich regions by strand specifically unmasking 20 nucleotide intervals centred at annotated 3’ gene ends. 3’ mRNA seq reads falling into those refined strand specific masks were then removed and the filtered reads reduced to the most distal nucleotide 3’ end nucleotide. Genome build: GRCz10/danRer10 Supplementary files format and content: bigWig files: genome coverage files mean from replicates single embryo head 32hpf   Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq 3’mRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. Zebrafish Danio rerio were raised according to standard protocols 28°C water temperature 14/10 hr light/dark cycle. TLAB fish generated by crossing zebrafish AB and the natural variant TL Tupfel Long fin stocks served as wild type zebrafish for all experiments. zPCF11 null and zPCF11 ∆PAS1 mutant zebrafish were generated by Cas9 mediated mutagenesis Gagnon et al. 2014 molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11deltaPAS1 / GSM3536472 GSM3536472: 3 primemRNAseq zPCF11deltaPAS1 hom e2; Danio rerio; RNA Seq GSM3536472   1 Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq three primemRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. GEO Accession:GSM3536472 RNA-Seq TRANSCRIPTOMIC cDNA SINGLE ILLUMINA Illumina HiSeq 2500   SRP175015     bam4__sel_CCGCAAATC.bam.fastq_trimmed.fastq.gz fastq 95347510.0 1083083.0 GSM3536472 r1 0:88.03 A:31277905;C:16917301;G:19217962;T:27934061;N:281 88       31277905 16917301 19217962 27934061 281 SRX5193228 SRS4199228 SRA828489 GEO Sir William Dunn School of Pathology, University of Oxford 1 0.84359   0.1281   0.77849   0.50658   89   B   usable mapping rate illumina hiseq_era 3prime poly_a lexogen bulk unknown unknown   United Kingdom 2019-01-02 Multi-stage Embryo Head Nervous System
50962 50962 SRR8383307 SRX5193228 SRS4199228 SRP175015 PRJNA512489 Selective roles of vertebrate PCF11 in premature and full length transcript termination zebrafish three prime mRNA seq GSE124555 Other The pervasive nature of RNA polymerase II Pol II transcription requires efficient termination. A key player in this process is the cleavage and polyadenylation CPA factor PCF11 which directly binds to the Pol II C terminal domain and dismantles elongating Pol II from DNA in vitro. We demonstrate that PCF11 mediated termination is essential for vertebrate development. A range of genomic analyses including: mNET seq three prime mRNA seq chromatin RNA seq and ChIP seq reveals that PCF11 enhances transcription termination and stimulates early polyadenylation genome wide. PCF11 binds preferentially between closely spaced genes where it prevents transcriptional interference and downstream gene silencing. Notably PCF11 is sub stoichiometric to the CPA complex. Low levels of PCF11 are maintained by an auto regulatory mechanism involving premature termination of its own transcript and are important for normal development. Both in human cell culture and during zebrafish development PCF11 selectively attenuates the expression of other transcriptional regulators by premature CPA and termination. Overall design: three prime mRNA seq in individual zebrafish embryo heads. Two types of mutants: zPCF11 null and zPCF11 with deletion of PAS1. Wild type wt +/+ heterozygous het +/ and homozygous mutant hom / embryos were analyzed. Wild type and heterozygous animals were phenotypically indistinguishable. parent bioproject:PRJNA507553 pubmed:30819644   3 primemRNAseq zPCF11deltaPAS1 hom e2 GSM3536472   tissue:single embryo head 32hpf|molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11deltaPAS1 / 3 primemRNAseq zPCF11deltaPAS1 hom e2 3’ mRNA seq data were mapped according to the guidelines of QuantSeq library kit manufacturer Lexogen. Unaligned bam files from HiSeq2500 were converted to FASTQ files with bam2fastx TopHat 2 component Kim et al. 2013. post overview with FastQC reads were trimmed with BBtools sourceforge.net/projects/bbmap/ script bbduk using following settings: k=13 ktrim=r useshortkmers=t mink=5 qtrim=r trimq=10 minlength=20 . post trimming control with FastQC curated reads were mapped with STAR2.5b Dobin et al. 2013 aligner. STAR index was generated with the GRCz10/danRer10 was used as the reference zebrafish genome together with the corresponding ENSEMBL annotation. Aligned 3’ mRNA seq reads were filtered to remove false positives due to internal priming of the QuantSeq assay on genome encoded polyA stretches. To do this first a crude genomic mask was generated that contained all loci harbouring 6 or more consecutive A bases as well as any 10 nucleotide windows containing more than 6 A bases. For genes expressed from the reverse strand an analogous T rich mask was generated. Those crude masks were then corrected to allow for detection of genuine PAS falling in A/T rich regions by strand specifically unmasking 20 nucleotide intervals centred at annotated 3’ gene ends. 3’ mRNA seq reads falling into those refined strand specific masks were then removed and the filtered reads reduced to the most distal nucleotide 3’ end nucleotide. Genome build: GRCz10/danRer10 Supplementary files format and content: bigWig files: genome coverage files mean from replicates single embryo head 32hpf   Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq 3’mRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. Zebrafish Danio rerio were raised according to standard protocols 28°C water temperature 14/10 hr light/dark cycle. TLAB fish generated by crossing zebrafish AB and the natural variant TL Tupfel Long fin stocks served as wild type zebrafish for all experiments. zPCF11 null and zPCF11 ∆PAS1 mutant zebrafish were generated by Cas9 mediated mutagenesis Gagnon et al. 2014 molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11deltaPAS1 / GSM3536472 GSM3536472: 3 primemRNAseq zPCF11deltaPAS1 hom e2; Danio rerio; RNA Seq GSM3536472   1 Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq three primemRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. GEO Accession:GSM3536472 RNA-Seq TRANSCRIPTOMIC cDNA SINGLE ILLUMINA Illumina HiSeq 2500   SRP175015     bam5__sel_CCGCAAATC.bam.fastq_trimmed.fastq.gz fastq 98289209.0 1116653.0 GSM3536472 r2 0:88.02 A:32271861;C:17428261;G:19780092;T:28808779;N:216 88       32271861 17428261 19780092 28808779 216 SRX5193228 SRS4199228 SRA828489 GEO Sir William Dunn School of Pathology, University of Oxford 1 0.84257   0.12826   0.77881   0.51159   89   B   usable mapping rate illumina hiseq_era 3prime poly_a lexogen bulk unknown unknown   United Kingdom 2019-01-02 Multi-stage Embryo Head Nervous System
50963 50963 SRR8383304 SRX5193227 SRS4199227 SRP175015 PRJNA512489 Selective roles of vertebrate PCF11 in premature and full length transcript termination zebrafish three prime mRNA seq GSE124555 Other The pervasive nature of RNA polymerase II Pol II transcription requires efficient termination. A key player in this process is the cleavage and polyadenylation CPA factor PCF11 which directly binds to the Pol II C terminal domain and dismantles elongating Pol II from DNA in vitro. We demonstrate that PCF11 mediated termination is essential for vertebrate development. A range of genomic analyses including: mNET seq three prime mRNA seq chromatin RNA seq and ChIP seq reveals that PCF11 enhances transcription termination and stimulates early polyadenylation genome wide. PCF11 binds preferentially between closely spaced genes where it prevents transcriptional interference and downstream gene silencing. Notably PCF11 is sub stoichiometric to the CPA complex. Low levels of PCF11 are maintained by an auto regulatory mechanism involving premature termination of its own transcript and are important for normal development. Both in human cell culture and during zebrafish development PCF11 selectively attenuates the expression of other transcriptional regulators by premature CPA and termination. Overall design: three prime mRNA seq in individual zebrafish embryo heads. Two types of mutants: zPCF11 null and zPCF11 with deletion of PAS1. Wild type wt +/+ heterozygous het +/ and homozygous mutant hom / embryos were analyzed. Wild type and heterozygous animals were phenotypically indistinguishable. parent bioproject:PRJNA507553 pubmed:30819644   3 primemRNAseq zPCF11deltaPAS1 hom e1 GSM3536471   tissue:single embryo head 32hpf|molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11deltaPAS1 / 3 primemRNAseq zPCF11deltaPAS1 hom e1 3’ mRNA seq data were mapped according to the guidelines of QuantSeq library kit manufacturer Lexogen. Unaligned bam files from HiSeq2500 were converted to FASTQ files with bam2fastx TopHat 2 component Kim et al. 2013. post overview with FastQC reads were trimmed with BBtools sourceforge.net/projects/bbmap/ script bbduk using following settings: k=13 ktrim=r useshortkmers=t mink=5 qtrim=r trimq=10 minlength=20 . post trimming control with FastQC curated reads were mapped with STAR2.5b Dobin et al. 2013 aligner. STAR index was generated with the GRCz10/danRer10 was used as the reference zebrafish genome together with the corresponding ENSEMBL annotation. Aligned 3’ mRNA seq reads were filtered to remove false positives due to internal priming of the QuantSeq assay on genome encoded polyA stretches. To do this first a crude genomic mask was generated that contained all loci harbouring 6 or more consecutive A bases as well as any 10 nucleotide windows containing more than 6 A bases. For genes expressed from the reverse strand an analogous T rich mask was generated. Those crude masks were then corrected to allow for detection of genuine PAS falling in A/T rich regions by strand specifically unmasking 20 nucleotide intervals centred at annotated 3’ gene ends. 3’ mRNA seq reads falling into those refined strand specific masks were then removed and the filtered reads reduced to the most distal nucleotide 3’ end nucleotide. Genome build: GRCz10/danRer10 Supplementary files format and content: bigWig files: genome coverage files mean from replicates single embryo head 32hpf   Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq 3’mRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. Zebrafish Danio rerio were raised according to standard protocols 28°C water temperature 14/10 hr light/dark cycle. TLAB fish generated by crossing zebrafish AB and the natural variant TL Tupfel Long fin stocks served as wild type zebrafish for all experiments. zPCF11 null and zPCF11 ∆PAS1 mutant zebrafish were generated by Cas9 mediated mutagenesis Gagnon et al. 2014 molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11deltaPAS1 / GSM3536471 GSM3536471: 3 primemRNAseq zPCF11deltaPAS1 hom e1; Danio rerio; RNA Seq GSM3536471   1 Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq three primemRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. GEO Accession:GSM3536471 RNA-Seq TRANSCRIPTOMIC cDNA SINGLE ILLUMINA Illumina HiSeq 2500   SRP175015     bam4__sel_CACACTATC.bam.fastq_trimmed.fastq.gz fastq 151964150.0 1729761.0 GSM3536471 r1 0:87.85 A:51182251;C:27047472;G:29597244;T:44136727;N:456 87       51182251 27047472 29597244 44136727 456 SRX5193227 SRS4199227 SRA828489 GEO Sir William Dunn School of Pathology, University of Oxford 1 0.83286   0.13372   0.78023   0.44685   89   B   usable mapping rate illumina hiseq_era 3prime poly_a lexogen bulk unknown unknown   United Kingdom 2019-01-02 Multi-stage Embryo Head Nervous System
50964 50964 SRR8383305 SRX5193227 SRS4199227 SRP175015 PRJNA512489 Selective roles of vertebrate PCF11 in premature and full length transcript termination zebrafish three prime mRNA seq GSE124555 Other The pervasive nature of RNA polymerase II Pol II transcription requires efficient termination. A key player in this process is the cleavage and polyadenylation CPA factor PCF11 which directly binds to the Pol II C terminal domain and dismantles elongating Pol II from DNA in vitro. We demonstrate that PCF11 mediated termination is essential for vertebrate development. A range of genomic analyses including: mNET seq three prime mRNA seq chromatin RNA seq and ChIP seq reveals that PCF11 enhances transcription termination and stimulates early polyadenylation genome wide. PCF11 binds preferentially between closely spaced genes where it prevents transcriptional interference and downstream gene silencing. Notably PCF11 is sub stoichiometric to the CPA complex. Low levels of PCF11 are maintained by an auto regulatory mechanism involving premature termination of its own transcript and are important for normal development. Both in human cell culture and during zebrafish development PCF11 selectively attenuates the expression of other transcriptional regulators by premature CPA and termination. Overall design: three prime mRNA seq in individual zebrafish embryo heads. Two types of mutants: zPCF11 null and zPCF11 with deletion of PAS1. Wild type wt +/+ heterozygous het +/ and homozygous mutant hom / embryos were analyzed. Wild type and heterozygous animals were phenotypically indistinguishable. parent bioproject:PRJNA507553 pubmed:30819644   3 primemRNAseq zPCF11deltaPAS1 hom e1 GSM3536471   tissue:single embryo head 32hpf|molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11deltaPAS1 / 3 primemRNAseq zPCF11deltaPAS1 hom e1 3’ mRNA seq data were mapped according to the guidelines of QuantSeq library kit manufacturer Lexogen. Unaligned bam files from HiSeq2500 were converted to FASTQ files with bam2fastx TopHat 2 component Kim et al. 2013. post overview with FastQC reads were trimmed with BBtools sourceforge.net/projects/bbmap/ script bbduk using following settings: k=13 ktrim=r useshortkmers=t mink=5 qtrim=r trimq=10 minlength=20 . post trimming control with FastQC curated reads were mapped with STAR2.5b Dobin et al. 2013 aligner. STAR index was generated with the GRCz10/danRer10 was used as the reference zebrafish genome together with the corresponding ENSEMBL annotation. Aligned 3’ mRNA seq reads were filtered to remove false positives due to internal priming of the QuantSeq assay on genome encoded polyA stretches. To do this first a crude genomic mask was generated that contained all loci harbouring 6 or more consecutive A bases as well as any 10 nucleotide windows containing more than 6 A bases. For genes expressed from the reverse strand an analogous T rich mask was generated. Those crude masks were then corrected to allow for detection of genuine PAS falling in A/T rich regions by strand specifically unmasking 20 nucleotide intervals centred at annotated 3’ gene ends. 3’ mRNA seq reads falling into those refined strand specific masks were then removed and the filtered reads reduced to the most distal nucleotide 3’ end nucleotide. Genome build: GRCz10/danRer10 Supplementary files format and content: bigWig files: genome coverage files mean from replicates single embryo head 32hpf   Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq 3’mRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. Zebrafish Danio rerio were raised according to standard protocols 28°C water temperature 14/10 hr light/dark cycle. TLAB fish generated by crossing zebrafish AB and the natural variant TL Tupfel Long fin stocks served as wild type zebrafish for all experiments. zPCF11 null and zPCF11 ∆PAS1 mutant zebrafish were generated by Cas9 mediated mutagenesis Gagnon et al. 2014 molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11deltaPAS1 / GSM3536471 GSM3536471: 3 primemRNAseq zPCF11deltaPAS1 hom e1; Danio rerio; RNA Seq GSM3536471   1 Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq three primemRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. GEO Accession:GSM3536471 RNA-Seq TRANSCRIPTOMIC cDNA SINGLE ILLUMINA Illumina HiSeq 2500   SRP175015     bam5__sel_CACACTATC.bam.fastq_trimmed.fastq.gz fastq 156746824.0 1784420.0 GSM3536471 r2 0:87.84 A:52870952;C:27860644;G:30505998;T:45508859;N:371 87       52870952 27860644 30505998 45508859 371 SRX5193227 SRS4199227 SRA828489 GEO Sir William Dunn School of Pathology, University of Oxford 1 0.83111   0.13281   0.78001   0.44609   89   B   usable mapping rate illumina hiseq_era 3prime poly_a lexogen bulk unknown unknown   United Kingdom 2019-01-02 Multi-stage Embryo Head Nervous System
50965 50965 SRR8383302 SRX5193226 SRS4199226 SRP175015 PRJNA512489 Selective roles of vertebrate PCF11 in premature and full length transcript termination zebrafish three prime mRNA seq GSE124555 Other The pervasive nature of RNA polymerase II Pol II transcription requires efficient termination. A key player in this process is the cleavage and polyadenylation CPA factor PCF11 which directly binds to the Pol II C terminal domain and dismantles elongating Pol II from DNA in vitro. We demonstrate that PCF11 mediated termination is essential for vertebrate development. A range of genomic analyses including: mNET seq three prime mRNA seq chromatin RNA seq and ChIP seq reveals that PCF11 enhances transcription termination and stimulates early polyadenylation genome wide. PCF11 binds preferentially between closely spaced genes where it prevents transcriptional interference and downstream gene silencing. Notably PCF11 is sub stoichiometric to the CPA complex. Low levels of PCF11 are maintained by an auto regulatory mechanism involving premature termination of its own transcript and are important for normal development. Both in human cell culture and during zebrafish development PCF11 selectively attenuates the expression of other transcriptional regulators by premature CPA and termination. Overall design: three prime mRNA seq in individual zebrafish embryo heads. Two types of mutants: zPCF11 null and zPCF11 with deletion of PAS1. Wild type wt +/+ heterozygous het +/ and homozygous mutant hom / embryos were analyzed. Wild type and heterozygous animals were phenotypically indistinguishable. parent bioproject:PRJNA507553 pubmed:30819644   3 primemRNAseq zPCF11deltaPAS1 het e5 GSM3536470   tissue:single embryo head 32hpf|molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11deltaPAS1 +/ 3 primemRNAseq zPCF11deltaPAS1 het e5 3’ mRNA seq data were mapped according to the guidelines of QuantSeq library kit manufacturer Lexogen. Unaligned bam files from HiSeq2500 were converted to FASTQ files with bam2fastx TopHat 2 component Kim et al. 2013. post overview with FastQC reads were trimmed with BBtools sourceforge.net/projects/bbmap/ script bbduk using following settings: k=13 ktrim=r useshortkmers=t mink=5 qtrim=r trimq=10 minlength=20 . post trimming control with FastQC curated reads were mapped with STAR2.5b Dobin et al. 2013 aligner. STAR index was generated with the GRCz10/danRer10 was used as the reference zebrafish genome together with the corresponding ENSEMBL annotation. Aligned 3’ mRNA seq reads were filtered to remove false positives due to internal priming of the QuantSeq assay on genome encoded polyA stretches. To do this first a crude genomic mask was generated that contained all loci harbouring 6 or more consecutive A bases as well as any 10 nucleotide windows containing more than 6 A bases. For genes expressed from the reverse strand an analogous T rich mask was generated. Those crude masks were then corrected to allow for detection of genuine PAS falling in A/T rich regions by strand specifically unmasking 20 nucleotide intervals centred at annotated 3’ gene ends. 3’ mRNA seq reads falling into those refined strand specific masks were then removed and the filtered reads reduced to the most distal nucleotide 3’ end nucleotide. Genome build: GRCz10/danRer10 Supplementary files format and content: bigWig files: genome coverage files mean from replicates single embryo head 32hpf   Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq 3’mRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. Zebrafish Danio rerio were raised according to standard protocols 28°C water temperature 14/10 hr light/dark cycle. TLAB fish generated by crossing zebrafish AB and the natural variant TL Tupfel Long fin stocks served as wild type zebrafish for all experiments. zPCF11 null and zPCF11 ∆PAS1 mutant zebrafish were generated by Cas9 mediated mutagenesis Gagnon et al. 2014 molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11deltaPAS1 +/ GSM3536470 GSM3536470: 3 primemRNAseq zPCF11deltaPAS1 het e5; Danio rerio; RNA Seq GSM3536470   1 Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq three primemRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. GEO Accession:GSM3536470 RNA-Seq TRANSCRIPTOMIC cDNA SINGLE ILLUMINA Illumina HiSeq 2500   SRP175015     bam4__sel_TACCTTATC.bam.fastq_trimmed.fastq.gz fastq 132241407.0 1505632.0 GSM3536470 r1 0:87.83 A:44701797;C:23272762;G:25927608;T:38338866;N:374 87       44701797 23272762 25927608 38338866 374 SRX5193226 SRS4199226 SRA828489 GEO Sir William Dunn School of Pathology, University of Oxford 1 0.82845   0.13469   0.7767   0.52442   89   B   usable mapping rate illumina hiseq_era 3prime poly_a lexogen bulk unknown unknown   United Kingdom 2019-01-02 Multi-stage Embryo Head Nervous System
50966 50966 SRR8383303 SRX5193226 SRS4199226 SRP175015 PRJNA512489 Selective roles of vertebrate PCF11 in premature and full length transcript termination zebrafish three prime mRNA seq GSE124555 Other The pervasive nature of RNA polymerase II Pol II transcription requires efficient termination. A key player in this process is the cleavage and polyadenylation CPA factor PCF11 which directly binds to the Pol II C terminal domain and dismantles elongating Pol II from DNA in vitro. We demonstrate that PCF11 mediated termination is essential for vertebrate development. A range of genomic analyses including: mNET seq three prime mRNA seq chromatin RNA seq and ChIP seq reveals that PCF11 enhances transcription termination and stimulates early polyadenylation genome wide. PCF11 binds preferentially between closely spaced genes where it prevents transcriptional interference and downstream gene silencing. Notably PCF11 is sub stoichiometric to the CPA complex. Low levels of PCF11 are maintained by an auto regulatory mechanism involving premature termination of its own transcript and are important for normal development. Both in human cell culture and during zebrafish development PCF11 selectively attenuates the expression of other transcriptional regulators by premature CPA and termination. Overall design: three prime mRNA seq in individual zebrafish embryo heads. Two types of mutants: zPCF11 null and zPCF11 with deletion of PAS1. Wild type wt +/+ heterozygous het +/ and homozygous mutant hom / embryos were analyzed. Wild type and heterozygous animals were phenotypically indistinguishable. parent bioproject:PRJNA507553 pubmed:30819644   3 primemRNAseq zPCF11deltaPAS1 het e5 GSM3536470   tissue:single embryo head 32hpf|molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11deltaPAS1 +/ 3 primemRNAseq zPCF11deltaPAS1 het e5 3’ mRNA seq data were mapped according to the guidelines of QuantSeq library kit manufacturer Lexogen. Unaligned bam files from HiSeq2500 were converted to FASTQ files with bam2fastx TopHat 2 component Kim et al. 2013. post overview with FastQC reads were trimmed with BBtools sourceforge.net/projects/bbmap/ script bbduk using following settings: k=13 ktrim=r useshortkmers=t mink=5 qtrim=r trimq=10 minlength=20 . post trimming control with FastQC curated reads were mapped with STAR2.5b Dobin et al. 2013 aligner. STAR index was generated with the GRCz10/danRer10 was used as the reference zebrafish genome together with the corresponding ENSEMBL annotation. Aligned 3’ mRNA seq reads were filtered to remove false positives due to internal priming of the QuantSeq assay on genome encoded polyA stretches. To do this first a crude genomic mask was generated that contained all loci harbouring 6 or more consecutive A bases as well as any 10 nucleotide windows containing more than 6 A bases. For genes expressed from the reverse strand an analogous T rich mask was generated. Those crude masks were then corrected to allow for detection of genuine PAS falling in A/T rich regions by strand specifically unmasking 20 nucleotide intervals centred at annotated 3’ gene ends. 3’ mRNA seq reads falling into those refined strand specific masks were then removed and the filtered reads reduced to the most distal nucleotide 3’ end nucleotide. Genome build: GRCz10/danRer10 Supplementary files format and content: bigWig files: genome coverage files mean from replicates single embryo head 32hpf   Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq 3’mRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. Zebrafish Danio rerio were raised according to standard protocols 28°C water temperature 14/10 hr light/dark cycle. TLAB fish generated by crossing zebrafish AB and the natural variant TL Tupfel Long fin stocks served as wild type zebrafish for all experiments. zPCF11 null and zPCF11 ∆PAS1 mutant zebrafish were generated by Cas9 mediated mutagenesis Gagnon et al. 2014 molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11deltaPAS1 +/ GSM3536470 GSM3536470: 3 primemRNAseq zPCF11deltaPAS1 het e5; Danio rerio; RNA Seq GSM3536470   1 Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq three primemRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. GEO Accession:GSM3536470 RNA-Seq TRANSCRIPTOMIC cDNA SINGLE ILLUMINA Illumina HiSeq 2500   SRP175015     bam5__sel_TACCTTATC.bam.fastq_trimmed.fastq.gz fastq 136234092.0 1551581.0 GSM3536470 r2 0:87.80 A:46139985;C:23944780;G:26663569;T:39485404;N:354 87       46139985 23944780 26663569 39485404 354 SRX5193226 SRS4199226 SRA828489 GEO Sir William Dunn School of Pathology, University of Oxford 1 0.82629   0.13474   0.7794   0.51749   89   B   usable mapping rate illumina hiseq_era 3prime poly_a lexogen bulk unknown unknown   United Kingdom 2019-01-02 Multi-stage Embryo Head Nervous System
50967 50967 SRR8383300 SRX5193225 SRS4199225 SRP175015 PRJNA512489 Selective roles of vertebrate PCF11 in premature and full length transcript termination zebrafish three prime mRNA seq GSE124555 Other The pervasive nature of RNA polymerase II Pol II transcription requires efficient termination. A key player in this process is the cleavage and polyadenylation CPA factor PCF11 which directly binds to the Pol II C terminal domain and dismantles elongating Pol II from DNA in vitro. We demonstrate that PCF11 mediated termination is essential for vertebrate development. A range of genomic analyses including: mNET seq three prime mRNA seq chromatin RNA seq and ChIP seq reveals that PCF11 enhances transcription termination and stimulates early polyadenylation genome wide. PCF11 binds preferentially between closely spaced genes where it prevents transcriptional interference and downstream gene silencing. Notably PCF11 is sub stoichiometric to the CPA complex. Low levels of PCF11 are maintained by an auto regulatory mechanism involving premature termination of its own transcript and are important for normal development. Both in human cell culture and during zebrafish development PCF11 selectively attenuates the expression of other transcriptional regulators by premature CPA and termination. Overall design: three prime mRNA seq in individual zebrafish embryo heads. Two types of mutants: zPCF11 null and zPCF11 with deletion of PAS1. Wild type wt +/+ heterozygous het +/ and homozygous mutant hom / embryos were analyzed. Wild type and heterozygous animals were phenotypically indistinguishable. parent bioproject:PRJNA507553 pubmed:30819644   3 primemRNAseq zPCF11deltaPAS1 het e4 GSM3536469   tissue:single embryo head 32hpf|molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11deltaPAS1 +/ 3 primemRNAseq zPCF11deltaPAS1 het e4 3’ mRNA seq data were mapped according to the guidelines of QuantSeq library kit manufacturer Lexogen. Unaligned bam files from HiSeq2500 were converted to FASTQ files with bam2fastx TopHat 2 component Kim et al. 2013. post overview with FastQC reads were trimmed with BBtools sourceforge.net/projects/bbmap/ script bbduk using following settings: k=13 ktrim=r useshortkmers=t mink=5 qtrim=r trimq=10 minlength=20 . post trimming control with FastQC curated reads were mapped with STAR2.5b Dobin et al. 2013 aligner. STAR index was generated with the GRCz10/danRer10 was used as the reference zebrafish genome together with the corresponding ENSEMBL annotation. Aligned 3’ mRNA seq reads were filtered to remove false positives due to internal priming of the QuantSeq assay on genome encoded polyA stretches. To do this first a crude genomic mask was generated that contained all loci harbouring 6 or more consecutive A bases as well as any 10 nucleotide windows containing more than 6 A bases. For genes expressed from the reverse strand an analogous T rich mask was generated. Those crude masks were then corrected to allow for detection of genuine PAS falling in A/T rich regions by strand specifically unmasking 20 nucleotide intervals centred at annotated 3’ gene ends. 3’ mRNA seq reads falling into those refined strand specific masks were then removed and the filtered reads reduced to the most distal nucleotide 3’ end nucleotide. Genome build: GRCz10/danRer10 Supplementary files format and content: bigWig files: genome coverage files mean from replicates single embryo head 32hpf   Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq 3’mRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. Zebrafish Danio rerio were raised according to standard protocols 28°C water temperature 14/10 hr light/dark cycle. TLAB fish generated by crossing zebrafish AB and the natural variant TL Tupfel Long fin stocks served as wild type zebrafish for all experiments. zPCF11 null and zPCF11 ∆PAS1 mutant zebrafish were generated by Cas9 mediated mutagenesis Gagnon et al. 2014 molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11deltaPAS1 +/ GSM3536469 GSM3536469: 3 primemRNAseq zPCF11deltaPAS1 het e4; Danio rerio; RNA Seq GSM3536469   1 Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq three primemRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. GEO Accession:GSM3536469 RNA-Seq TRANSCRIPTOMIC cDNA SINGLE ILLUMINA Illumina HiSeq 2500   SRP175015     bam4__sel_ATGAACATC.bam.fastq_trimmed.fastq.gz fastq 219395486.0 2496420.0 GSM3536469 r1 0:87.88 A:74049072;C:38996524;G:43402255;T:62946930;N:705 87       74049072 38996524 43402255 62946930 705 SRX5193225 SRS4199225 SRA828489 GEO Sir William Dunn School of Pathology, University of Oxford 1 0.83282   0.14125   0.78474   0.5102   42   B   usable mapping rate illumina hiseq_era 3prime poly_a lexogen bulk unknown unknown   United Kingdom 2019-01-02 Multi-stage Embryo Head Nervous System
50968 50968 SRR8383301 SRX5193225 SRS4199225 SRP175015 PRJNA512489 Selective roles of vertebrate PCF11 in premature and full length transcript termination zebrafish three prime mRNA seq GSE124555 Other The pervasive nature of RNA polymerase II Pol II transcription requires efficient termination. A key player in this process is the cleavage and polyadenylation CPA factor PCF11 which directly binds to the Pol II C terminal domain and dismantles elongating Pol II from DNA in vitro. We demonstrate that PCF11 mediated termination is essential for vertebrate development. A range of genomic analyses including: mNET seq three prime mRNA seq chromatin RNA seq and ChIP seq reveals that PCF11 enhances transcription termination and stimulates early polyadenylation genome wide. PCF11 binds preferentially between closely spaced genes where it prevents transcriptional interference and downstream gene silencing. Notably PCF11 is sub stoichiometric to the CPA complex. Low levels of PCF11 are maintained by an auto regulatory mechanism involving premature termination of its own transcript and are important for normal development. Both in human cell culture and during zebrafish development PCF11 selectively attenuates the expression of other transcriptional regulators by premature CPA and termination. Overall design: three prime mRNA seq in individual zebrafish embryo heads. Two types of mutants: zPCF11 null and zPCF11 with deletion of PAS1. Wild type wt +/+ heterozygous het +/ and homozygous mutant hom / embryos were analyzed. Wild type and heterozygous animals were phenotypically indistinguishable. parent bioproject:PRJNA507553 pubmed:30819644   3 primemRNAseq zPCF11deltaPAS1 het e4 GSM3536469   tissue:single embryo head 32hpf|molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11deltaPAS1 +/ 3 primemRNAseq zPCF11deltaPAS1 het e4 3’ mRNA seq data were mapped according to the guidelines of QuantSeq library kit manufacturer Lexogen. Unaligned bam files from HiSeq2500 were converted to FASTQ files with bam2fastx TopHat 2 component Kim et al. 2013. post overview with FastQC reads were trimmed with BBtools sourceforge.net/projects/bbmap/ script bbduk using following settings: k=13 ktrim=r useshortkmers=t mink=5 qtrim=r trimq=10 minlength=20 . post trimming control with FastQC curated reads were mapped with STAR2.5b Dobin et al. 2013 aligner. STAR index was generated with the GRCz10/danRer10 was used as the reference zebrafish genome together with the corresponding ENSEMBL annotation. Aligned 3’ mRNA seq reads were filtered to remove false positives due to internal priming of the QuantSeq assay on genome encoded polyA stretches. To do this first a crude genomic mask was generated that contained all loci harbouring 6 or more consecutive A bases as well as any 10 nucleotide windows containing more than 6 A bases. For genes expressed from the reverse strand an analogous T rich mask was generated. Those crude masks were then corrected to allow for detection of genuine PAS falling in A/T rich regions by strand specifically unmasking 20 nucleotide intervals centred at annotated 3’ gene ends. 3’ mRNA seq reads falling into those refined strand specific masks were then removed and the filtered reads reduced to the most distal nucleotide 3’ end nucleotide. Genome build: GRCz10/danRer10 Supplementary files format and content: bigWig files: genome coverage files mean from replicates single embryo head 32hpf   Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq 3’mRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. Zebrafish Danio rerio were raised according to standard protocols 28°C water temperature 14/10 hr light/dark cycle. TLAB fish generated by crossing zebrafish AB and the natural variant TL Tupfel Long fin stocks served as wild type zebrafish for all experiments. zPCF11 null and zPCF11 ∆PAS1 mutant zebrafish were generated by Cas9 mediated mutagenesis Gagnon et al. 2014 molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11deltaPAS1 +/ GSM3536469 GSM3536469: 3 primemRNAseq zPCF11deltaPAS1 het e4; Danio rerio; RNA Seq GSM3536469   1 Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq three primemRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. GEO Accession:GSM3536469 RNA-Seq TRANSCRIPTOMIC cDNA SINGLE ILLUMINA Illumina HiSeq 2500   SRP175015     bam5__sel_ATGAACATC.bam.fastq_trimmed.fastq.gz fastq 225177913.0 2562839.0 GSM3536469 r2 0:87.86 A:76113440;C:39951108;G:44487323;T:64625459;N:583 87       76113440 39951108 44487323 64625459 583 SRX5193225 SRS4199225 SRA828489 GEO Sir William Dunn School of Pathology, University of Oxford 1 0.82913   0.13953   0.78681   0.50967   89   B   usable mapping rate illumina hiseq_era 3prime poly_a lexogen bulk unknown unknown   United Kingdom 2019-01-02 Multi-stage Embryo Head Nervous System
50969 50969 SRR8383298 SRX5193224 SRS4199224 SRP175015 PRJNA512489 Selective roles of vertebrate PCF11 in premature and full length transcript termination zebrafish three prime mRNA seq GSE124555 Other The pervasive nature of RNA polymerase II Pol II transcription requires efficient termination. A key player in this process is the cleavage and polyadenylation CPA factor PCF11 which directly binds to the Pol II C terminal domain and dismantles elongating Pol II from DNA in vitro. We demonstrate that PCF11 mediated termination is essential for vertebrate development. A range of genomic analyses including: mNET seq three prime mRNA seq chromatin RNA seq and ChIP seq reveals that PCF11 enhances transcription termination and stimulates early polyadenylation genome wide. PCF11 binds preferentially between closely spaced genes where it prevents transcriptional interference and downstream gene silencing. Notably PCF11 is sub stoichiometric to the CPA complex. Low levels of PCF11 are maintained by an auto regulatory mechanism involving premature termination of its own transcript and are important for normal development. Both in human cell culture and during zebrafish development PCF11 selectively attenuates the expression of other transcriptional regulators by premature CPA and termination. Overall design: three prime mRNA seq in individual zebrafish embryo heads. Two types of mutants: zPCF11 null and zPCF11 with deletion of PAS1. Wild type wt +/+ heterozygous het +/ and homozygous mutant hom / embryos were analyzed. Wild type and heterozygous animals were phenotypically indistinguishable. parent bioproject:PRJNA507553 pubmed:30819644   3 primemRNAseq zPCF11deltaPAS1 het e3 GSM3536468   tissue:single embryo head 32hpf|molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11deltaPAS1 +/ 3 primemRNAseq zPCF11deltaPAS1 het e3 3’ mRNA seq data were mapped according to the guidelines of QuantSeq library kit manufacturer Lexogen. Unaligned bam files from HiSeq2500 were converted to FASTQ files with bam2fastx TopHat 2 component Kim et al. 2013. post overview with FastQC reads were trimmed with BBtools sourceforge.net/projects/bbmap/ script bbduk using following settings: k=13 ktrim=r useshortkmers=t mink=5 qtrim=r trimq=10 minlength=20 . post trimming control with FastQC curated reads were mapped with STAR2.5b Dobin et al. 2013 aligner. STAR index was generated with the GRCz10/danRer10 was used as the reference zebrafish genome together with the corresponding ENSEMBL annotation. Aligned 3’ mRNA seq reads were filtered to remove false positives due to internal priming of the QuantSeq assay on genome encoded polyA stretches. To do this first a crude genomic mask was generated that contained all loci harbouring 6 or more consecutive A bases as well as any 10 nucleotide windows containing more than 6 A bases. For genes expressed from the reverse strand an analogous T rich mask was generated. Those crude masks were then corrected to allow for detection of genuine PAS falling in A/T rich regions by strand specifically unmasking 20 nucleotide intervals centred at annotated 3’ gene ends. 3’ mRNA seq reads falling into those refined strand specific masks were then removed and the filtered reads reduced to the most distal nucleotide 3’ end nucleotide. Genome build: GRCz10/danRer10 Supplementary files format and content: bigWig files: genome coverage files mean from replicates single embryo head 32hpf   Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq 3’mRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. Zebrafish Danio rerio were raised according to standard protocols 28°C water temperature 14/10 hr light/dark cycle. TLAB fish generated by crossing zebrafish AB and the natural variant TL Tupfel Long fin stocks served as wild type zebrafish for all experiments. zPCF11 null and zPCF11 ∆PAS1 mutant zebrafish were generated by Cas9 mediated mutagenesis Gagnon et al. 2014 molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11deltaPAS1 +/ GSM3536468 GSM3536468: 3 primemRNAseq zPCF11deltaPAS1 het e3; Danio rerio; RNA Seq GSM3536468   1 Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq three primemRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. GEO Accession:GSM3536468 RNA-Seq TRANSCRIPTOMIC cDNA SINGLE ILLUMINA Illumina HiSeq 2500   SRP175015     bam4__sel_TTAACTATC.bam.fastq_trimmed.fastq.gz fastq 499775526.0 5688628.0 GSM3536468 r1 0:87.86 A:163216421;C:88556243;G:100895279;T:147106087;N:1496 87       163216421 88556243 100895279 147106087 1496 SRX5193224 SRS4199224 SRA828489 GEO Sir William Dunn School of Pathology, University of Oxford 1 0.84129   0.13912   0.78194   0.5187   89   B   usable mapping rate illumina hiseq_era 3prime poly_a lexogen bulk unknown unknown   United Kingdom 2019-01-02 Multi-stage Embryo Head Nervous System
50970 50970 SRR8383299 SRX5193224 SRS4199224 SRP175015 PRJNA512489 Selective roles of vertebrate PCF11 in premature and full length transcript termination zebrafish three prime mRNA seq GSE124555 Other The pervasive nature of RNA polymerase II Pol II transcription requires efficient termination. A key player in this process is the cleavage and polyadenylation CPA factor PCF11 which directly binds to the Pol II C terminal domain and dismantles elongating Pol II from DNA in vitro. We demonstrate that PCF11 mediated termination is essential for vertebrate development. A range of genomic analyses including: mNET seq three prime mRNA seq chromatin RNA seq and ChIP seq reveals that PCF11 enhances transcription termination and stimulates early polyadenylation genome wide. PCF11 binds preferentially between closely spaced genes where it prevents transcriptional interference and downstream gene silencing. Notably PCF11 is sub stoichiometric to the CPA complex. Low levels of PCF11 are maintained by an auto regulatory mechanism involving premature termination of its own transcript and are important for normal development. Both in human cell culture and during zebrafish development PCF11 selectively attenuates the expression of other transcriptional regulators by premature CPA and termination. Overall design: three prime mRNA seq in individual zebrafish embryo heads. Two types of mutants: zPCF11 null and zPCF11 with deletion of PAS1. Wild type wt +/+ heterozygous het +/ and homozygous mutant hom / embryos were analyzed. Wild type and heterozygous animals were phenotypically indistinguishable. parent bioproject:PRJNA507553 pubmed:30819644   3 primemRNAseq zPCF11deltaPAS1 het e3 GSM3536468   tissue:single embryo head 32hpf|molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11deltaPAS1 +/ 3 primemRNAseq zPCF11deltaPAS1 het e3 3’ mRNA seq data were mapped according to the guidelines of QuantSeq library kit manufacturer Lexogen. Unaligned bam files from HiSeq2500 were converted to FASTQ files with bam2fastx TopHat 2 component Kim et al. 2013. post overview with FastQC reads were trimmed with BBtools sourceforge.net/projects/bbmap/ script bbduk using following settings: k=13 ktrim=r useshortkmers=t mink=5 qtrim=r trimq=10 minlength=20 . post trimming control with FastQC curated reads were mapped with STAR2.5b Dobin et al. 2013 aligner. STAR index was generated with the GRCz10/danRer10 was used as the reference zebrafish genome together with the corresponding ENSEMBL annotation. Aligned 3’ mRNA seq reads were filtered to remove false positives due to internal priming of the QuantSeq assay on genome encoded polyA stretches. To do this first a crude genomic mask was generated that contained all loci harbouring 6 or more consecutive A bases as well as any 10 nucleotide windows containing more than 6 A bases. For genes expressed from the reverse strand an analogous T rich mask was generated. Those crude masks were then corrected to allow for detection of genuine PAS falling in A/T rich regions by strand specifically unmasking 20 nucleotide intervals centred at annotated 3’ gene ends. 3’ mRNA seq reads falling into those refined strand specific masks were then removed and the filtered reads reduced to the most distal nucleotide 3’ end nucleotide. Genome build: GRCz10/danRer10 Supplementary files format and content: bigWig files: genome coverage files mean from replicates single embryo head 32hpf   Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq 3’mRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. Zebrafish Danio rerio were raised according to standard protocols 28°C water temperature 14/10 hr light/dark cycle. TLAB fish generated by crossing zebrafish AB and the natural variant TL Tupfel Long fin stocks served as wild type zebrafish for all experiments. zPCF11 null and zPCF11 ∆PAS1 mutant zebrafish were generated by Cas9 mediated mutagenesis Gagnon et al. 2014 molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11deltaPAS1 +/ GSM3536468 GSM3536468: 3 primemRNAseq zPCF11deltaPAS1 het e3; Danio rerio; RNA Seq GSM3536468   1 Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq three primemRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. GEO Accession:GSM3536468 RNA-Seq TRANSCRIPTOMIC cDNA SINGLE ILLUMINA Illumina HiSeq 2500   SRP175015     bam5__sel_TTAACTATC.bam.fastq_trimmed.fastq.gz fastq 514657493.0 5859797.0 GSM3536468 r2 0:87.83 A:168321204;C:91084703;G:103742599;T:151507844;N:1143 87       168321204 91084703 103742599 151507844 1143 SRX5193224 SRS4199224 SRA828489 GEO Sir William Dunn School of Pathology, University of Oxford 1 0.83984   0.1389   0.78291   0.51811   89   B   usable mapping rate illumina hiseq_era 3prime poly_a lexogen bulk unknown unknown   United Kingdom 2019-01-02 Multi-stage Embryo Head Nervous System
50971 50971 SRR8383296 SRX5193223 SRS4199223 SRP175015 PRJNA512489 Selective roles of vertebrate PCF11 in premature and full length transcript termination zebrafish three prime mRNA seq GSE124555 Other The pervasive nature of RNA polymerase II Pol II transcription requires efficient termination. A key player in this process is the cleavage and polyadenylation CPA factor PCF11 which directly binds to the Pol II C terminal domain and dismantles elongating Pol II from DNA in vitro. We demonstrate that PCF11 mediated termination is essential for vertebrate development. A range of genomic analyses including: mNET seq three prime mRNA seq chromatin RNA seq and ChIP seq reveals that PCF11 enhances transcription termination and stimulates early polyadenylation genome wide. PCF11 binds preferentially between closely spaced genes where it prevents transcriptional interference and downstream gene silencing. Notably PCF11 is sub stoichiometric to the CPA complex. Low levels of PCF11 are maintained by an auto regulatory mechanism involving premature termination of its own transcript and are important for normal development. Both in human cell culture and during zebrafish development PCF11 selectively attenuates the expression of other transcriptional regulators by premature CPA and termination. Overall design: three prime mRNA seq in individual zebrafish embryo heads. Two types of mutants: zPCF11 null and zPCF11 with deletion of PAS1. Wild type wt +/+ heterozygous het +/ and homozygous mutant hom / embryos were analyzed. Wild type and heterozygous animals were phenotypically indistinguishable. parent bioproject:PRJNA507553 pubmed:30819644   3 primemRNAseq zPCF11deltaPAS1 het e2 GSM3536467   tissue:single embryo head 32hpf|molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11deltaPAS1 +/ 3 primemRNAseq zPCF11deltaPAS1 het e2 3’ mRNA seq data were mapped according to the guidelines of QuantSeq library kit manufacturer Lexogen. Unaligned bam files from HiSeq2500 were converted to FASTQ files with bam2fastx TopHat 2 component Kim et al. 2013. post overview with FastQC reads were trimmed with BBtools sourceforge.net/projects/bbmap/ script bbduk using following settings: k=13 ktrim=r useshortkmers=t mink=5 qtrim=r trimq=10 minlength=20 . post trimming control with FastQC curated reads were mapped with STAR2.5b Dobin et al. 2013 aligner. STAR index was generated with the GRCz10/danRer10 was used as the reference zebrafish genome together with the corresponding ENSEMBL annotation. Aligned 3’ mRNA seq reads were filtered to remove false positives due to internal priming of the QuantSeq assay on genome encoded polyA stretches. To do this first a crude genomic mask was generated that contained all loci harbouring 6 or more consecutive A bases as well as any 10 nucleotide windows containing more than 6 A bases. For genes expressed from the reverse strand an analogous T rich mask was generated. Those crude masks were then corrected to allow for detection of genuine PAS falling in A/T rich regions by strand specifically unmasking 20 nucleotide intervals centred at annotated 3’ gene ends. 3’ mRNA seq reads falling into those refined strand specific masks were then removed and the filtered reads reduced to the most distal nucleotide 3’ end nucleotide. Genome build: GRCz10/danRer10 Supplementary files format and content: bigWig files: genome coverage files mean from replicates single embryo head 32hpf   Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq 3’mRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. Zebrafish Danio rerio were raised according to standard protocols 28°C water temperature 14/10 hr light/dark cycle. TLAB fish generated by crossing zebrafish AB and the natural variant TL Tupfel Long fin stocks served as wild type zebrafish for all experiments. zPCF11 null and zPCF11 ∆PAS1 mutant zebrafish were generated by Cas9 mediated mutagenesis Gagnon et al. 2014 molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11deltaPAS1 +/ GSM3536467 GSM3536467: 3 primemRNAseq zPCF11deltaPAS1 het e2; Danio rerio; RNA Seq GSM3536467   1 Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq three primemRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. GEO Accession:GSM3536467 RNA-Seq TRANSCRIPTOMIC cDNA SINGLE ILLUMINA Illumina HiSeq 2500   SRP175015     bam4__sel_CGGTTAATC.bam.fastq_trimmed.fastq.gz fastq 628544705.0 7145719.0 GSM3536467 r1 0:87.96 A:208540640;C:110628846;G:124026622;T:185346588;N:2009 87       208540640 110628846 124026622 185346588 2009 SRX5193223 SRS4199223 SRA828489 GEO Sir William Dunn School of Pathology, University of Oxford 1 0.83647   0.11358   0.7821   0.50973   89   B   usable mapping rate illumina hiseq_era 3prime poly_a lexogen bulk unknown unknown   United Kingdom 2019-01-02 Multi-stage Embryo Head Nervous System
50972 50972 SRR8383297 SRX5193223 SRS4199223 SRP175015 PRJNA512489 Selective roles of vertebrate PCF11 in premature and full length transcript termination zebrafish three prime mRNA seq GSE124555 Other The pervasive nature of RNA polymerase II Pol II transcription requires efficient termination. A key player in this process is the cleavage and polyadenylation CPA factor PCF11 which directly binds to the Pol II C terminal domain and dismantles elongating Pol II from DNA in vitro. We demonstrate that PCF11 mediated termination is essential for vertebrate development. A range of genomic analyses including: mNET seq three prime mRNA seq chromatin RNA seq and ChIP seq reveals that PCF11 enhances transcription termination and stimulates early polyadenylation genome wide. PCF11 binds preferentially between closely spaced genes where it prevents transcriptional interference and downstream gene silencing. Notably PCF11 is sub stoichiometric to the CPA complex. Low levels of PCF11 are maintained by an auto regulatory mechanism involving premature termination of its own transcript and are important for normal development. Both in human cell culture and during zebrafish development PCF11 selectively attenuates the expression of other transcriptional regulators by premature CPA and termination. Overall design: three prime mRNA seq in individual zebrafish embryo heads. Two types of mutants: zPCF11 null and zPCF11 with deletion of PAS1. Wild type wt +/+ heterozygous het +/ and homozygous mutant hom / embryos were analyzed. Wild type and heterozygous animals were phenotypically indistinguishable. parent bioproject:PRJNA507553 pubmed:30819644   3 primemRNAseq zPCF11deltaPAS1 het e2 GSM3536467   tissue:single embryo head 32hpf|molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11deltaPAS1 +/ 3 primemRNAseq zPCF11deltaPAS1 het e2 3’ mRNA seq data were mapped according to the guidelines of QuantSeq library kit manufacturer Lexogen. Unaligned bam files from HiSeq2500 were converted to FASTQ files with bam2fastx TopHat 2 component Kim et al. 2013. post overview with FastQC reads were trimmed with BBtools sourceforge.net/projects/bbmap/ script bbduk using following settings: k=13 ktrim=r useshortkmers=t mink=5 qtrim=r trimq=10 minlength=20 . post trimming control with FastQC curated reads were mapped with STAR2.5b Dobin et al. 2013 aligner. STAR index was generated with the GRCz10/danRer10 was used as the reference zebrafish genome together with the corresponding ENSEMBL annotation. Aligned 3’ mRNA seq reads were filtered to remove false positives due to internal priming of the QuantSeq assay on genome encoded polyA stretches. To do this first a crude genomic mask was generated that contained all loci harbouring 6 or more consecutive A bases as well as any 10 nucleotide windows containing more than 6 A bases. For genes expressed from the reverse strand an analogous T rich mask was generated. Those crude masks were then corrected to allow for detection of genuine PAS falling in A/T rich regions by strand specifically unmasking 20 nucleotide intervals centred at annotated 3’ gene ends. 3’ mRNA seq reads falling into those refined strand specific masks were then removed and the filtered reads reduced to the most distal nucleotide 3’ end nucleotide. Genome build: GRCz10/danRer10 Supplementary files format and content: bigWig files: genome coverage files mean from replicates single embryo head 32hpf   Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq 3’mRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. Zebrafish Danio rerio were raised according to standard protocols 28°C water temperature 14/10 hr light/dark cycle. TLAB fish generated by crossing zebrafish AB and the natural variant TL Tupfel Long fin stocks served as wild type zebrafish for all experiments. zPCF11 null and zPCF11 ∆PAS1 mutant zebrafish were generated by Cas9 mediated mutagenesis Gagnon et al. 2014 molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11deltaPAS1 +/ GSM3536467 GSM3536467: 3 primemRNAseq zPCF11deltaPAS1 het e2; Danio rerio; RNA Seq GSM3536467   1 Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq three primemRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. GEO Accession:GSM3536467 RNA-Seq TRANSCRIPTOMIC cDNA SINGLE ILLUMINA Illumina HiSeq 2500   SRP175015     bam5__sel_CGGTTAATC.bam.fastq_trimmed.fastq.gz fastq 647232214.0 7359619.0 GSM3536467 r2 0:87.94 A:215025740;C:113794072;G:127525437;T:190885451;N:1514 87       215025740 113794072 127525437 190885451 1514 SRX5193223 SRS4199223 SRA828489 GEO Sir William Dunn School of Pathology, University of Oxford 1 0.83376   0.11387   0.78305   0.51943   79   B   usable mapping rate illumina hiseq_era 3prime poly_a lexogen bulk unknown unknown   United Kingdom 2019-01-02 Multi-stage Embryo Head Nervous System
50973 50973 SRR8383294 SRX5193222 SRS4199222 SRP175015 PRJNA512489 Selective roles of vertebrate PCF11 in premature and full length transcript termination zebrafish three prime mRNA seq GSE124555 Other The pervasive nature of RNA polymerase II Pol II transcription requires efficient termination. A key player in this process is the cleavage and polyadenylation CPA factor PCF11 which directly binds to the Pol II C terminal domain and dismantles elongating Pol II from DNA in vitro. We demonstrate that PCF11 mediated termination is essential for vertebrate development. A range of genomic analyses including: mNET seq three prime mRNA seq chromatin RNA seq and ChIP seq reveals that PCF11 enhances transcription termination and stimulates early polyadenylation genome wide. PCF11 binds preferentially between closely spaced genes where it prevents transcriptional interference and downstream gene silencing. Notably PCF11 is sub stoichiometric to the CPA complex. Low levels of PCF11 are maintained by an auto regulatory mechanism involving premature termination of its own transcript and are important for normal development. Both in human cell culture and during zebrafish development PCF11 selectively attenuates the expression of other transcriptional regulators by premature CPA and termination. Overall design: three prime mRNA seq in individual zebrafish embryo heads. Two types of mutants: zPCF11 null and zPCF11 with deletion of PAS1. Wild type wt +/+ heterozygous het +/ and homozygous mutant hom / embryos were analyzed. Wild type and heterozygous animals were phenotypically indistinguishable. parent bioproject:PRJNA507553 pubmed:30819644   3 primemRNAseq zPCF11deltaPAS1 het e1 GSM3536466   tissue:single embryo head 32hpf|molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11deltaPAS1 +/ 3 primemRNAseq zPCF11deltaPAS1 het e1 3’ mRNA seq data were mapped according to the guidelines of QuantSeq library kit manufacturer Lexogen. Unaligned bam files from HiSeq2500 were converted to FASTQ files with bam2fastx TopHat 2 component Kim et al. 2013. post overview with FastQC reads were trimmed with BBtools sourceforge.net/projects/bbmap/ script bbduk using following settings: k=13 ktrim=r useshortkmers=t mink=5 qtrim=r trimq=10 minlength=20 . post trimming control with FastQC curated reads were mapped with STAR2.5b Dobin et al. 2013 aligner. STAR index was generated with the GRCz10/danRer10 was used as the reference zebrafish genome together with the corresponding ENSEMBL annotation. Aligned 3’ mRNA seq reads were filtered to remove false positives due to internal priming of the QuantSeq assay on genome encoded polyA stretches. To do this first a crude genomic mask was generated that contained all loci harbouring 6 or more consecutive A bases as well as any 10 nucleotide windows containing more than 6 A bases. For genes expressed from the reverse strand an analogous T rich mask was generated. Those crude masks were then corrected to allow for detection of genuine PAS falling in A/T rich regions by strand specifically unmasking 20 nucleotide intervals centred at annotated 3’ gene ends. 3’ mRNA seq reads falling into those refined strand specific masks were then removed and the filtered reads reduced to the most distal nucleotide 3’ end nucleotide. Genome build: GRCz10/danRer10 Supplementary files format and content: bigWig files: genome coverage files mean from replicates single embryo head 32hpf   Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq 3’mRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. Zebrafish Danio rerio were raised according to standard protocols 28°C water temperature 14/10 hr light/dark cycle. TLAB fish generated by crossing zebrafish AB and the natural variant TL Tupfel Long fin stocks served as wild type zebrafish for all experiments. zPCF11 null and zPCF11 ∆PAS1 mutant zebrafish were generated by Cas9 mediated mutagenesis Gagnon et al. 2014 molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11deltaPAS1 +/ GSM3536466 GSM3536466: 3 primemRNAseq zPCF11deltaPAS1 het e1; Danio rerio; RNA Seq GSM3536466   1 Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq three primemRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. GEO Accession:GSM3536466 RNA-Seq TRANSCRIPTOMIC cDNA SINGLE ILLUMINA Illumina HiSeq 2500   SRP175015     bam4__sel_TCAGGAATC.bam.fastq_trimmed.fastq.gz fastq 789817010.0 8990777.0 GSM3536466 r1 0:87.85 A:255721217;C:143531700;G:162243420;T:228318196;N:2477 87       255721217 143531700 162243420 228318196 2477 SRX5193222 SRS4199222 SRA828489 GEO Sir William Dunn School of Pathology, University of Oxford 1 0.85167   0.14197   0.78813   0.51648   89   B   usable mapping rate illumina hiseq_era 3prime poly_a lexogen bulk unknown unknown   United Kingdom 2019-01-02 Multi-stage Embryo Head Nervous System
50974 50974 SRR8383295 SRX5193222 SRS4199222 SRP175015 PRJNA512489 Selective roles of vertebrate PCF11 in premature and full length transcript termination zebrafish three prime mRNA seq GSE124555 Other The pervasive nature of RNA polymerase II Pol II transcription requires efficient termination. A key player in this process is the cleavage and polyadenylation CPA factor PCF11 which directly binds to the Pol II C terminal domain and dismantles elongating Pol II from DNA in vitro. We demonstrate that PCF11 mediated termination is essential for vertebrate development. A range of genomic analyses including: mNET seq three prime mRNA seq chromatin RNA seq and ChIP seq reveals that PCF11 enhances transcription termination and stimulates early polyadenylation genome wide. PCF11 binds preferentially between closely spaced genes where it prevents transcriptional interference and downstream gene silencing. Notably PCF11 is sub stoichiometric to the CPA complex. Low levels of PCF11 are maintained by an auto regulatory mechanism involving premature termination of its own transcript and are important for normal development. Both in human cell culture and during zebrafish development PCF11 selectively attenuates the expression of other transcriptional regulators by premature CPA and termination. Overall design: three prime mRNA seq in individual zebrafish embryo heads. Two types of mutants: zPCF11 null and zPCF11 with deletion of PAS1. Wild type wt +/+ heterozygous het +/ and homozygous mutant hom / embryos were analyzed. Wild type and heterozygous animals were phenotypically indistinguishable. parent bioproject:PRJNA507553 pubmed:30819644   3 primemRNAseq zPCF11deltaPAS1 het e1 GSM3536466   tissue:single embryo head 32hpf|molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11deltaPAS1 +/ 3 primemRNAseq zPCF11deltaPAS1 het e1 3’ mRNA seq data were mapped according to the guidelines of QuantSeq library kit manufacturer Lexogen. Unaligned bam files from HiSeq2500 were converted to FASTQ files with bam2fastx TopHat 2 component Kim et al. 2013. post overview with FastQC reads were trimmed with BBtools sourceforge.net/projects/bbmap/ script bbduk using following settings: k=13 ktrim=r useshortkmers=t mink=5 qtrim=r trimq=10 minlength=20 . post trimming control with FastQC curated reads were mapped with STAR2.5b Dobin et al. 2013 aligner. STAR index was generated with the GRCz10/danRer10 was used as the reference zebrafish genome together with the corresponding ENSEMBL annotation. Aligned 3’ mRNA seq reads were filtered to remove false positives due to internal priming of the QuantSeq assay on genome encoded polyA stretches. To do this first a crude genomic mask was generated that contained all loci harbouring 6 or more consecutive A bases as well as any 10 nucleotide windows containing more than 6 A bases. For genes expressed from the reverse strand an analogous T rich mask was generated. Those crude masks were then corrected to allow for detection of genuine PAS falling in A/T rich regions by strand specifically unmasking 20 nucleotide intervals centred at annotated 3’ gene ends. 3’ mRNA seq reads falling into those refined strand specific masks were then removed and the filtered reads reduced to the most distal nucleotide 3’ end nucleotide. Genome build: GRCz10/danRer10 Supplementary files format and content: bigWig files: genome coverage files mean from replicates single embryo head 32hpf   Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq 3’mRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. Zebrafish Danio rerio were raised according to standard protocols 28°C water temperature 14/10 hr light/dark cycle. TLAB fish generated by crossing zebrafish AB and the natural variant TL Tupfel Long fin stocks served as wild type zebrafish for all experiments. zPCF11 null and zPCF11 ∆PAS1 mutant zebrafish were generated by Cas9 mediated mutagenesis Gagnon et al. 2014 molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11deltaPAS1 +/ GSM3536466 GSM3536466: 3 primemRNAseq zPCF11deltaPAS1 het e1; Danio rerio; RNA Seq GSM3536466   1 Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq three primemRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. GEO Accession:GSM3536466 RNA-Seq TRANSCRIPTOMIC cDNA SINGLE ILLUMINA Illumina HiSeq 2500   SRP175015     bam5__sel_TCAGGAATC.bam.fastq_trimmed.fastq.gz fastq 812333266.0 9249708.0 GSM3536466 r2 0:87.82 A:263342967;C:147474874;G:166670966;T:234842610;N:1849 87       263342967 147474874 166670966 234842610 1849 SRX5193222 SRS4199222 SRA828489 GEO Sir William Dunn School of Pathology, University of Oxford 1 0.85028   0.14374   0.78754   0.51112   89   B   usable mapping rate illumina hiseq_era 3prime poly_a lexogen bulk unknown unknown   United Kingdom 2019-01-02 Multi-stage Embryo Head Nervous System
50975 50975 SRR8383292 SRX5193221 SRS4199221 SRP175015 PRJNA512489 Selective roles of vertebrate PCF11 in premature and full length transcript termination zebrafish three prime mRNA seq GSE124555 Other The pervasive nature of RNA polymerase II Pol II transcription requires efficient termination. A key player in this process is the cleavage and polyadenylation CPA factor PCF11 which directly binds to the Pol II C terminal domain and dismantles elongating Pol II from DNA in vitro. We demonstrate that PCF11 mediated termination is essential for vertebrate development. A range of genomic analyses including: mNET seq three prime mRNA seq chromatin RNA seq and ChIP seq reveals that PCF11 enhances transcription termination and stimulates early polyadenylation genome wide. PCF11 binds preferentially between closely spaced genes where it prevents transcriptional interference and downstream gene silencing. Notably PCF11 is sub stoichiometric to the CPA complex. Low levels of PCF11 are maintained by an auto regulatory mechanism involving premature termination of its own transcript and are important for normal development. Both in human cell culture and during zebrafish development PCF11 selectively attenuates the expression of other transcriptional regulators by premature CPA and termination. Overall design: three prime mRNA seq in individual zebrafish embryo heads. Two types of mutants: zPCF11 null and zPCF11 with deletion of PAS1. Wild type wt +/+ heterozygous het +/ and homozygous mutant hom / embryos were analyzed. Wild type and heterozygous animals were phenotypically indistinguishable. parent bioproject:PRJNA507553 pubmed:30819644   3 primemRNAseq zPCF11deltaPAS1 wt e3 GSM3536465   tissue:single embryo head 32hpf|molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11deltaPAS1 +/+ 3 primemRNAseq zPCF11deltaPAS1 wt e3 3’ mRNA seq data were mapped according to the guidelines of QuantSeq library kit manufacturer Lexogen. Unaligned bam files from HiSeq2500 were converted to FASTQ files with bam2fastx TopHat 2 component Kim et al. 2013. post overview with FastQC reads were trimmed with BBtools sourceforge.net/projects/bbmap/ script bbduk using following settings: k=13 ktrim=r useshortkmers=t mink=5 qtrim=r trimq=10 minlength=20 . post trimming control with FastQC curated reads were mapped with STAR2.5b Dobin et al. 2013 aligner. STAR index was generated with the GRCz10/danRer10 was used as the reference zebrafish genome together with the corresponding ENSEMBL annotation. Aligned 3’ mRNA seq reads were filtered to remove false positives due to internal priming of the QuantSeq assay on genome encoded polyA stretches. To do this first a crude genomic mask was generated that contained all loci harbouring 6 or more consecutive A bases as well as any 10 nucleotide windows containing more than 6 A bases. For genes expressed from the reverse strand an analogous T rich mask was generated. Those crude masks were then corrected to allow for detection of genuine PAS falling in A/T rich regions by strand specifically unmasking 20 nucleotide intervals centred at annotated 3’ gene ends. 3’ mRNA seq reads falling into those refined strand specific masks were then removed and the filtered reads reduced to the most distal nucleotide 3’ end nucleotide. Genome build: GRCz10/danRer10 Supplementary files format and content: bigWig files: genome coverage files mean from replicates single embryo head 32hpf   Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq 3’mRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. Zebrafish Danio rerio were raised according to standard protocols 28°C water temperature 14/10 hr light/dark cycle. TLAB fish generated by crossing zebrafish AB and the natural variant TL Tupfel Long fin stocks served as wild type zebrafish for all experiments. zPCF11 null and zPCF11 ∆PAS1 mutant zebrafish were generated by Cas9 mediated mutagenesis Gagnon et al. 2014 molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11deltaPAS1 +/+ GSM3536465 GSM3536465: 3 primemRNAseq zPCF11deltaPAS1 wt e3; Danio rerio; RNA Seq GSM3536465   1 Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq three primemRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. GEO Accession:GSM3536465 RNA-Seq TRANSCRIPTOMIC cDNA SINGLE ILLUMINA Illumina HiSeq 2500   SRP175015     bam4__sel_GGCTGCATC.bam.fastq_trimmed.fastq.gz fastq 164145508.0 1870416.0 GSM3536465 r1 0:87.76 A:53096508;C:29983980;G:33483503;T:47581048;N:469 87       53096508 29983980 33483503 47581048 469 SRX5193221 SRS4199221 SRA828489 GEO Sir William Dunn School of Pathology, University of Oxford 1 0.85271   0.13713   0.78626   0.42953   89   B   usable mapping rate illumina hiseq_era 3prime poly_a lexogen bulk unknown unknown   United Kingdom 2019-01-02 Multi-stage Embryo Head Nervous System
50976 50976 SRR8383293 SRX5193221 SRS4199221 SRP175015 PRJNA512489 Selective roles of vertebrate PCF11 in premature and full length transcript termination zebrafish three prime mRNA seq GSE124555 Other The pervasive nature of RNA polymerase II Pol II transcription requires efficient termination. A key player in this process is the cleavage and polyadenylation CPA factor PCF11 which directly binds to the Pol II C terminal domain and dismantles elongating Pol II from DNA in vitro. We demonstrate that PCF11 mediated termination is essential for vertebrate development. A range of genomic analyses including: mNET seq three prime mRNA seq chromatin RNA seq and ChIP seq reveals that PCF11 enhances transcription termination and stimulates early polyadenylation genome wide. PCF11 binds preferentially between closely spaced genes where it prevents transcriptional interference and downstream gene silencing. Notably PCF11 is sub stoichiometric to the CPA complex. Low levels of PCF11 are maintained by an auto regulatory mechanism involving premature termination of its own transcript and are important for normal development. Both in human cell culture and during zebrafish development PCF11 selectively attenuates the expression of other transcriptional regulators by premature CPA and termination. Overall design: three prime mRNA seq in individual zebrafish embryo heads. Two types of mutants: zPCF11 null and zPCF11 with deletion of PAS1. Wild type wt +/+ heterozygous het +/ and homozygous mutant hom / embryos were analyzed. Wild type and heterozygous animals were phenotypically indistinguishable. parent bioproject:PRJNA507553 pubmed:30819644   3 primemRNAseq zPCF11deltaPAS1 wt e3 GSM3536465   tissue:single embryo head 32hpf|molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11deltaPAS1 +/+ 3 primemRNAseq zPCF11deltaPAS1 wt e3 3’ mRNA seq data were mapped according to the guidelines of QuantSeq library kit manufacturer Lexogen. Unaligned bam files from HiSeq2500 were converted to FASTQ files with bam2fastx TopHat 2 component Kim et al. 2013. post overview with FastQC reads were trimmed with BBtools sourceforge.net/projects/bbmap/ script bbduk using following settings: k=13 ktrim=r useshortkmers=t mink=5 qtrim=r trimq=10 minlength=20 . post trimming control with FastQC curated reads were mapped with STAR2.5b Dobin et al. 2013 aligner. STAR index was generated with the GRCz10/danRer10 was used as the reference zebrafish genome together with the corresponding ENSEMBL annotation. Aligned 3’ mRNA seq reads were filtered to remove false positives due to internal priming of the QuantSeq assay on genome encoded polyA stretches. To do this first a crude genomic mask was generated that contained all loci harbouring 6 or more consecutive A bases as well as any 10 nucleotide windows containing more than 6 A bases. For genes expressed from the reverse strand an analogous T rich mask was generated. Those crude masks were then corrected to allow for detection of genuine PAS falling in A/T rich regions by strand specifically unmasking 20 nucleotide intervals centred at annotated 3’ gene ends. 3’ mRNA seq reads falling into those refined strand specific masks were then removed and the filtered reads reduced to the most distal nucleotide 3’ end nucleotide. Genome build: GRCz10/danRer10 Supplementary files format and content: bigWig files: genome coverage files mean from replicates single embryo head 32hpf   Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq 3’mRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. Zebrafish Danio rerio were raised according to standard protocols 28°C water temperature 14/10 hr light/dark cycle. TLAB fish generated by crossing zebrafish AB and the natural variant TL Tupfel Long fin stocks served as wild type zebrafish for all experiments. zPCF11 null and zPCF11 ∆PAS1 mutant zebrafish were generated by Cas9 mediated mutagenesis Gagnon et al. 2014 molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11deltaPAS1 +/+ GSM3536465 GSM3536465: 3 primemRNAseq zPCF11deltaPAS1 wt e3; Danio rerio; RNA Seq GSM3536465   1 Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq three primemRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. GEO Accession:GSM3536465 RNA-Seq TRANSCRIPTOMIC cDNA SINGLE ILLUMINA Illumina HiSeq 2500   SRP175015     bam5__sel_GGCTGCATC.bam.fastq_trimmed.fastq.gz fastq 168774555.0 1923679.0 GSM3536465 r2 0:87.74 A:54636127;C:30795817;G:34408599;T:48933579;N:433 87       54636127 30795817 34408599 48933579 433 SRX5193221 SRS4199221 SRA828489 GEO Sir William Dunn School of Pathology, University of Oxford 1 0.85284   0.136   0.78595   0.51852   89   B   usable mapping rate illumina hiseq_era 3prime poly_a lexogen bulk unknown unknown   United Kingdom 2019-01-02 Multi-stage Embryo Head Nervous System
50977 50977 SRR8383290 SRX5193220 SRS4199220 SRP175015 PRJNA512489 Selective roles of vertebrate PCF11 in premature and full length transcript termination zebrafish three prime mRNA seq GSE124555 Other The pervasive nature of RNA polymerase II Pol II transcription requires efficient termination. A key player in this process is the cleavage and polyadenylation CPA factor PCF11 which directly binds to the Pol II C terminal domain and dismantles elongating Pol II from DNA in vitro. We demonstrate that PCF11 mediated termination is essential for vertebrate development. A range of genomic analyses including: mNET seq three prime mRNA seq chromatin RNA seq and ChIP seq reveals that PCF11 enhances transcription termination and stimulates early polyadenylation genome wide. PCF11 binds preferentially between closely spaced genes where it prevents transcriptional interference and downstream gene silencing. Notably PCF11 is sub stoichiometric to the CPA complex. Low levels of PCF11 are maintained by an auto regulatory mechanism involving premature termination of its own transcript and are important for normal development. Both in human cell culture and during zebrafish development PCF11 selectively attenuates the expression of other transcriptional regulators by premature CPA and termination. Overall design: three prime mRNA seq in individual zebrafish embryo heads. Two types of mutants: zPCF11 null and zPCF11 with deletion of PAS1. Wild type wt +/+ heterozygous het +/ and homozygous mutant hom / embryos were analyzed. Wild type and heterozygous animals were phenotypically indistinguishable. parent bioproject:PRJNA507553 pubmed:30819644   3 primemRNAseq zPCF11deltaPAS1 wt e2 GSM3536464   tissue:single embryo head 32hpf|molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11deltaPAS1 +/+ 3 primemRNAseq zPCF11deltaPAS1 wt e2 3’ mRNA seq data were mapped according to the guidelines of QuantSeq library kit manufacturer Lexogen. Unaligned bam files from HiSeq2500 were converted to FASTQ files with bam2fastx TopHat 2 component Kim et al. 2013. post overview with FastQC reads were trimmed with BBtools sourceforge.net/projects/bbmap/ script bbduk using following settings: k=13 ktrim=r useshortkmers=t mink=5 qtrim=r trimq=10 minlength=20 . post trimming control with FastQC curated reads were mapped with STAR2.5b Dobin et al. 2013 aligner. STAR index was generated with the GRCz10/danRer10 was used as the reference zebrafish genome together with the corresponding ENSEMBL annotation. Aligned 3’ mRNA seq reads were filtered to remove false positives due to internal priming of the QuantSeq assay on genome encoded polyA stretches. To do this first a crude genomic mask was generated that contained all loci harbouring 6 or more consecutive A bases as well as any 10 nucleotide windows containing more than 6 A bases. For genes expressed from the reverse strand an analogous T rich mask was generated. Those crude masks were then corrected to allow for detection of genuine PAS falling in A/T rich regions by strand specifically unmasking 20 nucleotide intervals centred at annotated 3’ gene ends. 3’ mRNA seq reads falling into those refined strand specific masks were then removed and the filtered reads reduced to the most distal nucleotide 3’ end nucleotide. Genome build: GRCz10/danRer10 Supplementary files format and content: bigWig files: genome coverage files mean from replicates single embryo head 32hpf   Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq 3’mRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. Zebrafish Danio rerio were raised according to standard protocols 28°C water temperature 14/10 hr light/dark cycle. TLAB fish generated by crossing zebrafish AB and the natural variant TL Tupfel Long fin stocks served as wild type zebrafish for all experiments. zPCF11 null and zPCF11 ∆PAS1 mutant zebrafish were generated by Cas9 mediated mutagenesis Gagnon et al. 2014 molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11deltaPAS1 +/+ GSM3536464 GSM3536464: 3 primemRNAseq zPCF11deltaPAS1 wt e2; Danio rerio; RNA Seq GSM3536464   1 Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq three primemRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. GEO Accession:GSM3536464 RNA-Seq TRANSCRIPTOMIC cDNA SINGLE ILLUMINA Illumina HiSeq 2500   SRP175015     bam4__sel_AATCCGATC.bam.fastq_trimmed.fastq.gz fastq 237761677.0 2716138.0 GSM3536464 r1 0:87.54 A:77086804;C:43323233;G:48823094;T:68527734;N:812 87       77086804 43323233 48823094 68527734 812 SRX5193220 SRS4199220 SRA828489 GEO Sir William Dunn School of Pathology, University of Oxford 1 0.84891   0.16038   0.78839   0.53051   89   B   usable mapping rate illumina hiseq_era 3prime poly_a lexogen bulk unknown unknown   United Kingdom 2019-01-02 Multi-stage Embryo Head Nervous System
50978 50978 SRR8383291 SRX5193220 SRS4199220 SRP175015 PRJNA512489 Selective roles of vertebrate PCF11 in premature and full length transcript termination zebrafish three prime mRNA seq GSE124555 Other The pervasive nature of RNA polymerase II Pol II transcription requires efficient termination. A key player in this process is the cleavage and polyadenylation CPA factor PCF11 which directly binds to the Pol II C terminal domain and dismantles elongating Pol II from DNA in vitro. We demonstrate that PCF11 mediated termination is essential for vertebrate development. A range of genomic analyses including: mNET seq three prime mRNA seq chromatin RNA seq and ChIP seq reveals that PCF11 enhances transcription termination and stimulates early polyadenylation genome wide. PCF11 binds preferentially between closely spaced genes where it prevents transcriptional interference and downstream gene silencing. Notably PCF11 is sub stoichiometric to the CPA complex. Low levels of PCF11 are maintained by an auto regulatory mechanism involving premature termination of its own transcript and are important for normal development. Both in human cell culture and during zebrafish development PCF11 selectively attenuates the expression of other transcriptional regulators by premature CPA and termination. Overall design: three prime mRNA seq in individual zebrafish embryo heads. Two types of mutants: zPCF11 null and zPCF11 with deletion of PAS1. Wild type wt +/+ heterozygous het +/ and homozygous mutant hom / embryos were analyzed. Wild type and heterozygous animals were phenotypically indistinguishable. parent bioproject:PRJNA507553 pubmed:30819644   3 primemRNAseq zPCF11deltaPAS1 wt e2 GSM3536464   tissue:single embryo head 32hpf|molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11deltaPAS1 +/+ 3 primemRNAseq zPCF11deltaPAS1 wt e2 3’ mRNA seq data were mapped according to the guidelines of QuantSeq library kit manufacturer Lexogen. Unaligned bam files from HiSeq2500 were converted to FASTQ files with bam2fastx TopHat 2 component Kim et al. 2013. post overview with FastQC reads were trimmed with BBtools sourceforge.net/projects/bbmap/ script bbduk using following settings: k=13 ktrim=r useshortkmers=t mink=5 qtrim=r trimq=10 minlength=20 . post trimming control with FastQC curated reads were mapped with STAR2.5b Dobin et al. 2013 aligner. STAR index was generated with the GRCz10/danRer10 was used as the reference zebrafish genome together with the corresponding ENSEMBL annotation. Aligned 3’ mRNA seq reads were filtered to remove false positives due to internal priming of the QuantSeq assay on genome encoded polyA stretches. To do this first a crude genomic mask was generated that contained all loci harbouring 6 or more consecutive A bases as well as any 10 nucleotide windows containing more than 6 A bases. For genes expressed from the reverse strand an analogous T rich mask was generated. Those crude masks were then corrected to allow for detection of genuine PAS falling in A/T rich regions by strand specifically unmasking 20 nucleotide intervals centred at annotated 3’ gene ends. 3’ mRNA seq reads falling into those refined strand specific masks were then removed and the filtered reads reduced to the most distal nucleotide 3’ end nucleotide. Genome build: GRCz10/danRer10 Supplementary files format and content: bigWig files: genome coverage files mean from replicates single embryo head 32hpf   Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq 3’mRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. Zebrafish Danio rerio were raised according to standard protocols 28°C water temperature 14/10 hr light/dark cycle. TLAB fish generated by crossing zebrafish AB and the natural variant TL Tupfel Long fin stocks served as wild type zebrafish for all experiments. zPCF11 null and zPCF11 ∆PAS1 mutant zebrafish were generated by Cas9 mediated mutagenesis Gagnon et al. 2014 molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11deltaPAS1 +/+ GSM3536464 GSM3536464: 3 primemRNAseq zPCF11deltaPAS1 wt e2; Danio rerio; RNA Seq GSM3536464   1 Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq three primemRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. GEO Accession:GSM3536464 RNA-Seq TRANSCRIPTOMIC cDNA SINGLE ILLUMINA Illumina HiSeq 2500   SRP175015     bam5__sel_AATCCGATC.bam.fastq_trimmed.fastq.gz fastq 244669202.0 2795847.0 GSM3536464 r2 0:87.51 A:79391766;C:44532003;G:50184304;T:70560584;N:545 87       79391766 44532003 50184304 70560584 545 SRX5193220 SRS4199220 SRA828489 GEO Sir William Dunn School of Pathology, University of Oxford 1 0.84722   0.16156   0.79011   0.53158   89   B   usable mapping rate illumina hiseq_era 3prime poly_a lexogen bulk unknown unknown   United Kingdom 2019-01-02 Multi-stage Embryo Head Nervous System
50979 50979 SRR8383288 SRX5193219 SRS4199219 SRP175015 PRJNA512489 Selective roles of vertebrate PCF11 in premature and full length transcript termination zebrafish three prime mRNA seq GSE124555 Other The pervasive nature of RNA polymerase II Pol II transcription requires efficient termination. A key player in this process is the cleavage and polyadenylation CPA factor PCF11 which directly binds to the Pol II C terminal domain and dismantles elongating Pol II from DNA in vitro. We demonstrate that PCF11 mediated termination is essential for vertebrate development. A range of genomic analyses including: mNET seq three prime mRNA seq chromatin RNA seq and ChIP seq reveals that PCF11 enhances transcription termination and stimulates early polyadenylation genome wide. PCF11 binds preferentially between closely spaced genes where it prevents transcriptional interference and downstream gene silencing. Notably PCF11 is sub stoichiometric to the CPA complex. Low levels of PCF11 are maintained by an auto regulatory mechanism involving premature termination of its own transcript and are important for normal development. Both in human cell culture and during zebrafish development PCF11 selectively attenuates the expression of other transcriptional regulators by premature CPA and termination. Overall design: three prime mRNA seq in individual zebrafish embryo heads. Two types of mutants: zPCF11 null and zPCF11 with deletion of PAS1. Wild type wt +/+ heterozygous het +/ and homozygous mutant hom / embryos were analyzed. Wild type and heterozygous animals were phenotypically indistinguishable. parent bioproject:PRJNA507553 pubmed:30819644   3 primemRNAseq zPCF11deltaPAS1 wt e1 GSM3536463   tissue:single embryo head 32hpf|molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11deltaPAS1 +/+ 3 primemRNAseq zPCF11deltaPAS1 wt e1 3’ mRNA seq data were mapped according to the guidelines of QuantSeq library kit manufacturer Lexogen. Unaligned bam files from HiSeq2500 were converted to FASTQ files with bam2fastx TopHat 2 component Kim et al. 2013. post overview with FastQC reads were trimmed with BBtools sourceforge.net/projects/bbmap/ script bbduk using following settings: k=13 ktrim=r useshortkmers=t mink=5 qtrim=r trimq=10 minlength=20 . post trimming control with FastQC curated reads were mapped with STAR2.5b Dobin et al. 2013 aligner. STAR index was generated with the GRCz10/danRer10 was used as the reference zebrafish genome together with the corresponding ENSEMBL annotation. Aligned 3’ mRNA seq reads were filtered to remove false positives due to internal priming of the QuantSeq assay on genome encoded polyA stretches. To do this first a crude genomic mask was generated that contained all loci harbouring 6 or more consecutive A bases as well as any 10 nucleotide windows containing more than 6 A bases. For genes expressed from the reverse strand an analogous T rich mask was generated. Those crude masks were then corrected to allow for detection of genuine PAS falling in A/T rich regions by strand specifically unmasking 20 nucleotide intervals centred at annotated 3’ gene ends. 3’ mRNA seq reads falling into those refined strand specific masks were then removed and the filtered reads reduced to the most distal nucleotide 3’ end nucleotide. Genome build: GRCz10/danRer10 Supplementary files format and content: bigWig files: genome coverage files mean from replicates single embryo head 32hpf   Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq 3’mRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. Zebrafish Danio rerio were raised according to standard protocols 28°C water temperature 14/10 hr light/dark cycle. TLAB fish generated by crossing zebrafish AB and the natural variant TL Tupfel Long fin stocks served as wild type zebrafish for all experiments. zPCF11 null and zPCF11 ∆PAS1 mutant zebrafish were generated by Cas9 mediated mutagenesis Gagnon et al. 2014 molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11deltaPAS1 +/+ GSM3536463 GSM3536463: 3 primemRNAseq zPCF11deltaPAS1 wt e1; Danio rerio; RNA Seq GSM3536463   1 Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq three primemRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. GEO Accession:GSM3536463 RNA-Seq TRANSCRIPTOMIC cDNA SINGLE ILLUMINA Illumina HiSeq 2500   SRP175015     bam4__sel_CCTAAGATC.bam.fastq_trimmed.fastq.gz fastq 129332543.0 1472338.0 GSM3536463 r1 0:87.84 A:43564845;C:22890586;G:25561204;T:37315459;N:449 87       43564845 22890586 25561204 37315459 449 SRX5193219 SRS4199219 SRA828489 GEO Sir William Dunn School of Pathology, University of Oxford 1 0.83187   0.13547   0.78234   0.52271   89   B   usable mapping rate illumina hiseq_era 3prime poly_a lexogen bulk unknown unknown   United Kingdom 2019-01-02 Multi-stage Embryo Head Nervous System
50980 50980 SRR8383289 SRX5193219 SRS4199219 SRP175015 PRJNA512489 Selective roles of vertebrate PCF11 in premature and full length transcript termination zebrafish three prime mRNA seq GSE124555 Other The pervasive nature of RNA polymerase II Pol II transcription requires efficient termination. A key player in this process is the cleavage and polyadenylation CPA factor PCF11 which directly binds to the Pol II C terminal domain and dismantles elongating Pol II from DNA in vitro. We demonstrate that PCF11 mediated termination is essential for vertebrate development. A range of genomic analyses including: mNET seq three prime mRNA seq chromatin RNA seq and ChIP seq reveals that PCF11 enhances transcription termination and stimulates early polyadenylation genome wide. PCF11 binds preferentially between closely spaced genes where it prevents transcriptional interference and downstream gene silencing. Notably PCF11 is sub stoichiometric to the CPA complex. Low levels of PCF11 are maintained by an auto regulatory mechanism involving premature termination of its own transcript and are important for normal development. Both in human cell culture and during zebrafish development PCF11 selectively attenuates the expression of other transcriptional regulators by premature CPA and termination. Overall design: three prime mRNA seq in individual zebrafish embryo heads. Two types of mutants: zPCF11 null and zPCF11 with deletion of PAS1. Wild type wt +/+ heterozygous het +/ and homozygous mutant hom / embryos were analyzed. Wild type and heterozygous animals were phenotypically indistinguishable. parent bioproject:PRJNA507553 pubmed:30819644   3 primemRNAseq zPCF11deltaPAS1 wt e1 GSM3536463   tissue:single embryo head 32hpf|molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11deltaPAS1 +/+ 3 primemRNAseq zPCF11deltaPAS1 wt e1 3’ mRNA seq data were mapped according to the guidelines of QuantSeq library kit manufacturer Lexogen. Unaligned bam files from HiSeq2500 were converted to FASTQ files with bam2fastx TopHat 2 component Kim et al. 2013. post overview with FastQC reads were trimmed with BBtools sourceforge.net/projects/bbmap/ script bbduk using following settings: k=13 ktrim=r useshortkmers=t mink=5 qtrim=r trimq=10 minlength=20 . post trimming control with FastQC curated reads were mapped with STAR2.5b Dobin et al. 2013 aligner. STAR index was generated with the GRCz10/danRer10 was used as the reference zebrafish genome together with the corresponding ENSEMBL annotation. Aligned 3’ mRNA seq reads were filtered to remove false positives due to internal priming of the QuantSeq assay on genome encoded polyA stretches. To do this first a crude genomic mask was generated that contained all loci harbouring 6 or more consecutive A bases as well as any 10 nucleotide windows containing more than 6 A bases. For genes expressed from the reverse strand an analogous T rich mask was generated. Those crude masks were then corrected to allow for detection of genuine PAS falling in A/T rich regions by strand specifically unmasking 20 nucleotide intervals centred at annotated 3’ gene ends. 3’ mRNA seq reads falling into those refined strand specific masks were then removed and the filtered reads reduced to the most distal nucleotide 3’ end nucleotide. Genome build: GRCz10/danRer10 Supplementary files format and content: bigWig files: genome coverage files mean from replicates single embryo head 32hpf   Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq 3’mRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. Zebrafish Danio rerio were raised according to standard protocols 28°C water temperature 14/10 hr light/dark cycle. TLAB fish generated by crossing zebrafish AB and the natural variant TL Tupfel Long fin stocks served as wild type zebrafish for all experiments. zPCF11 null and zPCF11 ∆PAS1 mutant zebrafish were generated by Cas9 mediated mutagenesis Gagnon et al. 2014 molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11deltaPAS1 +/+ GSM3536463 GSM3536463: 3 primemRNAseq zPCF11deltaPAS1 wt e1; Danio rerio; RNA Seq GSM3536463   1 Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq three primemRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. GEO Accession:GSM3536463 RNA-Seq TRANSCRIPTOMIC cDNA SINGLE ILLUMINA Illumina HiSeq 2500   SRP175015     bam5__sel_CCTAAGATC.bam.fastq_trimmed.fastq.gz fastq 133356093.0 1518522.0 GSM3536463 r2 0:87.82 A:44962657;C:23585729;G:26335793;T:38471613;N:301 87       44962657 23585729 26335793 38471613 301 SRX5193219 SRS4199219 SRA828489 GEO Sir William Dunn School of Pathology, University of Oxford 1 0.83065   0.13598   0.78378   0.53247   89   B   usable mapping rate illumina hiseq_era 3prime poly_a lexogen bulk unknown unknown   United Kingdom 2019-01-02 Multi-stage Embryo Head Nervous System
50981 50981 SRR8383286 SRX5193218 SRS4199218 SRP175015 PRJNA512489 Selective roles of vertebrate PCF11 in premature and full length transcript termination zebrafish three prime mRNA seq GSE124555 Other The pervasive nature of RNA polymerase II Pol II transcription requires efficient termination. A key player in this process is the cleavage and polyadenylation CPA factor PCF11 which directly binds to the Pol II C terminal domain and dismantles elongating Pol II from DNA in vitro. We demonstrate that PCF11 mediated termination is essential for vertebrate development. A range of genomic analyses including: mNET seq three prime mRNA seq chromatin RNA seq and ChIP seq reveals that PCF11 enhances transcription termination and stimulates early polyadenylation genome wide. PCF11 binds preferentially between closely spaced genes where it prevents transcriptional interference and downstream gene silencing. Notably PCF11 is sub stoichiometric to the CPA complex. Low levels of PCF11 are maintained by an auto regulatory mechanism involving premature termination of its own transcript and are important for normal development. Both in human cell culture and during zebrafish development PCF11 selectively attenuates the expression of other transcriptional regulators by premature CPA and termination. Overall design: three prime mRNA seq in individual zebrafish embryo heads. Two types of mutants: zPCF11 null and zPCF11 with deletion of PAS1. Wild type wt +/+ heterozygous het +/ and homozygous mutant hom / embryos were analyzed. Wild type and heterozygous animals were phenotypically indistinguishable. parent bioproject:PRJNA507553 pubmed:30819644   3 primemRNAseq zPCF11null hom e6 GSM3536462   tissue:single embryo head 19hpf|molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11null / 3 primemRNAseq zPCF11null hom e6 3’ mRNA seq data were mapped according to the guidelines of QuantSeq library kit manufacturer Lexogen. Unaligned bam files from HiSeq2500 were converted to FASTQ files with bam2fastx TopHat 2 component Kim et al. 2013. post overview with FastQC reads were trimmed with BBtools sourceforge.net/projects/bbmap/ script bbduk using following settings: k=13 ktrim=r useshortkmers=t mink=5 qtrim=r trimq=10 minlength=20 . post trimming control with FastQC curated reads were mapped with STAR2.5b Dobin et al. 2013 aligner. STAR index was generated with the GRCz10/danRer10 was used as the reference zebrafish genome together with the corresponding ENSEMBL annotation. Aligned 3’ mRNA seq reads were filtered to remove false positives due to internal priming of the QuantSeq assay on genome encoded polyA stretches. To do this first a crude genomic mask was generated that contained all loci harbouring 6 or more consecutive A bases as well as any 10 nucleotide windows containing more than 6 A bases. For genes expressed from the reverse strand an analogous T rich mask was generated. Those crude masks were then corrected to allow for detection of genuine PAS falling in A/T rich regions by strand specifically unmasking 20 nucleotide intervals centred at annotated 3’ gene ends. 3’ mRNA seq reads falling into those refined strand specific masks were then removed and the filtered reads reduced to the most distal nucleotide 3’ end nucleotide. Genome build: GRCz10/danRer10 Supplementary files format and content: bigWig files: genome coverage files mean from replicates single embryo head 19hpf   Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq 3’mRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. Zebrafish Danio rerio were raised according to standard protocols 28°C water temperature 14/10 hr light/dark cycle. TLAB fish generated by crossing zebrafish AB and the natural variant TL Tupfel Long fin stocks served as wild type zebrafish for all experiments. zPCF11 null and zPCF11 ∆PAS1 mutant zebrafish were generated by Cas9 mediated mutagenesis Gagnon et al. 2014 molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11null / GSM3536462 GSM3536462: 3 primemRNAseq zPCF11null hom e6; Danio rerio; RNA Seq GSM3536462   1 Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq three primemRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. GEO Accession:GSM3536462 RNA-Seq TRANSCRIPTOMIC cDNA SINGLE ILLUMINA Illumina HiSeq 2500   SRP175015     bam4__sel_TCGAGGATC.bam.fastq_trimmed.fastq.gz fastq 685570157.0 7880276.0 GSM3536462 r1 0:87.00 A:220749091;C:129576519;G:144120777;T:191121622;N:2148 87       220749091 129576519 144120777 191121622 2148 SRX5193218 SRS4199218 SRA828489 GEO Sir William Dunn School of Pathology, University of Oxford 1 0.82107   0.23078   0.81941   0.59068   89   B   usable mapping rate illumina hiseq_era 3prime poly_a lexogen bulk unknown unknown   United Kingdom 2019-01-02 Multi-stage Embryo Head Nervous System
50982 50982 SRR8383287 SRX5193218 SRS4199218 SRP175015 PRJNA512489 Selective roles of vertebrate PCF11 in premature and full length transcript termination zebrafish three prime mRNA seq GSE124555 Other The pervasive nature of RNA polymerase II Pol II transcription requires efficient termination. A key player in this process is the cleavage and polyadenylation CPA factor PCF11 which directly binds to the Pol II C terminal domain and dismantles elongating Pol II from DNA in vitro. We demonstrate that PCF11 mediated termination is essential for vertebrate development. A range of genomic analyses including: mNET seq three prime mRNA seq chromatin RNA seq and ChIP seq reveals that PCF11 enhances transcription termination and stimulates early polyadenylation genome wide. PCF11 binds preferentially between closely spaced genes where it prevents transcriptional interference and downstream gene silencing. Notably PCF11 is sub stoichiometric to the CPA complex. Low levels of PCF11 are maintained by an auto regulatory mechanism involving premature termination of its own transcript and are important for normal development. Both in human cell culture and during zebrafish development PCF11 selectively attenuates the expression of other transcriptional regulators by premature CPA and termination. Overall design: three prime mRNA seq in individual zebrafish embryo heads. Two types of mutants: zPCF11 null and zPCF11 with deletion of PAS1. Wild type wt +/+ heterozygous het +/ and homozygous mutant hom / embryos were analyzed. Wild type and heterozygous animals were phenotypically indistinguishable. parent bioproject:PRJNA507553 pubmed:30819644   3 primemRNAseq zPCF11null hom e6 GSM3536462   tissue:single embryo head 19hpf|molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11null / 3 primemRNAseq zPCF11null hom e6 3’ mRNA seq data were mapped according to the guidelines of QuantSeq library kit manufacturer Lexogen. Unaligned bam files from HiSeq2500 were converted to FASTQ files with bam2fastx TopHat 2 component Kim et al. 2013. post overview with FastQC reads were trimmed with BBtools sourceforge.net/projects/bbmap/ script bbduk using following settings: k=13 ktrim=r useshortkmers=t mink=5 qtrim=r trimq=10 minlength=20 . post trimming control with FastQC curated reads were mapped with STAR2.5b Dobin et al. 2013 aligner. STAR index was generated with the GRCz10/danRer10 was used as the reference zebrafish genome together with the corresponding ENSEMBL annotation. Aligned 3’ mRNA seq reads were filtered to remove false positives due to internal priming of the QuantSeq assay on genome encoded polyA stretches. To do this first a crude genomic mask was generated that contained all loci harbouring 6 or more consecutive A bases as well as any 10 nucleotide windows containing more than 6 A bases. For genes expressed from the reverse strand an analogous T rich mask was generated. Those crude masks were then corrected to allow for detection of genuine PAS falling in A/T rich regions by strand specifically unmasking 20 nucleotide intervals centred at annotated 3’ gene ends. 3’ mRNA seq reads falling into those refined strand specific masks were then removed and the filtered reads reduced to the most distal nucleotide 3’ end nucleotide. Genome build: GRCz10/danRer10 Supplementary files format and content: bigWig files: genome coverage files mean from replicates single embryo head 19hpf   Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq 3’mRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. Zebrafish Danio rerio were raised according to standard protocols 28°C water temperature 14/10 hr light/dark cycle. TLAB fish generated by crossing zebrafish AB and the natural variant TL Tupfel Long fin stocks served as wild type zebrafish for all experiments. zPCF11 null and zPCF11 ∆PAS1 mutant zebrafish were generated by Cas9 mediated mutagenesis Gagnon et al. 2014 molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11null / GSM3536462 GSM3536462: 3 primemRNAseq zPCF11null hom e6; Danio rerio; RNA Seq GSM3536462   1 Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq three primemRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. GEO Accession:GSM3536462 RNA-Seq TRANSCRIPTOMIC cDNA SINGLE ILLUMINA Illumina HiSeq 2500   SRP175015     bam5__sel_TCGAGGATC.bam.fastq_trimmed.fastq.gz fastq 704276360.0 8098980.0 GSM3536462 r2 0:86.96 A:227042988;C:132957237;G:147852510;T:196421958;N:1667 86       227042988 132957237 147852510 196421958 1667 SRX5193218 SRS4199218 SRA828489 GEO Sir William Dunn School of Pathology, University of Oxford 1 0.81976   0.23195   0.82014   0.58764   89   B   usable mapping rate illumina hiseq_era 3prime poly_a lexogen bulk unknown unknown   United Kingdom 2019-01-02 Multi-stage Embryo Head Nervous System
50983 50983 SRR8383284 SRX5193217 SRS4199217 SRP175015 PRJNA512489 Selective roles of vertebrate PCF11 in premature and full length transcript termination zebrafish three prime mRNA seq GSE124555 Other The pervasive nature of RNA polymerase II Pol II transcription requires efficient termination. A key player in this process is the cleavage and polyadenylation CPA factor PCF11 which directly binds to the Pol II C terminal domain and dismantles elongating Pol II from DNA in vitro. We demonstrate that PCF11 mediated termination is essential for vertebrate development. A range of genomic analyses including: mNET seq three prime mRNA seq chromatin RNA seq and ChIP seq reveals that PCF11 enhances transcription termination and stimulates early polyadenylation genome wide. PCF11 binds preferentially between closely spaced genes where it prevents transcriptional interference and downstream gene silencing. Notably PCF11 is sub stoichiometric to the CPA complex. Low levels of PCF11 are maintained by an auto regulatory mechanism involving premature termination of its own transcript and are important for normal development. Both in human cell culture and during zebrafish development PCF11 selectively attenuates the expression of other transcriptional regulators by premature CPA and termination. Overall design: three prime mRNA seq in individual zebrafish embryo heads. Two types of mutants: zPCF11 null and zPCF11 with deletion of PAS1. Wild type wt +/+ heterozygous het +/ and homozygous mutant hom / embryos were analyzed. Wild type and heterozygous animals were phenotypically indistinguishable. parent bioproject:PRJNA507553 pubmed:30819644   3 primemRNAseq zPCF11null hom e5 GSM3536461   tissue:single embryo head 19hpf|molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11null / 3 primemRNAseq zPCF11null hom e5 3’ mRNA seq data were mapped according to the guidelines of QuantSeq library kit manufacturer Lexogen. Unaligned bam files from HiSeq2500 were converted to FASTQ files with bam2fastx TopHat 2 component Kim et al. 2013. post overview with FastQC reads were trimmed with BBtools sourceforge.net/projects/bbmap/ script bbduk using following settings: k=13 ktrim=r useshortkmers=t mink=5 qtrim=r trimq=10 minlength=20 . post trimming control with FastQC curated reads were mapped with STAR2.5b Dobin et al. 2013 aligner. STAR index was generated with the GRCz10/danRer10 was used as the reference zebrafish genome together with the corresponding ENSEMBL annotation. Aligned 3’ mRNA seq reads were filtered to remove false positives due to internal priming of the QuantSeq assay on genome encoded polyA stretches. To do this first a crude genomic mask was generated that contained all loci harbouring 6 or more consecutive A bases as well as any 10 nucleotide windows containing more than 6 A bases. For genes expressed from the reverse strand an analogous T rich mask was generated. Those crude masks were then corrected to allow for detection of genuine PAS falling in A/T rich regions by strand specifically unmasking 20 nucleotide intervals centred at annotated 3’ gene ends. 3’ mRNA seq reads falling into those refined strand specific masks were then removed and the filtered reads reduced to the most distal nucleotide 3’ end nucleotide. Genome build: GRCz10/danRer10 Supplementary files format and content: bigWig files: genome coverage files mean from replicates single embryo head 19hpf   Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq 3’mRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. Zebrafish Danio rerio were raised according to standard protocols 28°C water temperature 14/10 hr light/dark cycle. TLAB fish generated by crossing zebrafish AB and the natural variant TL Tupfel Long fin stocks served as wild type zebrafish for all experiments. zPCF11 null and zPCF11 ∆PAS1 mutant zebrafish were generated by Cas9 mediated mutagenesis Gagnon et al. 2014 molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11null / GSM3536461 GSM3536461: 3 primemRNAseq zPCF11null hom e5; Danio rerio; RNA Seq GSM3536461   1 Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq three primemRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. GEO Accession:GSM3536461 RNA-Seq TRANSCRIPTOMIC cDNA SINGLE ILLUMINA Illumina HiSeq 2500   SRP175015     bam4__sel_GTGCCAATC.bam.fastq_trimmed.fastq.gz fastq 770954082.0 8859450.0 GSM3536461 r1 0:87.02 A:257152925;C:141557495;G:158071178;T:214170140;N:2344 87       257152925 141557495 158071178 214170140 2344 SRX5193217 SRS4199217 SRA828489 GEO Sir William Dunn School of Pathology, University of Oxford 1 0.80635   0.19837   0.81355   0.40061   89   B   usable mapping rate illumina hiseq_era 3prime poly_a lexogen bulk unknown unknown   United Kingdom 2019-01-02 Multi-stage Embryo Head Nervous System
50984 50984 SRR8383285 SRX5193217 SRS4199217 SRP175015 PRJNA512489 Selective roles of vertebrate PCF11 in premature and full length transcript termination zebrafish three prime mRNA seq GSE124555 Other The pervasive nature of RNA polymerase II Pol II transcription requires efficient termination. A key player in this process is the cleavage and polyadenylation CPA factor PCF11 which directly binds to the Pol II C terminal domain and dismantles elongating Pol II from DNA in vitro. We demonstrate that PCF11 mediated termination is essential for vertebrate development. A range of genomic analyses including: mNET seq three prime mRNA seq chromatin RNA seq and ChIP seq reveals that PCF11 enhances transcription termination and stimulates early polyadenylation genome wide. PCF11 binds preferentially between closely spaced genes where it prevents transcriptional interference and downstream gene silencing. Notably PCF11 is sub stoichiometric to the CPA complex. Low levels of PCF11 are maintained by an auto regulatory mechanism involving premature termination of its own transcript and are important for normal development. Both in human cell culture and during zebrafish development PCF11 selectively attenuates the expression of other transcriptional regulators by premature CPA and termination. Overall design: three prime mRNA seq in individual zebrafish embryo heads. Two types of mutants: zPCF11 null and zPCF11 with deletion of PAS1. Wild type wt +/+ heterozygous het +/ and homozygous mutant hom / embryos were analyzed. Wild type and heterozygous animals were phenotypically indistinguishable. parent bioproject:PRJNA507553 pubmed:30819644   3 primemRNAseq zPCF11null hom e5 GSM3536461   tissue:single embryo head 19hpf|molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11null / 3 primemRNAseq zPCF11null hom e5 3’ mRNA seq data were mapped according to the guidelines of QuantSeq library kit manufacturer Lexogen. Unaligned bam files from HiSeq2500 were converted to FASTQ files with bam2fastx TopHat 2 component Kim et al. 2013. post overview with FastQC reads were trimmed with BBtools sourceforge.net/projects/bbmap/ script bbduk using following settings: k=13 ktrim=r useshortkmers=t mink=5 qtrim=r trimq=10 minlength=20 . post trimming control with FastQC curated reads were mapped with STAR2.5b Dobin et al. 2013 aligner. STAR index was generated with the GRCz10/danRer10 was used as the reference zebrafish genome together with the corresponding ENSEMBL annotation. Aligned 3’ mRNA seq reads were filtered to remove false positives due to internal priming of the QuantSeq assay on genome encoded polyA stretches. To do this first a crude genomic mask was generated that contained all loci harbouring 6 or more consecutive A bases as well as any 10 nucleotide windows containing more than 6 A bases. For genes expressed from the reverse strand an analogous T rich mask was generated. Those crude masks were then corrected to allow for detection of genuine PAS falling in A/T rich regions by strand specifically unmasking 20 nucleotide intervals centred at annotated 3’ gene ends. 3’ mRNA seq reads falling into those refined strand specific masks were then removed and the filtered reads reduced to the most distal nucleotide 3’ end nucleotide. Genome build: GRCz10/danRer10 Supplementary files format and content: bigWig files: genome coverage files mean from replicates single embryo head 19hpf   Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq 3’mRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. Zebrafish Danio rerio were raised according to standard protocols 28°C water temperature 14/10 hr light/dark cycle. TLAB fish generated by crossing zebrafish AB and the natural variant TL Tupfel Long fin stocks served as wild type zebrafish for all experiments. zPCF11 null and zPCF11 ∆PAS1 mutant zebrafish were generated by Cas9 mediated mutagenesis Gagnon et al. 2014 molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11null / GSM3536461 GSM3536461: 3 primemRNAseq zPCF11null hom e5; Danio rerio; RNA Seq GSM3536461   1 Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq three primemRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. GEO Accession:GSM3536461 RNA-Seq TRANSCRIPTOMIC cDNA SINGLE ILLUMINA Illumina HiSeq 2500   SRP175015     bam5__sel_GTGCCAATC.bam.fastq_trimmed.fastq.gz fastq 792682872.0 9113148.0 GSM3536461 r2 0:86.98 A:264818153;C:145303306;G:162291530;T:220268109;N:1774 86       264818153 145303306 162291530 220268109 1774 SRX5193217 SRS4199217 SRA828489 GEO Sir William Dunn School of Pathology, University of Oxford 1 0.80446   0.1978   0.81397   0.57793   89   B   usable mapping rate illumina hiseq_era 3prime poly_a lexogen bulk unknown unknown   United Kingdom 2019-01-02 Multi-stage Embryo Head Nervous System
50985 50985 SRR8383282 SRX5193216 SRS4199216 SRP175015 PRJNA512489 Selective roles of vertebrate PCF11 in premature and full length transcript termination zebrafish three prime mRNA seq GSE124555 Other The pervasive nature of RNA polymerase II Pol II transcription requires efficient termination. A key player in this process is the cleavage and polyadenylation CPA factor PCF11 which directly binds to the Pol II C terminal domain and dismantles elongating Pol II from DNA in vitro. We demonstrate that PCF11 mediated termination is essential for vertebrate development. A range of genomic analyses including: mNET seq three prime mRNA seq chromatin RNA seq and ChIP seq reveals that PCF11 enhances transcription termination and stimulates early polyadenylation genome wide. PCF11 binds preferentially between closely spaced genes where it prevents transcriptional interference and downstream gene silencing. Notably PCF11 is sub stoichiometric to the CPA complex. Low levels of PCF11 are maintained by an auto regulatory mechanism involving premature termination of its own transcript and are important for normal development. Both in human cell culture and during zebrafish development PCF11 selectively attenuates the expression of other transcriptional regulators by premature CPA and termination. Overall design: three prime mRNA seq in individual zebrafish embryo heads. Two types of mutants: zPCF11 null and zPCF11 with deletion of PAS1. Wild type wt +/+ heterozygous het +/ and homozygous mutant hom / embryos were analyzed. Wild type and heterozygous animals were phenotypically indistinguishable. parent bioproject:PRJNA507553 pubmed:30819644   3 primemRNAseq zPCF11null hom e4 GSM3536460   tissue:single embryo head 19hpf|molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11null / 3 primemRNAseq zPCF11null hom e4 3’ mRNA seq data were mapped according to the guidelines of QuantSeq library kit manufacturer Lexogen. Unaligned bam files from HiSeq2500 were converted to FASTQ files with bam2fastx TopHat 2 component Kim et al. 2013. post overview with FastQC reads were trimmed with BBtools sourceforge.net/projects/bbmap/ script bbduk using following settings: k=13 ktrim=r useshortkmers=t mink=5 qtrim=r trimq=10 minlength=20 . post trimming control with FastQC curated reads were mapped with STAR2.5b Dobin et al. 2013 aligner. STAR index was generated with the GRCz10/danRer10 was used as the reference zebrafish genome together with the corresponding ENSEMBL annotation. Aligned 3’ mRNA seq reads were filtered to remove false positives due to internal priming of the QuantSeq assay on genome encoded polyA stretches. To do this first a crude genomic mask was generated that contained all loci harbouring 6 or more consecutive A bases as well as any 10 nucleotide windows containing more than 6 A bases. For genes expressed from the reverse strand an analogous T rich mask was generated. Those crude masks were then corrected to allow for detection of genuine PAS falling in A/T rich regions by strand specifically unmasking 20 nucleotide intervals centred at annotated 3’ gene ends. 3’ mRNA seq reads falling into those refined strand specific masks were then removed and the filtered reads reduced to the most distal nucleotide 3’ end nucleotide. Genome build: GRCz10/danRer10 Supplementary files format and content: bigWig files: genome coverage files mean from replicates single embryo head 19hpf   Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq 3’mRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. Zebrafish Danio rerio were raised according to standard protocols 28°C water temperature 14/10 hr light/dark cycle. TLAB fish generated by crossing zebrafish AB and the natural variant TL Tupfel Long fin stocks served as wild type zebrafish for all experiments. zPCF11 null and zPCF11 ∆PAS1 mutant zebrafish were generated by Cas9 mediated mutagenesis Gagnon et al. 2014 molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11null / GSM3536460 GSM3536460: 3 primemRNAseq zPCF11null hom e4; Danio rerio; RNA Seq GSM3536460   1 Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq three primemRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. GEO Accession:GSM3536460 RNA-Seq TRANSCRIPTOMIC cDNA SINGLE ILLUMINA Illumina HiSeq 2500   SRP175015     bam4__sel_AATAGCATC.bam.fastq_trimmed.fastq.gz fastq 128394352.0 1469184.0 GSM3536460 r1 0:87.39 A:41878409;C:23656471;G:26363280;T:36495822;N:370 87       41878409 23656471 26363280 36495822 370 SRX5193216 SRS4199216 SRA828489 GEO Sir William Dunn School of Pathology, University of Oxford 1 0.8404   0.20794   0.81635   0.57453   89   B   usable mapping rate illumina hiseq_era 3prime poly_a lexogen bulk unknown unknown   United Kingdom 2019-01-02 Multi-stage Embryo Head Nervous System
50986 50986 SRR8383283 SRX5193216 SRS4199216 SRP175015 PRJNA512489 Selective roles of vertebrate PCF11 in premature and full length transcript termination zebrafish three prime mRNA seq GSE124555 Other The pervasive nature of RNA polymerase II Pol II transcription requires efficient termination. A key player in this process is the cleavage and polyadenylation CPA factor PCF11 which directly binds to the Pol II C terminal domain and dismantles elongating Pol II from DNA in vitro. We demonstrate that PCF11 mediated termination is essential for vertebrate development. A range of genomic analyses including: mNET seq three prime mRNA seq chromatin RNA seq and ChIP seq reveals that PCF11 enhances transcription termination and stimulates early polyadenylation genome wide. PCF11 binds preferentially between closely spaced genes where it prevents transcriptional interference and downstream gene silencing. Notably PCF11 is sub stoichiometric to the CPA complex. Low levels of PCF11 are maintained by an auto regulatory mechanism involving premature termination of its own transcript and are important for normal development. Both in human cell culture and during zebrafish development PCF11 selectively attenuates the expression of other transcriptional regulators by premature CPA and termination. Overall design: three prime mRNA seq in individual zebrafish embryo heads. Two types of mutants: zPCF11 null and zPCF11 with deletion of PAS1. Wild type wt +/+ heterozygous het +/ and homozygous mutant hom / embryos were analyzed. Wild type and heterozygous animals were phenotypically indistinguishable. parent bioproject:PRJNA507553 pubmed:30819644   3 primemRNAseq zPCF11null hom e4 GSM3536460   tissue:single embryo head 19hpf|molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11null / 3 primemRNAseq zPCF11null hom e4 3’ mRNA seq data were mapped according to the guidelines of QuantSeq library kit manufacturer Lexogen. Unaligned bam files from HiSeq2500 were converted to FASTQ files with bam2fastx TopHat 2 component Kim et al. 2013. post overview with FastQC reads were trimmed with BBtools sourceforge.net/projects/bbmap/ script bbduk using following settings: k=13 ktrim=r useshortkmers=t mink=5 qtrim=r trimq=10 minlength=20 . post trimming control with FastQC curated reads were mapped with STAR2.5b Dobin et al. 2013 aligner. STAR index was generated with the GRCz10/danRer10 was used as the reference zebrafish genome together with the corresponding ENSEMBL annotation. Aligned 3’ mRNA seq reads were filtered to remove false positives due to internal priming of the QuantSeq assay on genome encoded polyA stretches. To do this first a crude genomic mask was generated that contained all loci harbouring 6 or more consecutive A bases as well as any 10 nucleotide windows containing more than 6 A bases. For genes expressed from the reverse strand an analogous T rich mask was generated. Those crude masks were then corrected to allow for detection of genuine PAS falling in A/T rich regions by strand specifically unmasking 20 nucleotide intervals centred at annotated 3’ gene ends. 3’ mRNA seq reads falling into those refined strand specific masks were then removed and the filtered reads reduced to the most distal nucleotide 3’ end nucleotide. Genome build: GRCz10/danRer10 Supplementary files format and content: bigWig files: genome coverage files mean from replicates single embryo head 19hpf   Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq 3’mRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. Zebrafish Danio rerio were raised according to standard protocols 28°C water temperature 14/10 hr light/dark cycle. TLAB fish generated by crossing zebrafish AB and the natural variant TL Tupfel Long fin stocks served as wild type zebrafish for all experiments. zPCF11 null and zPCF11 ∆PAS1 mutant zebrafish were generated by Cas9 mediated mutagenesis Gagnon et al. 2014 molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11null / GSM3536460 GSM3536460: 3 primemRNAseq zPCF11null hom e4; Danio rerio; RNA Seq GSM3536460   1 Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq three primemRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. GEO Accession:GSM3536460 RNA-Seq TRANSCRIPTOMIC cDNA SINGLE ILLUMINA Illumina HiSeq 2500   SRP175015     bam5__sel_AATAGCATC.bam.fastq_trimmed.fastq.gz fastq 131964907.0 1510456.0 GSM3536460 r2 0:87.37 A:43071448;C:24301835;G:27045591;T:37545716;N:317 87       43071448 24301835 27045591 37545716 317 SRX5193216 SRS4199216 SRA828489 GEO Sir William Dunn School of Pathology, University of Oxford 1 0.84091   0.21002   0.8168   0.57732   89   B   usable mapping rate illumina hiseq_era 3prime poly_a lexogen bulk unknown unknown   United Kingdom 2019-01-02 Multi-stage Embryo Head Nervous System
50987 50987 SRR8383280 SRX5193215 SRS4199215 SRP175015 PRJNA512489 Selective roles of vertebrate PCF11 in premature and full length transcript termination zebrafish three prime mRNA seq GSE124555 Other The pervasive nature of RNA polymerase II Pol II transcription requires efficient termination. A key player in this process is the cleavage and polyadenylation CPA factor PCF11 which directly binds to the Pol II C terminal domain and dismantles elongating Pol II from DNA in vitro. We demonstrate that PCF11 mediated termination is essential for vertebrate development. A range of genomic analyses including: mNET seq three prime mRNA seq chromatin RNA seq and ChIP seq reveals that PCF11 enhances transcription termination and stimulates early polyadenylation genome wide. PCF11 binds preferentially between closely spaced genes where it prevents transcriptional interference and downstream gene silencing. Notably PCF11 is sub stoichiometric to the CPA complex. Low levels of PCF11 are maintained by an auto regulatory mechanism involving premature termination of its own transcript and are important for normal development. Both in human cell culture and during zebrafish development PCF11 selectively attenuates the expression of other transcriptional regulators by premature CPA and termination. Overall design: three prime mRNA seq in individual zebrafish embryo heads. Two types of mutants: zPCF11 null and zPCF11 with deletion of PAS1. Wild type wt +/+ heterozygous het +/ and homozygous mutant hom / embryos were analyzed. Wild type and heterozygous animals were phenotypically indistinguishable. parent bioproject:PRJNA507553 pubmed:30819644   3 primemRNAseq zPCF11null hom e3 GSM3536459   tissue:single embryo head 19hpf|molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11null / 3 primemRNAseq zPCF11null hom e3 3’ mRNA seq data were mapped according to the guidelines of QuantSeq library kit manufacturer Lexogen. Unaligned bam files from HiSeq2500 were converted to FASTQ files with bam2fastx TopHat 2 component Kim et al. 2013. post overview with FastQC reads were trimmed with BBtools sourceforge.net/projects/bbmap/ script bbduk using following settings: k=13 ktrim=r useshortkmers=t mink=5 qtrim=r trimq=10 minlength=20 . post trimming control with FastQC curated reads were mapped with STAR2.5b Dobin et al. 2013 aligner. STAR index was generated with the GRCz10/danRer10 was used as the reference zebrafish genome together with the corresponding ENSEMBL annotation. Aligned 3’ mRNA seq reads were filtered to remove false positives due to internal priming of the QuantSeq assay on genome encoded polyA stretches. To do this first a crude genomic mask was generated that contained all loci harbouring 6 or more consecutive A bases as well as any 10 nucleotide windows containing more than 6 A bases. For genes expressed from the reverse strand an analogous T rich mask was generated. Those crude masks were then corrected to allow for detection of genuine PAS falling in A/T rich regions by strand specifically unmasking 20 nucleotide intervals centred at annotated 3’ gene ends. 3’ mRNA seq reads falling into those refined strand specific masks were then removed and the filtered reads reduced to the most distal nucleotide 3’ end nucleotide. Genome build: GRCz10/danRer10 Supplementary files format and content: bigWig files: genome coverage files mean from replicates single embryo head 19hpf   Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq 3’mRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. Zebrafish Danio rerio were raised according to standard protocols 28°C water temperature 14/10 hr light/dark cycle. TLAB fish generated by crossing zebrafish AB and the natural variant TL Tupfel Long fin stocks served as wild type zebrafish for all experiments. zPCF11 null and zPCF11 ∆PAS1 mutant zebrafish were generated by Cas9 mediated mutagenesis Gagnon et al. 2014 molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11null / GSM3536459 GSM3536459: 3 primemRNAseq zPCF11null hom e3; Danio rerio; RNA Seq GSM3536459   1 Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq three primemRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. GEO Accession:GSM3536459 RNA-Seq TRANSCRIPTOMIC cDNA SINGLE ILLUMINA Illumina HiSeq 2500   SRP175015     bam4__sel_GCATGGATC.bam.fastq_trimmed.fastq.gz fastq 198921603.0 2281764.0 GSM3536459 r1 0:87.18 A:67933819;C:36044297;G:40047977;T:54894929;N:581 87       67933819 36044297 40047977 54894929 581 SRX5193215 SRS4199215 SRA828489 GEO Sir William Dunn School of Pathology, University of Oxford 1 0.80512   0.21831   0.81856   0.58328   89   B   usable mapping rate illumina hiseq_era 3prime poly_a lexogen bulk unknown unknown   United Kingdom 2019-01-02 Multi-stage Embryo Head Nervous System
50988 50988 SRR8383281 SRX5193215 SRS4199215 SRP175015 PRJNA512489 Selective roles of vertebrate PCF11 in premature and full length transcript termination zebrafish three prime mRNA seq GSE124555 Other The pervasive nature of RNA polymerase II Pol II transcription requires efficient termination. A key player in this process is the cleavage and polyadenylation CPA factor PCF11 which directly binds to the Pol II C terminal domain and dismantles elongating Pol II from DNA in vitro. We demonstrate that PCF11 mediated termination is essential for vertebrate development. A range of genomic analyses including: mNET seq three prime mRNA seq chromatin RNA seq and ChIP seq reveals that PCF11 enhances transcription termination and stimulates early polyadenylation genome wide. PCF11 binds preferentially between closely spaced genes where it prevents transcriptional interference and downstream gene silencing. Notably PCF11 is sub stoichiometric to the CPA complex. Low levels of PCF11 are maintained by an auto regulatory mechanism involving premature termination of its own transcript and are important for normal development. Both in human cell culture and during zebrafish development PCF11 selectively attenuates the expression of other transcriptional regulators by premature CPA and termination. Overall design: three prime mRNA seq in individual zebrafish embryo heads. Two types of mutants: zPCF11 null and zPCF11 with deletion of PAS1. Wild type wt +/+ heterozygous het +/ and homozygous mutant hom / embryos were analyzed. Wild type and heterozygous animals were phenotypically indistinguishable. parent bioproject:PRJNA507553 pubmed:30819644   3 primemRNAseq zPCF11null hom e3 GSM3536459   tissue:single embryo head 19hpf|molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11null / 3 primemRNAseq zPCF11null hom e3 3’ mRNA seq data were mapped according to the guidelines of QuantSeq library kit manufacturer Lexogen. Unaligned bam files from HiSeq2500 were converted to FASTQ files with bam2fastx TopHat 2 component Kim et al. 2013. post overview with FastQC reads were trimmed with BBtools sourceforge.net/projects/bbmap/ script bbduk using following settings: k=13 ktrim=r useshortkmers=t mink=5 qtrim=r trimq=10 minlength=20 . post trimming control with FastQC curated reads were mapped with STAR2.5b Dobin et al. 2013 aligner. STAR index was generated with the GRCz10/danRer10 was used as the reference zebrafish genome together with the corresponding ENSEMBL annotation. Aligned 3’ mRNA seq reads were filtered to remove false positives due to internal priming of the QuantSeq assay on genome encoded polyA stretches. To do this first a crude genomic mask was generated that contained all loci harbouring 6 or more consecutive A bases as well as any 10 nucleotide windows containing more than 6 A bases. For genes expressed from the reverse strand an analogous T rich mask was generated. Those crude masks were then corrected to allow for detection of genuine PAS falling in A/T rich regions by strand specifically unmasking 20 nucleotide intervals centred at annotated 3’ gene ends. 3’ mRNA seq reads falling into those refined strand specific masks were then removed and the filtered reads reduced to the most distal nucleotide 3’ end nucleotide. Genome build: GRCz10/danRer10 Supplementary files format and content: bigWig files: genome coverage files mean from replicates single embryo head 19hpf   Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq 3’mRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. Zebrafish Danio rerio were raised according to standard protocols 28°C water temperature 14/10 hr light/dark cycle. TLAB fish generated by crossing zebrafish AB and the natural variant TL Tupfel Long fin stocks served as wild type zebrafish for all experiments. zPCF11 null and zPCF11 ∆PAS1 mutant zebrafish were generated by Cas9 mediated mutagenesis Gagnon et al. 2014 molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11null / GSM3536459 GSM3536459: 3 primemRNAseq zPCF11null hom e3; Danio rerio; RNA Seq GSM3536459   1 Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq three primemRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. GEO Accession:GSM3536459 RNA-Seq TRANSCRIPTOMIC cDNA SINGLE ILLUMINA Illumina HiSeq 2500   SRP175015     bam5__sel_GCATGGATC.bam.fastq_trimmed.fastq.gz fastq 204339172.0 2344861.0 GSM3536459 r2 0:87.14 A:69852605;C:36969411;G:41077998;T:56438756;N:402 87       69852605 36969411 41077998 56438756 402 SRX5193215 SRS4199215 SRA828489 GEO Sir William Dunn School of Pathology, University of Oxford 1 0.80367   0.21762   0.81858   0.58312   89   B   usable mapping rate illumina hiseq_era 3prime poly_a lexogen bulk unknown unknown   United Kingdom 2019-01-02 Multi-stage Embryo Head Nervous System
50989 50989 SRR8383278 SRX5193214 SRS4199214 SRP175015 PRJNA512489 Selective roles of vertebrate PCF11 in premature and full length transcript termination zebrafish three prime mRNA seq GSE124555 Other The pervasive nature of RNA polymerase II Pol II transcription requires efficient termination. A key player in this process is the cleavage and polyadenylation CPA factor PCF11 which directly binds to the Pol II C terminal domain and dismantles elongating Pol II from DNA in vitro. We demonstrate that PCF11 mediated termination is essential for vertebrate development. A range of genomic analyses including: mNET seq three prime mRNA seq chromatin RNA seq and ChIP seq reveals that PCF11 enhances transcription termination and stimulates early polyadenylation genome wide. PCF11 binds preferentially between closely spaced genes where it prevents transcriptional interference and downstream gene silencing. Notably PCF11 is sub stoichiometric to the CPA complex. Low levels of PCF11 are maintained by an auto regulatory mechanism involving premature termination of its own transcript and are important for normal development. Both in human cell culture and during zebrafish development PCF11 selectively attenuates the expression of other transcriptional regulators by premature CPA and termination. Overall design: three prime mRNA seq in individual zebrafish embryo heads. Two types of mutants: zPCF11 null and zPCF11 with deletion of PAS1. Wild type wt +/+ heterozygous het +/ and homozygous mutant hom / embryos were analyzed. Wild type and heterozygous animals were phenotypically indistinguishable. parent bioproject:PRJNA507553 pubmed:30819644   3 primemRNAseq zPCF11null hom e2 GSM3536458   tissue:single embryo head 19hpf|molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11null / 3 primemRNAseq zPCF11null hom e2 3’ mRNA seq data were mapped according to the guidelines of QuantSeq library kit manufacturer Lexogen. Unaligned bam files from HiSeq2500 were converted to FASTQ files with bam2fastx TopHat 2 component Kim et al. 2013. post overview with FastQC reads were trimmed with BBtools sourceforge.net/projects/bbmap/ script bbduk using following settings: k=13 ktrim=r useshortkmers=t mink=5 qtrim=r trimq=10 minlength=20 . post trimming control with FastQC curated reads were mapped with STAR2.5b Dobin et al. 2013 aligner. STAR index was generated with the GRCz10/danRer10 was used as the reference zebrafish genome together with the corresponding ENSEMBL annotation. Aligned 3’ mRNA seq reads were filtered to remove false positives due to internal priming of the QuantSeq assay on genome encoded polyA stretches. To do this first a crude genomic mask was generated that contained all loci harbouring 6 or more consecutive A bases as well as any 10 nucleotide windows containing more than 6 A bases. For genes expressed from the reverse strand an analogous T rich mask was generated. Those crude masks were then corrected to allow for detection of genuine PAS falling in A/T rich regions by strand specifically unmasking 20 nucleotide intervals centred at annotated 3’ gene ends. 3’ mRNA seq reads falling into those refined strand specific masks were then removed and the filtered reads reduced to the most distal nucleotide 3’ end nucleotide. Genome build: GRCz10/danRer10 Supplementary files format and content: bigWig files: genome coverage files mean from replicates single embryo head 19hpf   Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq 3’mRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. Zebrafish Danio rerio were raised according to standard protocols 28°C water temperature 14/10 hr light/dark cycle. TLAB fish generated by crossing zebrafish AB and the natural variant TL Tupfel Long fin stocks served as wild type zebrafish for all experiments. zPCF11 null and zPCF11 ∆PAS1 mutant zebrafish were generated by Cas9 mediated mutagenesis Gagnon et al. 2014 molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11null / GSM3536458 GSM3536458: 3 primemRNAseq zPCF11null hom e2; Danio rerio; RNA Seq GSM3536458   1 Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq three primemRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. GEO Accession:GSM3536458 RNA-Seq TRANSCRIPTOMIC cDNA SINGLE ILLUMINA Illumina HiSeq 2500   SRP175015     bam4__sel_CTCCATATC.bam.fastq_trimmed.fastq.gz fastq 243678478.0 2785241.0 GSM3536458 r1 0:87.49 A:80921391;C:44962028;G:49483835;T:68310471;N:753 87       80921391 44962028 49483835 68310471 753 SRX5193214 SRS4199214 SRA828489 GEO Sir William Dunn School of Pathology, University of Oxford 1 0.8288   0.19506   0.82674   0.5695   89   B   usable mapping rate illumina hiseq_era 3prime poly_a lexogen bulk unknown unknown   United Kingdom 2019-01-02 Multi-stage Embryo Head Nervous System
50990 50990 SRR8383279 SRX5193214 SRS4199214 SRP175015 PRJNA512489 Selective roles of vertebrate PCF11 in premature and full length transcript termination zebrafish three prime mRNA seq GSE124555 Other The pervasive nature of RNA polymerase II Pol II transcription requires efficient termination. A key player in this process is the cleavage and polyadenylation CPA factor PCF11 which directly binds to the Pol II C terminal domain and dismantles elongating Pol II from DNA in vitro. We demonstrate that PCF11 mediated termination is essential for vertebrate development. A range of genomic analyses including: mNET seq three prime mRNA seq chromatin RNA seq and ChIP seq reveals that PCF11 enhances transcription termination and stimulates early polyadenylation genome wide. PCF11 binds preferentially between closely spaced genes where it prevents transcriptional interference and downstream gene silencing. Notably PCF11 is sub stoichiometric to the CPA complex. Low levels of PCF11 are maintained by an auto regulatory mechanism involving premature termination of its own transcript and are important for normal development. Both in human cell culture and during zebrafish development PCF11 selectively attenuates the expression of other transcriptional regulators by premature CPA and termination. Overall design: three prime mRNA seq in individual zebrafish embryo heads. Two types of mutants: zPCF11 null and zPCF11 with deletion of PAS1. Wild type wt +/+ heterozygous het +/ and homozygous mutant hom / embryos were analyzed. Wild type and heterozygous animals were phenotypically indistinguishable. parent bioproject:PRJNA507553 pubmed:30819644   3 primemRNAseq zPCF11null hom e2 GSM3536458   tissue:single embryo head 19hpf|molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11null / 3 primemRNAseq zPCF11null hom e2 3’ mRNA seq data were mapped according to the guidelines of QuantSeq library kit manufacturer Lexogen. Unaligned bam files from HiSeq2500 were converted to FASTQ files with bam2fastx TopHat 2 component Kim et al. 2013. post overview with FastQC reads were trimmed with BBtools sourceforge.net/projects/bbmap/ script bbduk using following settings: k=13 ktrim=r useshortkmers=t mink=5 qtrim=r trimq=10 minlength=20 . post trimming control with FastQC curated reads were mapped with STAR2.5b Dobin et al. 2013 aligner. STAR index was generated with the GRCz10/danRer10 was used as the reference zebrafish genome together with the corresponding ENSEMBL annotation. Aligned 3’ mRNA seq reads were filtered to remove false positives due to internal priming of the QuantSeq assay on genome encoded polyA stretches. To do this first a crude genomic mask was generated that contained all loci harbouring 6 or more consecutive A bases as well as any 10 nucleotide windows containing more than 6 A bases. For genes expressed from the reverse strand an analogous T rich mask was generated. Those crude masks were then corrected to allow for detection of genuine PAS falling in A/T rich regions by strand specifically unmasking 20 nucleotide intervals centred at annotated 3’ gene ends. 3’ mRNA seq reads falling into those refined strand specific masks were then removed and the filtered reads reduced to the most distal nucleotide 3’ end nucleotide. Genome build: GRCz10/danRer10 Supplementary files format and content: bigWig files: genome coverage files mean from replicates single embryo head 19hpf   Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq 3’mRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. Zebrafish Danio rerio were raised according to standard protocols 28°C water temperature 14/10 hr light/dark cycle. TLAB fish generated by crossing zebrafish AB and the natural variant TL Tupfel Long fin stocks served as wild type zebrafish for all experiments. zPCF11 null and zPCF11 ∆PAS1 mutant zebrafish were generated by Cas9 mediated mutagenesis Gagnon et al. 2014 molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11null / GSM3536458 GSM3536458: 3 primemRNAseq zPCF11null hom e2; Danio rerio; RNA Seq GSM3536458   1 Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq three primemRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. GEO Accession:GSM3536458 RNA-Seq TRANSCRIPTOMIC cDNA SINGLE ILLUMINA Illumina HiSeq 2500   SRP175015     bam5__sel_CTCCATATC.bam.fastq_trimmed.fastq.gz fastq 251121623.0 2871387.0 GSM3536458 r2 0:87.46 A:83477676;C:46277942;G:50927013;T:70438422;N:570 87       83477676 46277942 50927013 70438422 570 SRX5193214 SRS4199214 SRA828489 GEO Sir William Dunn School of Pathology, University of Oxford 1 0.82553   0.19377   0.82674   0.56696   89   B   usable mapping rate illumina hiseq_era 3prime poly_a lexogen bulk unknown unknown   United Kingdom 2019-01-02 Multi-stage Embryo Head Nervous System
50991 50991 SRR8383276 SRX5193213 SRS4199213 SRP175015 PRJNA512489 Selective roles of vertebrate PCF11 in premature and full length transcript termination zebrafish three prime mRNA seq GSE124555 Other The pervasive nature of RNA polymerase II Pol II transcription requires efficient termination. A key player in this process is the cleavage and polyadenylation CPA factor PCF11 which directly binds to the Pol II C terminal domain and dismantles elongating Pol II from DNA in vitro. We demonstrate that PCF11 mediated termination is essential for vertebrate development. A range of genomic analyses including: mNET seq three prime mRNA seq chromatin RNA seq and ChIP seq reveals that PCF11 enhances transcription termination and stimulates early polyadenylation genome wide. PCF11 binds preferentially between closely spaced genes where it prevents transcriptional interference and downstream gene silencing. Notably PCF11 is sub stoichiometric to the CPA complex. Low levels of PCF11 are maintained by an auto regulatory mechanism involving premature termination of its own transcript and are important for normal development. Both in human cell culture and during zebrafish development PCF11 selectively attenuates the expression of other transcriptional regulators by premature CPA and termination. Overall design: three prime mRNA seq in individual zebrafish embryo heads. Two types of mutants: zPCF11 null and zPCF11 with deletion of PAS1. Wild type wt +/+ heterozygous het +/ and homozygous mutant hom / embryos were analyzed. Wild type and heterozygous animals were phenotypically indistinguishable. parent bioproject:PRJNA507553 pubmed:30819644   3 primemRNAseq zPCF11null hom e1 GSM3536457   tissue:single embryo head 19hpf|molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11null / 3 primemRNAseq zPCF11null hom e1 3’ mRNA seq data were mapped according to the guidelines of QuantSeq library kit manufacturer Lexogen. Unaligned bam files from HiSeq2500 were converted to FASTQ files with bam2fastx TopHat 2 component Kim et al. 2013. post overview with FastQC reads were trimmed with BBtools sourceforge.net/projects/bbmap/ script bbduk using following settings: k=13 ktrim=r useshortkmers=t mink=5 qtrim=r trimq=10 minlength=20 . post trimming control with FastQC curated reads were mapped with STAR2.5b Dobin et al. 2013 aligner. STAR index was generated with the GRCz10/danRer10 was used as the reference zebrafish genome together with the corresponding ENSEMBL annotation. Aligned 3’ mRNA seq reads were filtered to remove false positives due to internal priming of the QuantSeq assay on genome encoded polyA stretches. To do this first a crude genomic mask was generated that contained all loci harbouring 6 or more consecutive A bases as well as any 10 nucleotide windows containing more than 6 A bases. For genes expressed from the reverse strand an analogous T rich mask was generated. Those crude masks were then corrected to allow for detection of genuine PAS falling in A/T rich regions by strand specifically unmasking 20 nucleotide intervals centred at annotated 3’ gene ends. 3’ mRNA seq reads falling into those refined strand specific masks were then removed and the filtered reads reduced to the most distal nucleotide 3’ end nucleotide. Genome build: GRCz10/danRer10 Supplementary files format and content: bigWig files: genome coverage files mean from replicates single embryo head 19hpf   Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq 3’mRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. Zebrafish Danio rerio were raised according to standard protocols 28°C water temperature 14/10 hr light/dark cycle. TLAB fish generated by crossing zebrafish AB and the natural variant TL Tupfel Long fin stocks served as wild type zebrafish for all experiments. zPCF11 null and zPCF11 ∆PAS1 mutant zebrafish were generated by Cas9 mediated mutagenesis Gagnon et al. 2014 molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11null / GSM3536457 GSM3536457: 3 primemRNAseq zPCF11null hom e1; Danio rerio; RNA Seq GSM3536457   1 Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq three primemRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. GEO Accession:GSM3536457 RNA-Seq TRANSCRIPTOMIC cDNA SINGLE ILLUMINA Illumina HiSeq 2500   SRP175015     bam4__sel_TAGGCTATC.bam.fastq_trimmed.fastq.gz fastq 125156975.0 1440450.0 GSM3536457 r1 0:86.89 A:41125276;C:23385340;G:26101084;T:34544902;N:373 86       41125276 23385340 26101084 34544902 373 SRX5193213 SRS4199213 SRA828489 GEO Sir William Dunn School of Pathology, University of Oxford 1 0.82681   0.23851   0.82162   0.59477   89   B   usable mapping rate illumina hiseq_era 3prime poly_a lexogen bulk unknown unknown   United Kingdom 2019-01-02 Multi-stage Embryo Head Nervous System
50992 50992 SRR8383277 SRX5193213 SRS4199213 SRP175015 PRJNA512489 Selective roles of vertebrate PCF11 in premature and full length transcript termination zebrafish three prime mRNA seq GSE124555 Other The pervasive nature of RNA polymerase II Pol II transcription requires efficient termination. A key player in this process is the cleavage and polyadenylation CPA factor PCF11 which directly binds to the Pol II C terminal domain and dismantles elongating Pol II from DNA in vitro. We demonstrate that PCF11 mediated termination is essential for vertebrate development. A range of genomic analyses including: mNET seq three prime mRNA seq chromatin RNA seq and ChIP seq reveals that PCF11 enhances transcription termination and stimulates early polyadenylation genome wide. PCF11 binds preferentially between closely spaced genes where it prevents transcriptional interference and downstream gene silencing. Notably PCF11 is sub stoichiometric to the CPA complex. Low levels of PCF11 are maintained by an auto regulatory mechanism involving premature termination of its own transcript and are important for normal development. Both in human cell culture and during zebrafish development PCF11 selectively attenuates the expression of other transcriptional regulators by premature CPA and termination. Overall design: three prime mRNA seq in individual zebrafish embryo heads. Two types of mutants: zPCF11 null and zPCF11 with deletion of PAS1. Wild type wt +/+ heterozygous het +/ and homozygous mutant hom / embryos were analyzed. Wild type and heterozygous animals were phenotypically indistinguishable. parent bioproject:PRJNA507553 pubmed:30819644   3 primemRNAseq zPCF11null hom e1 GSM3536457   tissue:single embryo head 19hpf|molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11null / 3 primemRNAseq zPCF11null hom e1 3’ mRNA seq data were mapped according to the guidelines of QuantSeq library kit manufacturer Lexogen. Unaligned bam files from HiSeq2500 were converted to FASTQ files with bam2fastx TopHat 2 component Kim et al. 2013. post overview with FastQC reads were trimmed with BBtools sourceforge.net/projects/bbmap/ script bbduk using following settings: k=13 ktrim=r useshortkmers=t mink=5 qtrim=r trimq=10 minlength=20 . post trimming control with FastQC curated reads were mapped with STAR2.5b Dobin et al. 2013 aligner. STAR index was generated with the GRCz10/danRer10 was used as the reference zebrafish genome together with the corresponding ENSEMBL annotation. Aligned 3’ mRNA seq reads were filtered to remove false positives due to internal priming of the QuantSeq assay on genome encoded polyA stretches. To do this first a crude genomic mask was generated that contained all loci harbouring 6 or more consecutive A bases as well as any 10 nucleotide windows containing more than 6 A bases. For genes expressed from the reverse strand an analogous T rich mask was generated. Those crude masks were then corrected to allow for detection of genuine PAS falling in A/T rich regions by strand specifically unmasking 20 nucleotide intervals centred at annotated 3’ gene ends. 3’ mRNA seq reads falling into those refined strand specific masks were then removed and the filtered reads reduced to the most distal nucleotide 3’ end nucleotide. Genome build: GRCz10/danRer10 Supplementary files format and content: bigWig files: genome coverage files mean from replicates single embryo head 19hpf   Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq 3’mRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. Zebrafish Danio rerio were raised according to standard protocols 28°C water temperature 14/10 hr light/dark cycle. TLAB fish generated by crossing zebrafish AB and the natural variant TL Tupfel Long fin stocks served as wild type zebrafish for all experiments. zPCF11 null and zPCF11 ∆PAS1 mutant zebrafish were generated by Cas9 mediated mutagenesis Gagnon et al. 2014 molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11null / GSM3536457 GSM3536457: 3 primemRNAseq zPCF11null hom e1; Danio rerio; RNA Seq GSM3536457   1 Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq three primemRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. GEO Accession:GSM3536457 RNA-Seq TRANSCRIPTOMIC cDNA SINGLE ILLUMINA Illumina HiSeq 2500   SRP175015     bam5__sel_TAGGCTATC.bam.fastq_trimmed.fastq.gz fastq 128831164.0 1483314.0 GSM3536457 r2 0:86.85 A:42399550;C:24025044;G:26817005;T:35589296;N:269 86       42399550 24025044 26817005 35589296 269 SRX5193213 SRS4199213 SRA828489 GEO Sir William Dunn School of Pathology, University of Oxford 1 0.82598   0.23646   0.82207   0.59052   89   B   usable mapping rate illumina hiseq_era 3prime poly_a lexogen bulk unknown unknown   United Kingdom 2019-01-02 Multi-stage Embryo Head Nervous System
50993 50993 SRR8383274 SRX5193212 SRS4199212 SRP175015 PRJNA512489 Selective roles of vertebrate PCF11 in premature and full length transcript termination zebrafish three prime mRNA seq GSE124555 Other The pervasive nature of RNA polymerase II Pol II transcription requires efficient termination. A key player in this process is the cleavage and polyadenylation CPA factor PCF11 which directly binds to the Pol II C terminal domain and dismantles elongating Pol II from DNA in vitro. We demonstrate that PCF11 mediated termination is essential for vertebrate development. A range of genomic analyses including: mNET seq three prime mRNA seq chromatin RNA seq and ChIP seq reveals that PCF11 enhances transcription termination and stimulates early polyadenylation genome wide. PCF11 binds preferentially between closely spaced genes where it prevents transcriptional interference and downstream gene silencing. Notably PCF11 is sub stoichiometric to the CPA complex. Low levels of PCF11 are maintained by an auto regulatory mechanism involving premature termination of its own transcript and are important for normal development. Both in human cell culture and during zebrafish development PCF11 selectively attenuates the expression of other transcriptional regulators by premature CPA and termination. Overall design: three prime mRNA seq in individual zebrafish embryo heads. Two types of mutants: zPCF11 null and zPCF11 with deletion of PAS1. Wild type wt +/+ heterozygous het +/ and homozygous mutant hom / embryos were analyzed. Wild type and heterozygous animals were phenotypically indistinguishable. parent bioproject:PRJNA507553 pubmed:30819644   3 primemRNAseq zPCF11null het e4 GSM3536456   tissue:single embryo head 19hpf|molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11null +/ 3 primemRNAseq zPCF11null het e4 3’ mRNA seq data were mapped according to the guidelines of QuantSeq library kit manufacturer Lexogen. Unaligned bam files from HiSeq2500 were converted to FASTQ files with bam2fastx TopHat 2 component Kim et al. 2013. post overview with FastQC reads were trimmed with BBtools sourceforge.net/projects/bbmap/ script bbduk using following settings: k=13 ktrim=r useshortkmers=t mink=5 qtrim=r trimq=10 minlength=20 . post trimming control with FastQC curated reads were mapped with STAR2.5b Dobin et al. 2013 aligner. STAR index was generated with the GRCz10/danRer10 was used as the reference zebrafish genome together with the corresponding ENSEMBL annotation. Aligned 3’ mRNA seq reads were filtered to remove false positives due to internal priming of the QuantSeq assay on genome encoded polyA stretches. To do this first a crude genomic mask was generated that contained all loci harbouring 6 or more consecutive A bases as well as any 10 nucleotide windows containing more than 6 A bases. For genes expressed from the reverse strand an analogous T rich mask was generated. Those crude masks were then corrected to allow for detection of genuine PAS falling in A/T rich regions by strand specifically unmasking 20 nucleotide intervals centred at annotated 3’ gene ends. 3’ mRNA seq reads falling into those refined strand specific masks were then removed and the filtered reads reduced to the most distal nucleotide 3’ end nucleotide. Genome build: GRCz10/danRer10 Supplementary files format and content: bigWig files: genome coverage files mean from replicates single embryo head 19hpf   Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq 3’mRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. Zebrafish Danio rerio were raised according to standard protocols 28°C water temperature 14/10 hr light/dark cycle. TLAB fish generated by crossing zebrafish AB and the natural variant TL Tupfel Long fin stocks served as wild type zebrafish for all experiments. zPCF11 null and zPCF11 ∆PAS1 mutant zebrafish were generated by Cas9 mediated mutagenesis Gagnon et al. 2014 molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11null +/ GSM3536456 GSM3536456: 3 primemRNAseq zPCF11null het e4; Danio rerio; RNA Seq GSM3536456   1 Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq three primemRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. GEO Accession:GSM3536456 RNA-Seq TRANSCRIPTOMIC cDNA SINGLE ILLUMINA Illumina HiSeq 2500   SRP175015     bam4__sel_CGAAGGATC.bam.fastq_trimmed.fastq.gz fastq 118253929.0 1353894.0 GSM3536456 r1 0:87.34 A:41106016;C:21057430;G:23427525;T:32662627;N:331 87       41106016 21057430 23427525 32662627 331 SRX5193212 SRS4199212 SRA828489 GEO Sir William Dunn School of Pathology, University of Oxford 1 0.81195   0.18309   0.82191   0.55215   89   B   usable mapping rate illumina hiseq_era 3prime poly_a lexogen bulk unknown unknown   United Kingdom 2019-01-02 Multi-stage Embryo Head Nervous System
50994 50994 SRR8383275 SRX5193212 SRS4199212 SRP175015 PRJNA512489 Selective roles of vertebrate PCF11 in premature and full length transcript termination zebrafish three prime mRNA seq GSE124555 Other The pervasive nature of RNA polymerase II Pol II transcription requires efficient termination. A key player in this process is the cleavage and polyadenylation CPA factor PCF11 which directly binds to the Pol II C terminal domain and dismantles elongating Pol II from DNA in vitro. We demonstrate that PCF11 mediated termination is essential for vertebrate development. A range of genomic analyses including: mNET seq three prime mRNA seq chromatin RNA seq and ChIP seq reveals that PCF11 enhances transcription termination and stimulates early polyadenylation genome wide. PCF11 binds preferentially between closely spaced genes where it prevents transcriptional interference and downstream gene silencing. Notably PCF11 is sub stoichiometric to the CPA complex. Low levels of PCF11 are maintained by an auto regulatory mechanism involving premature termination of its own transcript and are important for normal development. Both in human cell culture and during zebrafish development PCF11 selectively attenuates the expression of other transcriptional regulators by premature CPA and termination. Overall design: three prime mRNA seq in individual zebrafish embryo heads. Two types of mutants: zPCF11 null and zPCF11 with deletion of PAS1. Wild type wt +/+ heterozygous het +/ and homozygous mutant hom / embryos were analyzed. Wild type and heterozygous animals were phenotypically indistinguishable. parent bioproject:PRJNA507553 pubmed:30819644   3 primemRNAseq zPCF11null het e4 GSM3536456   tissue:single embryo head 19hpf|molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11null +/ 3 primemRNAseq zPCF11null het e4 3’ mRNA seq data were mapped according to the guidelines of QuantSeq library kit manufacturer Lexogen. Unaligned bam files from HiSeq2500 were converted to FASTQ files with bam2fastx TopHat 2 component Kim et al. 2013. post overview with FastQC reads were trimmed with BBtools sourceforge.net/projects/bbmap/ script bbduk using following settings: k=13 ktrim=r useshortkmers=t mink=5 qtrim=r trimq=10 minlength=20 . post trimming control with FastQC curated reads were mapped with STAR2.5b Dobin et al. 2013 aligner. STAR index was generated with the GRCz10/danRer10 was used as the reference zebrafish genome together with the corresponding ENSEMBL annotation. Aligned 3’ mRNA seq reads were filtered to remove false positives due to internal priming of the QuantSeq assay on genome encoded polyA stretches. To do this first a crude genomic mask was generated that contained all loci harbouring 6 or more consecutive A bases as well as any 10 nucleotide windows containing more than 6 A bases. For genes expressed from the reverse strand an analogous T rich mask was generated. Those crude masks were then corrected to allow for detection of genuine PAS falling in A/T rich regions by strand specifically unmasking 20 nucleotide intervals centred at annotated 3’ gene ends. 3’ mRNA seq reads falling into those refined strand specific masks were then removed and the filtered reads reduced to the most distal nucleotide 3’ end nucleotide. Genome build: GRCz10/danRer10 Supplementary files format and content: bigWig files: genome coverage files mean from replicates single embryo head 19hpf   Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq 3’mRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. Zebrafish Danio rerio were raised according to standard protocols 28°C water temperature 14/10 hr light/dark cycle. TLAB fish generated by crossing zebrafish AB and the natural variant TL Tupfel Long fin stocks served as wild type zebrafish for all experiments. zPCF11 null and zPCF11 ∆PAS1 mutant zebrafish were generated by Cas9 mediated mutagenesis Gagnon et al. 2014 molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11null +/ GSM3536456 GSM3536456: 3 primemRNAseq zPCF11null het e4; Danio rerio; RNA Seq GSM3536456   1 Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq three primemRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. GEO Accession:GSM3536456 RNA-Seq TRANSCRIPTOMIC cDNA SINGLE ILLUMINA Illumina HiSeq 2500   SRP175015     bam5__sel_CGAAGGATC.bam.fastq_trimmed.fastq.gz fastq 121725680.0 1394241.0 GSM3536456 r2                       SRX5193212 SRS4199212 SRA828489 GEO Sir William Dunn School of Pathology, University of Oxford 1 0.76785   0.17246   0.81962   0.55625   89   B   usable mapping rate illumina hiseq_era 3prime poly_a lexogen bulk unknown unknown   United Kingdom 2019-01-02 Multi-stage Embryo Head Nervous System
50995 50995 SRR8383272 SRX5193211 SRS4199211 SRP175015 PRJNA512489 Selective roles of vertebrate PCF11 in premature and full length transcript termination zebrafish three prime mRNA seq GSE124555 Other The pervasive nature of RNA polymerase II Pol II transcription requires efficient termination. A key player in this process is the cleavage and polyadenylation CPA factor PCF11 which directly binds to the Pol II C terminal domain and dismantles elongating Pol II from DNA in vitro. We demonstrate that PCF11 mediated termination is essential for vertebrate development. A range of genomic analyses including: mNET seq three prime mRNA seq chromatin RNA seq and ChIP seq reveals that PCF11 enhances transcription termination and stimulates early polyadenylation genome wide. PCF11 binds preferentially between closely spaced genes where it prevents transcriptional interference and downstream gene silencing. Notably PCF11 is sub stoichiometric to the CPA complex. Low levels of PCF11 are maintained by an auto regulatory mechanism involving premature termination of its own transcript and are important for normal development. Both in human cell culture and during zebrafish development PCF11 selectively attenuates the expression of other transcriptional regulators by premature CPA and termination. Overall design: three prime mRNA seq in individual zebrafish embryo heads. Two types of mutants: zPCF11 null and zPCF11 with deletion of PAS1. Wild type wt +/+ heterozygous het +/ and homozygous mutant hom / embryos were analyzed. Wild type and heterozygous animals were phenotypically indistinguishable. parent bioproject:PRJNA507553 pubmed:30819644   3 primemRNAseq zPCF11null het e3 GSM3536455   tissue:single embryo head 19hpf|molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11null +/ 3 primemRNAseq zPCF11null het e3 3’ mRNA seq data were mapped according to the guidelines of QuantSeq library kit manufacturer Lexogen. Unaligned bam files from HiSeq2500 were converted to FASTQ files with bam2fastx TopHat 2 component Kim et al. 2013. post overview with FastQC reads were trimmed with BBtools sourceforge.net/projects/bbmap/ script bbduk using following settings: k=13 ktrim=r useshortkmers=t mink=5 qtrim=r trimq=10 minlength=20 . post trimming control with FastQC curated reads were mapped with STAR2.5b Dobin et al. 2013 aligner. STAR index was generated with the GRCz10/danRer10 was used as the reference zebrafish genome together with the corresponding ENSEMBL annotation. Aligned 3’ mRNA seq reads were filtered to remove false positives due to internal priming of the QuantSeq assay on genome encoded polyA stretches. To do this first a crude genomic mask was generated that contained all loci harbouring 6 or more consecutive A bases as well as any 10 nucleotide windows containing more than 6 A bases. For genes expressed from the reverse strand an analogous T rich mask was generated. Those crude masks were then corrected to allow for detection of genuine PAS falling in A/T rich regions by strand specifically unmasking 20 nucleotide intervals centred at annotated 3’ gene ends. 3’ mRNA seq reads falling into those refined strand specific masks were then removed and the filtered reads reduced to the most distal nucleotide 3’ end nucleotide. Genome build: GRCz10/danRer10 Supplementary files format and content: bigWig files: genome coverage files mean from replicates single embryo head 19hpf   Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq 3’mRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. Zebrafish Danio rerio were raised according to standard protocols 28°C water temperature 14/10 hr light/dark cycle. TLAB fish generated by crossing zebrafish AB and the natural variant TL Tupfel Long fin stocks served as wild type zebrafish for all experiments. zPCF11 null and zPCF11 ∆PAS1 mutant zebrafish were generated by Cas9 mediated mutagenesis Gagnon et al. 2014 molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11null +/ GSM3536455 GSM3536455: 3 primemRNAseq zPCF11null het e3; Danio rerio; RNA Seq GSM3536455   1 Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq three primemRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. GEO Accession:GSM3536455 RNA-Seq TRANSCRIPTOMIC cDNA SINGLE ILLUMINA Illumina HiSeq 2500   SRP175015     bam4__sel_ACCTACATC.bam.fastq_trimmed.fastq.gz fastq 186888249.0 2140393.0 GSM3536455 r1 0:87.31 A:65786002;C:32923065;G:36796601;T:51382007;N:574 87       65786002 32923065 36796601 51382007 574 SRX5193211 SRS4199211 SRA828489 GEO Sir William Dunn School of Pathology, University of Oxford 1 0.80995   0.19928   0.8033   0.5594   89   B   usable mapping rate illumina hiseq_era 3prime poly_a lexogen bulk unknown unknown   United Kingdom 2019-01-02 Multi-stage Embryo Head Nervous System
50996 50996 SRR8383273 SRX5193211 SRS4199211 SRP175015 PRJNA512489 Selective roles of vertebrate PCF11 in premature and full length transcript termination zebrafish three prime mRNA seq GSE124555 Other The pervasive nature of RNA polymerase II Pol II transcription requires efficient termination. A key player in this process is the cleavage and polyadenylation CPA factor PCF11 which directly binds to the Pol II C terminal domain and dismantles elongating Pol II from DNA in vitro. We demonstrate that PCF11 mediated termination is essential for vertebrate development. A range of genomic analyses including: mNET seq three prime mRNA seq chromatin RNA seq and ChIP seq reveals that PCF11 enhances transcription termination and stimulates early polyadenylation genome wide. PCF11 binds preferentially between closely spaced genes where it prevents transcriptional interference and downstream gene silencing. Notably PCF11 is sub stoichiometric to the CPA complex. Low levels of PCF11 are maintained by an auto regulatory mechanism involving premature termination of its own transcript and are important for normal development. Both in human cell culture and during zebrafish development PCF11 selectively attenuates the expression of other transcriptional regulators by premature CPA and termination. Overall design: three prime mRNA seq in individual zebrafish embryo heads. Two types of mutants: zPCF11 null and zPCF11 with deletion of PAS1. Wild type wt +/+ heterozygous het +/ and homozygous mutant hom / embryos were analyzed. Wild type and heterozygous animals were phenotypically indistinguishable. parent bioproject:PRJNA507553 pubmed:30819644   3 primemRNAseq zPCF11null het e3 GSM3536455   tissue:single embryo head 19hpf|molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11null +/ 3 primemRNAseq zPCF11null het e3 3’ mRNA seq data were mapped according to the guidelines of QuantSeq library kit manufacturer Lexogen. Unaligned bam files from HiSeq2500 were converted to FASTQ files with bam2fastx TopHat 2 component Kim et al. 2013. post overview with FastQC reads were trimmed with BBtools sourceforge.net/projects/bbmap/ script bbduk using following settings: k=13 ktrim=r useshortkmers=t mink=5 qtrim=r trimq=10 minlength=20 . post trimming control with FastQC curated reads were mapped with STAR2.5b Dobin et al. 2013 aligner. STAR index was generated with the GRCz10/danRer10 was used as the reference zebrafish genome together with the corresponding ENSEMBL annotation. Aligned 3’ mRNA seq reads were filtered to remove false positives due to internal priming of the QuantSeq assay on genome encoded polyA stretches. To do this first a crude genomic mask was generated that contained all loci harbouring 6 or more consecutive A bases as well as any 10 nucleotide windows containing more than 6 A bases. For genes expressed from the reverse strand an analogous T rich mask was generated. Those crude masks were then corrected to allow for detection of genuine PAS falling in A/T rich regions by strand specifically unmasking 20 nucleotide intervals centred at annotated 3’ gene ends. 3’ mRNA seq reads falling into those refined strand specific masks were then removed and the filtered reads reduced to the most distal nucleotide 3’ end nucleotide. Genome build: GRCz10/danRer10 Supplementary files format and content: bigWig files: genome coverage files mean from replicates single embryo head 19hpf   Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq 3’mRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. Zebrafish Danio rerio were raised according to standard protocols 28°C water temperature 14/10 hr light/dark cycle. TLAB fish generated by crossing zebrafish AB and the natural variant TL Tupfel Long fin stocks served as wild type zebrafish for all experiments. zPCF11 null and zPCF11 ∆PAS1 mutant zebrafish were generated by Cas9 mediated mutagenesis Gagnon et al. 2014 molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11null +/ GSM3536455 GSM3536455: 3 primemRNAseq zPCF11null het e3; Danio rerio; RNA Seq GSM3536455   1 Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq three primemRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. GEO Accession:GSM3536455 RNA-Seq TRANSCRIPTOMIC cDNA SINGLE ILLUMINA Illumina HiSeq 2500   SRP175015     bam5__sel_ACCTACATC.bam.fastq_trimmed.fastq.gz fastq 192642130.0 2206994.0 GSM3536455 r2 0:87.29 A:67912529;C:33877053;G:37899068;T:52953002;N:478 87       67912529 33877053 37899068 52953002 478 SRX5193211 SRS4199211 SRA828489 GEO Sir William Dunn School of Pathology, University of Oxford 1 0.80759   0.19797   0.80348   0.55867   89   B   usable mapping rate illumina hiseq_era 3prime poly_a lexogen bulk unknown unknown   United Kingdom 2019-01-02 Multi-stage Embryo Head Nervous System
50997 50997 SRR8383270 SRX5193210 SRS4199210 SRP175015 PRJNA512489 Selective roles of vertebrate PCF11 in premature and full length transcript termination zebrafish three prime mRNA seq GSE124555 Other The pervasive nature of RNA polymerase II Pol II transcription requires efficient termination. A key player in this process is the cleavage and polyadenylation CPA factor PCF11 which directly binds to the Pol II C terminal domain and dismantles elongating Pol II from DNA in vitro. We demonstrate that PCF11 mediated termination is essential for vertebrate development. A range of genomic analyses including: mNET seq three prime mRNA seq chromatin RNA seq and ChIP seq reveals that PCF11 enhances transcription termination and stimulates early polyadenylation genome wide. PCF11 binds preferentially between closely spaced genes where it prevents transcriptional interference and downstream gene silencing. Notably PCF11 is sub stoichiometric to the CPA complex. Low levels of PCF11 are maintained by an auto regulatory mechanism involving premature termination of its own transcript and are important for normal development. Both in human cell culture and during zebrafish development PCF11 selectively attenuates the expression of other transcriptional regulators by premature CPA and termination. Overall design: three prime mRNA seq in individual zebrafish embryo heads. Two types of mutants: zPCF11 null and zPCF11 with deletion of PAS1. Wild type wt +/+ heterozygous het +/ and homozygous mutant hom / embryos were analyzed. Wild type and heterozygous animals were phenotypically indistinguishable. parent bioproject:PRJNA507553 pubmed:30819644   3 primemRNAseq zPCF11null het e2 GSM3536454   tissue:single embryo head 19hpf|molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11null +/ 3 primemRNAseq zPCF11null het e2 3’ mRNA seq data were mapped according to the guidelines of QuantSeq library kit manufacturer Lexogen. Unaligned bam files from HiSeq2500 were converted to FASTQ files with bam2fastx TopHat 2 component Kim et al. 2013. post overview with FastQC reads were trimmed with BBtools sourceforge.net/projects/bbmap/ script bbduk using following settings: k=13 ktrim=r useshortkmers=t mink=5 qtrim=r trimq=10 minlength=20 . post trimming control with FastQC curated reads were mapped with STAR2.5b Dobin et al. 2013 aligner. STAR index was generated with the GRCz10/danRer10 was used as the reference zebrafish genome together with the corresponding ENSEMBL annotation. Aligned 3’ mRNA seq reads were filtered to remove false positives due to internal priming of the QuantSeq assay on genome encoded polyA stretches. To do this first a crude genomic mask was generated that contained all loci harbouring 6 or more consecutive A bases as well as any 10 nucleotide windows containing more than 6 A bases. For genes expressed from the reverse strand an analogous T rich mask was generated. Those crude masks were then corrected to allow for detection of genuine PAS falling in A/T rich regions by strand specifically unmasking 20 nucleotide intervals centred at annotated 3’ gene ends. 3’ mRNA seq reads falling into those refined strand specific masks were then removed and the filtered reads reduced to the most distal nucleotide 3’ end nucleotide. Genome build: GRCz10/danRer10 Supplementary files format and content: bigWig files: genome coverage files mean from replicates single embryo head 19hpf   Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq 3’mRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. Zebrafish Danio rerio were raised according to standard protocols 28°C water temperature 14/10 hr light/dark cycle. TLAB fish generated by crossing zebrafish AB and the natural variant TL Tupfel Long fin stocks served as wild type zebrafish for all experiments. zPCF11 null and zPCF11 ∆PAS1 mutant zebrafish were generated by Cas9 mediated mutagenesis Gagnon et al. 2014 molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11null +/ GSM3536454 GSM3536454: 3 primemRNAseq zPCF11null het e2; Danio rerio; RNA Seq GSM3536454   1 Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq three primemRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. GEO Accession:GSM3536454 RNA-Seq TRANSCRIPTOMIC cDNA SINGLE ILLUMINA Illumina HiSeq 2500   SRP175015     bam4__sel_TGGATTATC.bam.fastq_trimmed.fastq.gz fastq 198506304.0 2271548.0 GSM3536454 r1 0:87.39 A:65363370;C:36248554;G:40646664;T:56247077;N:639 87       65363370 36248554 40646664 56247077 639 SRX5193210 SRS4199210 SRA828489 GEO Sir William Dunn School of Pathology, University of Oxford 1 0.81892   0.1808   0.81558   0.55265   89   B   usable mapping rate illumina hiseq_era 3prime poly_a lexogen bulk unknown unknown   United Kingdom 2019-01-02 Multi-stage Embryo Head Nervous System
50998 50998 SRR8383271 SRX5193210 SRS4199210 SRP175015 PRJNA512489 Selective roles of vertebrate PCF11 in premature and full length transcript termination zebrafish three prime mRNA seq GSE124555 Other The pervasive nature of RNA polymerase II Pol II transcription requires efficient termination. A key player in this process is the cleavage and polyadenylation CPA factor PCF11 which directly binds to the Pol II C terminal domain and dismantles elongating Pol II from DNA in vitro. We demonstrate that PCF11 mediated termination is essential for vertebrate development. A range of genomic analyses including: mNET seq three prime mRNA seq chromatin RNA seq and ChIP seq reveals that PCF11 enhances transcription termination and stimulates early polyadenylation genome wide. PCF11 binds preferentially between closely spaced genes where it prevents transcriptional interference and downstream gene silencing. Notably PCF11 is sub stoichiometric to the CPA complex. Low levels of PCF11 are maintained by an auto regulatory mechanism involving premature termination of its own transcript and are important for normal development. Both in human cell culture and during zebrafish development PCF11 selectively attenuates the expression of other transcriptional regulators by premature CPA and termination. Overall design: three prime mRNA seq in individual zebrafish embryo heads. Two types of mutants: zPCF11 null and zPCF11 with deletion of PAS1. Wild type wt +/+ heterozygous het +/ and homozygous mutant hom / embryos were analyzed. Wild type and heterozygous animals were phenotypically indistinguishable. parent bioproject:PRJNA507553 pubmed:30819644   3 primemRNAseq zPCF11null het e2 GSM3536454   tissue:single embryo head 19hpf|molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11null +/ 3 primemRNAseq zPCF11null het e2 3’ mRNA seq data were mapped according to the guidelines of QuantSeq library kit manufacturer Lexogen. Unaligned bam files from HiSeq2500 were converted to FASTQ files with bam2fastx TopHat 2 component Kim et al. 2013. post overview with FastQC reads were trimmed with BBtools sourceforge.net/projects/bbmap/ script bbduk using following settings: k=13 ktrim=r useshortkmers=t mink=5 qtrim=r trimq=10 minlength=20 . post trimming control with FastQC curated reads were mapped with STAR2.5b Dobin et al. 2013 aligner. STAR index was generated with the GRCz10/danRer10 was used as the reference zebrafish genome together with the corresponding ENSEMBL annotation. Aligned 3’ mRNA seq reads were filtered to remove false positives due to internal priming of the QuantSeq assay on genome encoded polyA stretches. To do this first a crude genomic mask was generated that contained all loci harbouring 6 or more consecutive A bases as well as any 10 nucleotide windows containing more than 6 A bases. For genes expressed from the reverse strand an analogous T rich mask was generated. Those crude masks were then corrected to allow for detection of genuine PAS falling in A/T rich regions by strand specifically unmasking 20 nucleotide intervals centred at annotated 3’ gene ends. 3’ mRNA seq reads falling into those refined strand specific masks were then removed and the filtered reads reduced to the most distal nucleotide 3’ end nucleotide. Genome build: GRCz10/danRer10 Supplementary files format and content: bigWig files: genome coverage files mean from replicates single embryo head 19hpf   Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq 3’mRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. Zebrafish Danio rerio were raised according to standard protocols 28°C water temperature 14/10 hr light/dark cycle. TLAB fish generated by crossing zebrafish AB and the natural variant TL Tupfel Long fin stocks served as wild type zebrafish for all experiments. zPCF11 null and zPCF11 ∆PAS1 mutant zebrafish were generated by Cas9 mediated mutagenesis Gagnon et al. 2014 molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11null +/ GSM3536454 GSM3536454: 3 primemRNAseq zPCF11null het e2; Danio rerio; RNA Seq GSM3536454   1 Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq three primemRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. GEO Accession:GSM3536454 RNA-Seq TRANSCRIPTOMIC cDNA SINGLE ILLUMINA Illumina HiSeq 2500   SRP175015     bam5__sel_TGGATTATC.bam.fastq_trimmed.fastq.gz fastq 204546039.0 2341528.0 GSM3536454 r2 0:87.36 A:67442570;C:37300418;G:41843482;T:57959051;N:518 87       67442570 37300418 41843482 57959051 518 SRX5193210 SRS4199210 SRA828489 GEO Sir William Dunn School of Pathology, University of Oxford 1 0.81597   0.18014   0.81714   0.55071   89   B   usable mapping rate illumina hiseq_era 3prime poly_a lexogen bulk unknown unknown   United Kingdom 2019-01-02 Multi-stage Embryo Head Nervous System
50999 50999 SRR8383268 SRX5193209 SRS4199209 SRP175015 PRJNA512489 Selective roles of vertebrate PCF11 in premature and full length transcript termination zebrafish three prime mRNA seq GSE124555 Other The pervasive nature of RNA polymerase II Pol II transcription requires efficient termination. A key player in this process is the cleavage and polyadenylation CPA factor PCF11 which directly binds to the Pol II C terminal domain and dismantles elongating Pol II from DNA in vitro. We demonstrate that PCF11 mediated termination is essential for vertebrate development. A range of genomic analyses including: mNET seq three prime mRNA seq chromatin RNA seq and ChIP seq reveals that PCF11 enhances transcription termination and stimulates early polyadenylation genome wide. PCF11 binds preferentially between closely spaced genes where it prevents transcriptional interference and downstream gene silencing. Notably PCF11 is sub stoichiometric to the CPA complex. Low levels of PCF11 are maintained by an auto regulatory mechanism involving premature termination of its own transcript and are important for normal development. Both in human cell culture and during zebrafish development PCF11 selectively attenuates the expression of other transcriptional regulators by premature CPA and termination. Overall design: three prime mRNA seq in individual zebrafish embryo heads. Two types of mutants: zPCF11 null and zPCF11 with deletion of PAS1. Wild type wt +/+ heterozygous het +/ and homozygous mutant hom / embryos were analyzed. Wild type and heterozygous animals were phenotypically indistinguishable. parent bioproject:PRJNA507553 pubmed:30819644   3 primemRNAseq zPCF11null het e1 GSM3536453   tissue:single embryo head 19hpf|molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11null +/ 3 primemRNAseq zPCF11null het e1 3’ mRNA seq data were mapped according to the guidelines of QuantSeq library kit manufacturer Lexogen. Unaligned bam files from HiSeq2500 were converted to FASTQ files with bam2fastx TopHat 2 component Kim et al. 2013. post overview with FastQC reads were trimmed with BBtools sourceforge.net/projects/bbmap/ script bbduk using following settings: k=13 ktrim=r useshortkmers=t mink=5 qtrim=r trimq=10 minlength=20 . post trimming control with FastQC curated reads were mapped with STAR2.5b Dobin et al. 2013 aligner. STAR index was generated with the GRCz10/danRer10 was used as the reference zebrafish genome together with the corresponding ENSEMBL annotation. Aligned 3’ mRNA seq reads were filtered to remove false positives due to internal priming of the QuantSeq assay on genome encoded polyA stretches. To do this first a crude genomic mask was generated that contained all loci harbouring 6 or more consecutive A bases as well as any 10 nucleotide windows containing more than 6 A bases. For genes expressed from the reverse strand an analogous T rich mask was generated. Those crude masks were then corrected to allow for detection of genuine PAS falling in A/T rich regions by strand specifically unmasking 20 nucleotide intervals centred at annotated 3’ gene ends. 3’ mRNA seq reads falling into those refined strand specific masks were then removed and the filtered reads reduced to the most distal nucleotide 3’ end nucleotide. Genome build: GRCz10/danRer10 Supplementary files format and content: bigWig files: genome coverage files mean from replicates single embryo head 19hpf   Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq 3’mRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. Zebrafish Danio rerio were raised according to standard protocols 28°C water temperature 14/10 hr light/dark cycle. TLAB fish generated by crossing zebrafish AB and the natural variant TL Tupfel Long fin stocks served as wild type zebrafish for all experiments. zPCF11 null and zPCF11 ∆PAS1 mutant zebrafish were generated by Cas9 mediated mutagenesis Gagnon et al. 2014 molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11null +/ GSM3536453 GSM3536453: 3 primemRNAseq zPCF11null het e1; Danio rerio; RNA Seq GSM3536453   1 Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq three primemRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. GEO Accession:GSM3536453 RNA-Seq TRANSCRIPTOMIC cDNA SINGLE ILLUMINA Illumina HiSeq 2500   SRP175015     bam4__sel_GCGAATATC.bam.fastq_trimmed.fastq.gz fastq 166454199.0 1896918.0 GSM3536453 r1 0:87.75 A:54362704;C:30210406;G:33769933;T:48110651;N:505 87       54362704 30210406 33769933 48110651 505 SRX5193209 SRS4199209 SRA828489 GEO Sir William Dunn School of Pathology, University of Oxford 1 0.83471   0.17434   0.80231   0.55417   89   B   usable mapping rate illumina hiseq_era 3prime poly_a lexogen bulk unknown unknown   United Kingdom 2019-01-02 Multi-stage Embryo Head Nervous System
51000 51000 SRR8383269 SRX5193209 SRS4199209 SRP175015 PRJNA512489 Selective roles of vertebrate PCF11 in premature and full length transcript termination zebrafish three prime mRNA seq GSE124555 Other The pervasive nature of RNA polymerase II Pol II transcription requires efficient termination. A key player in this process is the cleavage and polyadenylation CPA factor PCF11 which directly binds to the Pol II C terminal domain and dismantles elongating Pol II from DNA in vitro. We demonstrate that PCF11 mediated termination is essential for vertebrate development. A range of genomic analyses including: mNET seq three prime mRNA seq chromatin RNA seq and ChIP seq reveals that PCF11 enhances transcription termination and stimulates early polyadenylation genome wide. PCF11 binds preferentially between closely spaced genes where it prevents transcriptional interference and downstream gene silencing. Notably PCF11 is sub stoichiometric to the CPA complex. Low levels of PCF11 are maintained by an auto regulatory mechanism involving premature termination of its own transcript and are important for normal development. Both in human cell culture and during zebrafish development PCF11 selectively attenuates the expression of other transcriptional regulators by premature CPA and termination. Overall design: three prime mRNA seq in individual zebrafish embryo heads. Two types of mutants: zPCF11 null and zPCF11 with deletion of PAS1. Wild type wt +/+ heterozygous het +/ and homozygous mutant hom / embryos were analyzed. Wild type and heterozygous animals were phenotypically indistinguishable. parent bioproject:PRJNA507553 pubmed:30819644   3 primemRNAseq zPCF11null het e1 GSM3536453   tissue:single embryo head 19hpf|molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11null +/ 3 primemRNAseq zPCF11null het e1 3’ mRNA seq data were mapped according to the guidelines of QuantSeq library kit manufacturer Lexogen. Unaligned bam files from HiSeq2500 were converted to FASTQ files with bam2fastx TopHat 2 component Kim et al. 2013. post overview with FastQC reads were trimmed with BBtools sourceforge.net/projects/bbmap/ script bbduk using following settings: k=13 ktrim=r useshortkmers=t mink=5 qtrim=r trimq=10 minlength=20 . post trimming control with FastQC curated reads were mapped with STAR2.5b Dobin et al. 2013 aligner. STAR index was generated with the GRCz10/danRer10 was used as the reference zebrafish genome together with the corresponding ENSEMBL annotation. Aligned 3’ mRNA seq reads were filtered to remove false positives due to internal priming of the QuantSeq assay on genome encoded polyA stretches. To do this first a crude genomic mask was generated that contained all loci harbouring 6 or more consecutive A bases as well as any 10 nucleotide windows containing more than 6 A bases. For genes expressed from the reverse strand an analogous T rich mask was generated. Those crude masks were then corrected to allow for detection of genuine PAS falling in A/T rich regions by strand specifically unmasking 20 nucleotide intervals centred at annotated 3’ gene ends. 3’ mRNA seq reads falling into those refined strand specific masks were then removed and the filtered reads reduced to the most distal nucleotide 3’ end nucleotide. Genome build: GRCz10/danRer10 Supplementary files format and content: bigWig files: genome coverage files mean from replicates single embryo head 19hpf   Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq 3’mRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. Zebrafish Danio rerio were raised according to standard protocols 28°C water temperature 14/10 hr light/dark cycle. TLAB fish generated by crossing zebrafish AB and the natural variant TL Tupfel Long fin stocks served as wild type zebrafish for all experiments. zPCF11 null and zPCF11 ∆PAS1 mutant zebrafish were generated by Cas9 mediated mutagenesis Gagnon et al. 2014 molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11null +/ GSM3536453 GSM3536453: 3 primemRNAseq zPCF11null het e1; Danio rerio; RNA Seq GSM3536453   1 Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq three primemRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. GEO Accession:GSM3536453 RNA-Seq TRANSCRIPTOMIC cDNA SINGLE ILLUMINA Illumina HiSeq 2500   SRP175015     bam5__sel_GCGAATATC.bam.fastq_trimmed.fastq.gz fastq 171370619.0 1953554.0 GSM3536453 r2 0:87.72 A:56037972;C:31055338;G:34710723;T:49566184;N:402 87       56037972 31055338 34710723 49566184 402 SRX5193209 SRS4199209 SRA828489 GEO Sir William Dunn School of Pathology, University of Oxford 1 0.83503   0.17275   0.80389   0.54802   89   B   usable mapping rate illumina hiseq_era 3prime poly_a lexogen bulk unknown unknown   United Kingdom 2019-01-02 Multi-stage Embryo Head Nervous System
51001 51001 SRR8383266 SRX5193208 SRS4199208 SRP175015 PRJNA512489 Selective roles of vertebrate PCF11 in premature and full length transcript termination zebrafish three prime mRNA seq GSE124555 Other The pervasive nature of RNA polymerase II Pol II transcription requires efficient termination. A key player in this process is the cleavage and polyadenylation CPA factor PCF11 which directly binds to the Pol II C terminal domain and dismantles elongating Pol II from DNA in vitro. We demonstrate that PCF11 mediated termination is essential for vertebrate development. A range of genomic analyses including: mNET seq three prime mRNA seq chromatin RNA seq and ChIP seq reveals that PCF11 enhances transcription termination and stimulates early polyadenylation genome wide. PCF11 binds preferentially between closely spaced genes where it prevents transcriptional interference and downstream gene silencing. Notably PCF11 is sub stoichiometric to the CPA complex. Low levels of PCF11 are maintained by an auto regulatory mechanism involving premature termination of its own transcript and are important for normal development. Both in human cell culture and during zebrafish development PCF11 selectively attenuates the expression of other transcriptional regulators by premature CPA and termination. Overall design: three prime mRNA seq in individual zebrafish embryo heads. Two types of mutants: zPCF11 null and zPCF11 with deletion of PAS1. Wild type wt +/+ heterozygous het +/ and homozygous mutant hom / embryos were analyzed. Wild type and heterozygous animals were phenotypically indistinguishable. parent bioproject:PRJNA507553 pubmed:30819644   3 primemRNAseq zPCF11null wt e4 GSM3536452   tissue:single embryo head 19hpf|molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11null +/+ 3 primemRNAseq zPCF11null wt e4 3’ mRNA seq data were mapped according to the guidelines of QuantSeq library kit manufacturer Lexogen. Unaligned bam files from HiSeq2500 were converted to FASTQ files with bam2fastx TopHat 2 component Kim et al. 2013. post overview with FastQC reads were trimmed with BBtools sourceforge.net/projects/bbmap/ script bbduk using following settings: k=13 ktrim=r useshortkmers=t mink=5 qtrim=r trimq=10 minlength=20 . post trimming control with FastQC curated reads were mapped with STAR2.5b Dobin et al. 2013 aligner. STAR index was generated with the GRCz10/danRer10 was used as the reference zebrafish genome together with the corresponding ENSEMBL annotation. Aligned 3’ mRNA seq reads were filtered to remove false positives due to internal priming of the QuantSeq assay on genome encoded polyA stretches. To do this first a crude genomic mask was generated that contained all loci harbouring 6 or more consecutive A bases as well as any 10 nucleotide windows containing more than 6 A bases. For genes expressed from the reverse strand an analogous T rich mask was generated. Those crude masks were then corrected to allow for detection of genuine PAS falling in A/T rich regions by strand specifically unmasking 20 nucleotide intervals centred at annotated 3’ gene ends. 3’ mRNA seq reads falling into those refined strand specific masks were then removed and the filtered reads reduced to the most distal nucleotide 3’ end nucleotide. Genome build: GRCz10/danRer10 Supplementary files format and content: bigWig files: genome coverage files mean from replicates single embryo head 19hpf   Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq 3’mRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. Zebrafish Danio rerio were raised according to standard protocols 28°C water temperature 14/10 hr light/dark cycle. TLAB fish generated by crossing zebrafish AB and the natural variant TL Tupfel Long fin stocks served as wild type zebrafish for all experiments. zPCF11 null and zPCF11 ∆PAS1 mutant zebrafish were generated by Cas9 mediated mutagenesis Gagnon et al. 2014 molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11null +/+ GSM3536452 GSM3536452: 3 primemRNAseq zPCF11null wt e4; Danio rerio; RNA Seq GSM3536452   1 Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq three primemRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. GEO Accession:GSM3536452 RNA-Seq TRANSCRIPTOMIC cDNA SINGLE ILLUMINA Illumina HiSeq 2500   SRP175015     bam4__sel_CGCAACATC.bam.fastq_trimmed.fastq.gz fastq 174611360.0 2002483.0 GSM3536452 r1 0:87.20 A:56929522;C:32074265;G:35774482;T:49832562;N:529 87       56929522 32074265 35774482 49832562 529 SRX5193208 SRS4199208 SRA828489 GEO Sir William Dunn School of Pathology, University of Oxford 1 0.83372   0.18461   0.80811   0.54823   52   B   usable mapping rate illumina hiseq_era 3prime poly_a lexogen bulk unknown unknown   United Kingdom 2019-01-02 Multi-stage Embryo Head Nervous System
51002 51002 SRR8383267 SRX5193208 SRS4199208 SRP175015 PRJNA512489 Selective roles of vertebrate PCF11 in premature and full length transcript termination zebrafish three prime mRNA seq GSE124555 Other The pervasive nature of RNA polymerase II Pol II transcription requires efficient termination. A key player in this process is the cleavage and polyadenylation CPA factor PCF11 which directly binds to the Pol II C terminal domain and dismantles elongating Pol II from DNA in vitro. We demonstrate that PCF11 mediated termination is essential for vertebrate development. A range of genomic analyses including: mNET seq three prime mRNA seq chromatin RNA seq and ChIP seq reveals that PCF11 enhances transcription termination and stimulates early polyadenylation genome wide. PCF11 binds preferentially between closely spaced genes where it prevents transcriptional interference and downstream gene silencing. Notably PCF11 is sub stoichiometric to the CPA complex. Low levels of PCF11 are maintained by an auto regulatory mechanism involving premature termination of its own transcript and are important for normal development. Both in human cell culture and during zebrafish development PCF11 selectively attenuates the expression of other transcriptional regulators by premature CPA and termination. Overall design: three prime mRNA seq in individual zebrafish embryo heads. Two types of mutants: zPCF11 null and zPCF11 with deletion of PAS1. Wild type wt +/+ heterozygous het +/ and homozygous mutant hom / embryos were analyzed. Wild type and heterozygous animals were phenotypically indistinguishable. parent bioproject:PRJNA507553 pubmed:30819644   3 primemRNAseq zPCF11null wt e4 GSM3536452   tissue:single embryo head 19hpf|molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11null +/+ 3 primemRNAseq zPCF11null wt e4 3’ mRNA seq data were mapped according to the guidelines of QuantSeq library kit manufacturer Lexogen. Unaligned bam files from HiSeq2500 were converted to FASTQ files with bam2fastx TopHat 2 component Kim et al. 2013. post overview with FastQC reads were trimmed with BBtools sourceforge.net/projects/bbmap/ script bbduk using following settings: k=13 ktrim=r useshortkmers=t mink=5 qtrim=r trimq=10 minlength=20 . post trimming control with FastQC curated reads were mapped with STAR2.5b Dobin et al. 2013 aligner. STAR index was generated with the GRCz10/danRer10 was used as the reference zebrafish genome together with the corresponding ENSEMBL annotation. Aligned 3’ mRNA seq reads were filtered to remove false positives due to internal priming of the QuantSeq assay on genome encoded polyA stretches. To do this first a crude genomic mask was generated that contained all loci harbouring 6 or more consecutive A bases as well as any 10 nucleotide windows containing more than 6 A bases. For genes expressed from the reverse strand an analogous T rich mask was generated. Those crude masks were then corrected to allow for detection of genuine PAS falling in A/T rich regions by strand specifically unmasking 20 nucleotide intervals centred at annotated 3’ gene ends. 3’ mRNA seq reads falling into those refined strand specific masks were then removed and the filtered reads reduced to the most distal nucleotide 3’ end nucleotide. Genome build: GRCz10/danRer10 Supplementary files format and content: bigWig files: genome coverage files mean from replicates single embryo head 19hpf   Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq 3’mRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. Zebrafish Danio rerio were raised according to standard protocols 28°C water temperature 14/10 hr light/dark cycle. TLAB fish generated by crossing zebrafish AB and the natural variant TL Tupfel Long fin stocks served as wild type zebrafish for all experiments. zPCF11 null and zPCF11 ∆PAS1 mutant zebrafish were generated by Cas9 mediated mutagenesis Gagnon et al. 2014 molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11null +/+ GSM3536452 GSM3536452: 3 primemRNAseq zPCF11null wt e4; Danio rerio; RNA Seq GSM3536452   1 Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq three primemRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. GEO Accession:GSM3536452 RNA-Seq TRANSCRIPTOMIC cDNA SINGLE ILLUMINA Illumina HiSeq 2500   SRP175015     bam5__sel_CGCAACATC.bam.fastq_trimmed.fastq.gz fastq 179927114.0 2064019.0 GSM3536452 r2 0:87.17 A:58698795;C:32998345;G:36824253;T:51405334;N:387 87       58698795 32998345 36824253 51405334 387 SRX5193208 SRS4199208 SRA828489 GEO Sir William Dunn School of Pathology, University of Oxford 1 0.83291   0.18273   0.80898   0.54951   89   B   usable mapping rate illumina hiseq_era 3prime poly_a lexogen bulk unknown unknown   United Kingdom 2019-01-02 Multi-stage Embryo Head Nervous System
51003 51003 SRR8383264 SRX5193207 SRS4199207 SRP175015 PRJNA512489 Selective roles of vertebrate PCF11 in premature and full length transcript termination zebrafish three prime mRNA seq GSE124555 Other The pervasive nature of RNA polymerase II Pol II transcription requires efficient termination. A key player in this process is the cleavage and polyadenylation CPA factor PCF11 which directly binds to the Pol II C terminal domain and dismantles elongating Pol II from DNA in vitro. We demonstrate that PCF11 mediated termination is essential for vertebrate development. A range of genomic analyses including: mNET seq three prime mRNA seq chromatin RNA seq and ChIP seq reveals that PCF11 enhances transcription termination and stimulates early polyadenylation genome wide. PCF11 binds preferentially between closely spaced genes where it prevents transcriptional interference and downstream gene silencing. Notably PCF11 is sub stoichiometric to the CPA complex. Low levels of PCF11 are maintained by an auto regulatory mechanism involving premature termination of its own transcript and are important for normal development. Both in human cell culture and during zebrafish development PCF11 selectively attenuates the expression of other transcriptional regulators by premature CPA and termination. Overall design: three prime mRNA seq in individual zebrafish embryo heads. Two types of mutants: zPCF11 null and zPCF11 with deletion of PAS1. Wild type wt +/+ heterozygous het +/ and homozygous mutant hom / embryos were analyzed. Wild type and heterozygous animals were phenotypically indistinguishable. parent bioproject:PRJNA507553 pubmed:30819644   3 primemRNAseq zPCF11null wt e3 GSM3536451   tissue:single embryo head 19hpf|molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11null +/+ 3 primemRNAseq zPCF11null wt e3 3’ mRNA seq data were mapped according to the guidelines of QuantSeq library kit manufacturer Lexogen. Unaligned bam files from HiSeq2500 were converted to FASTQ files with bam2fastx TopHat 2 component Kim et al. 2013. post overview with FastQC reads were trimmed with BBtools sourceforge.net/projects/bbmap/ script bbduk using following settings: k=13 ktrim=r useshortkmers=t mink=5 qtrim=r trimq=10 minlength=20 . post trimming control with FastQC curated reads were mapped with STAR2.5b Dobin et al. 2013 aligner. STAR index was generated with the GRCz10/danRer10 was used as the reference zebrafish genome together with the corresponding ENSEMBL annotation. Aligned 3’ mRNA seq reads were filtered to remove false positives due to internal priming of the QuantSeq assay on genome encoded polyA stretches. To do this first a crude genomic mask was generated that contained all loci harbouring 6 or more consecutive A bases as well as any 10 nucleotide windows containing more than 6 A bases. For genes expressed from the reverse strand an analogous T rich mask was generated. Those crude masks were then corrected to allow for detection of genuine PAS falling in A/T rich regions by strand specifically unmasking 20 nucleotide intervals centred at annotated 3’ gene ends. 3’ mRNA seq reads falling into those refined strand specific masks were then removed and the filtered reads reduced to the most distal nucleotide 3’ end nucleotide. Genome build: GRCz10/danRer10 Supplementary files format and content: bigWig files: genome coverage files mean from replicates single embryo head 19hpf   Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq 3’mRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. Zebrafish Danio rerio were raised according to standard protocols 28°C water temperature 14/10 hr light/dark cycle. TLAB fish generated by crossing zebrafish AB and the natural variant TL Tupfel Long fin stocks served as wild type zebrafish for all experiments. zPCF11 null and zPCF11 ∆PAS1 mutant zebrafish were generated by Cas9 mediated mutagenesis Gagnon et al. 2014 molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11null +/+ GSM3536451 GSM3536451: 3 primemRNAseq zPCF11null wt e3; Danio rerio; RNA Seq GSM3536451   1 Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq three primemRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. GEO Accession:GSM3536451 RNA-Seq TRANSCRIPTOMIC cDNA SINGLE ILLUMINA Illumina HiSeq 2500   SRP175015     bam4__sel_GTTACCATC.bam.fastq_trimmed.fastq.gz fastq 423907322.0 4857964.0 GSM3536451 r1 0:87.26 A:142119526;C:76370356;G:86122715;T:119293410;N:1315 87       142119526 76370356 86122715 119293410 1315 SRX5193207 SRS4199207 SRA828489 GEO Sir William Dunn School of Pathology, University of Oxford 1 0.82515   0.17866   0.80598   0.5424   89   B   usable mapping rate illumina hiseq_era 3prime poly_a lexogen bulk unknown unknown   United Kingdom 2019-01-02 Multi-stage Embryo Head Nervous System
51004 51004 SRR8383265 SRX5193207 SRS4199207 SRP175015 PRJNA512489 Selective roles of vertebrate PCF11 in premature and full length transcript termination zebrafish three prime mRNA seq GSE124555 Other The pervasive nature of RNA polymerase II Pol II transcription requires efficient termination. A key player in this process is the cleavage and polyadenylation CPA factor PCF11 which directly binds to the Pol II C terminal domain and dismantles elongating Pol II from DNA in vitro. We demonstrate that PCF11 mediated termination is essential for vertebrate development. A range of genomic analyses including: mNET seq three prime mRNA seq chromatin RNA seq and ChIP seq reveals that PCF11 enhances transcription termination and stimulates early polyadenylation genome wide. PCF11 binds preferentially between closely spaced genes where it prevents transcriptional interference and downstream gene silencing. Notably PCF11 is sub stoichiometric to the CPA complex. Low levels of PCF11 are maintained by an auto regulatory mechanism involving premature termination of its own transcript and are important for normal development. Both in human cell culture and during zebrafish development PCF11 selectively attenuates the expression of other transcriptional regulators by premature CPA and termination. Overall design: three prime mRNA seq in individual zebrafish embryo heads. Two types of mutants: zPCF11 null and zPCF11 with deletion of PAS1. Wild type wt +/+ heterozygous het +/ and homozygous mutant hom / embryos were analyzed. Wild type and heterozygous animals were phenotypically indistinguishable. parent bioproject:PRJNA507553 pubmed:30819644   3 primemRNAseq zPCF11null wt e3 GSM3536451   tissue:single embryo head 19hpf|molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11null +/+ 3 primemRNAseq zPCF11null wt e3 3’ mRNA seq data were mapped according to the guidelines of QuantSeq library kit manufacturer Lexogen. Unaligned bam files from HiSeq2500 were converted to FASTQ files with bam2fastx TopHat 2 component Kim et al. 2013. post overview with FastQC reads were trimmed with BBtools sourceforge.net/projects/bbmap/ script bbduk using following settings: k=13 ktrim=r useshortkmers=t mink=5 qtrim=r trimq=10 minlength=20 . post trimming control with FastQC curated reads were mapped with STAR2.5b Dobin et al. 2013 aligner. STAR index was generated with the GRCz10/danRer10 was used as the reference zebrafish genome together with the corresponding ENSEMBL annotation. Aligned 3’ mRNA seq reads were filtered to remove false positives due to internal priming of the QuantSeq assay on genome encoded polyA stretches. To do this first a crude genomic mask was generated that contained all loci harbouring 6 or more consecutive A bases as well as any 10 nucleotide windows containing more than 6 A bases. For genes expressed from the reverse strand an analogous T rich mask was generated. Those crude masks were then corrected to allow for detection of genuine PAS falling in A/T rich regions by strand specifically unmasking 20 nucleotide intervals centred at annotated 3’ gene ends. 3’ mRNA seq reads falling into those refined strand specific masks were then removed and the filtered reads reduced to the most distal nucleotide 3’ end nucleotide. Genome build: GRCz10/danRer10 Supplementary files format and content: bigWig files: genome coverage files mean from replicates single embryo head 19hpf   Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq 3’mRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. Zebrafish Danio rerio were raised according to standard protocols 28°C water temperature 14/10 hr light/dark cycle. TLAB fish generated by crossing zebrafish AB and the natural variant TL Tupfel Long fin stocks served as wild type zebrafish for all experiments. zPCF11 null and zPCF11 ∆PAS1 mutant zebrafish were generated by Cas9 mediated mutagenesis Gagnon et al. 2014 molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11null +/+ GSM3536451 GSM3536451: 3 primemRNAseq zPCF11null wt e3; Danio rerio; RNA Seq GSM3536451   1 Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq three primemRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. GEO Accession:GSM3536451 RNA-Seq TRANSCRIPTOMIC cDNA SINGLE ILLUMINA Illumina HiSeq 2500   SRP175015     bam5__sel_GTTACCATC.bam.fastq_trimmed.fastq.gz fastq 436230914.0 5000909.0 GSM3536451 r2 0:87.23 A:146479519;C:78476816;G:88511770;T:122761779;N:1030 87       146479519 78476816 88511770 122761779 1030 SRX5193207 SRS4199207 SRA828489 GEO Sir William Dunn School of Pathology, University of Oxford 1 0.82369   0.17724   0.80799   0.53506   89   B   usable mapping rate illumina hiseq_era 3prime poly_a lexogen bulk unknown unknown   United Kingdom 2019-01-02 Multi-stage Embryo Head Nervous System
51005 51005 SRR8383262 SRX5193206 SRS4199206 SRP175015 PRJNA512489 Selective roles of vertebrate PCF11 in premature and full length transcript termination zebrafish three prime mRNA seq GSE124555 Other The pervasive nature of RNA polymerase II Pol II transcription requires efficient termination. A key player in this process is the cleavage and polyadenylation CPA factor PCF11 which directly binds to the Pol II C terminal domain and dismantles elongating Pol II from DNA in vitro. We demonstrate that PCF11 mediated termination is essential for vertebrate development. A range of genomic analyses including: mNET seq three prime mRNA seq chromatin RNA seq and ChIP seq reveals that PCF11 enhances transcription termination and stimulates early polyadenylation genome wide. PCF11 binds preferentially between closely spaced genes where it prevents transcriptional interference and downstream gene silencing. Notably PCF11 is sub stoichiometric to the CPA complex. Low levels of PCF11 are maintained by an auto regulatory mechanism involving premature termination of its own transcript and are important for normal development. Both in human cell culture and during zebrafish development PCF11 selectively attenuates the expression of other transcriptional regulators by premature CPA and termination. Overall design: three prime mRNA seq in individual zebrafish embryo heads. Two types of mutants: zPCF11 null and zPCF11 with deletion of PAS1. Wild type wt +/+ heterozygous het +/ and homozygous mutant hom / embryos were analyzed. Wild type and heterozygous animals were phenotypically indistinguishable. parent bioproject:PRJNA507553 pubmed:30819644   3 primemRNAseq zPCF11null wt e2 GSM3536450   tissue:single embryo head 19hpf|molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11null +/+ 3 primemRNAseq zPCF11null wt e2 3’ mRNA seq data were mapped according to the guidelines of QuantSeq library kit manufacturer Lexogen. Unaligned bam files from HiSeq2500 were converted to FASTQ files with bam2fastx TopHat 2 component Kim et al. 2013. post overview with FastQC reads were trimmed with BBtools sourceforge.net/projects/bbmap/ script bbduk using following settings: k=13 ktrim=r useshortkmers=t mink=5 qtrim=r trimq=10 minlength=20 . post trimming control with FastQC curated reads were mapped with STAR2.5b Dobin et al. 2013 aligner. STAR index was generated with the GRCz10/danRer10 was used as the reference zebrafish genome together with the corresponding ENSEMBL annotation. Aligned 3’ mRNA seq reads were filtered to remove false positives due to internal priming of the QuantSeq assay on genome encoded polyA stretches. To do this first a crude genomic mask was generated that contained all loci harbouring 6 or more consecutive A bases as well as any 10 nucleotide windows containing more than 6 A bases. For genes expressed from the reverse strand an analogous T rich mask was generated. Those crude masks were then corrected to allow for detection of genuine PAS falling in A/T rich regions by strand specifically unmasking 20 nucleotide intervals centred at annotated 3’ gene ends. 3’ mRNA seq reads falling into those refined strand specific masks were then removed and the filtered reads reduced to the most distal nucleotide 3’ end nucleotide. Genome build: GRCz10/danRer10 Supplementary files format and content: bigWig files: genome coverage files mean from replicates single embryo head 19hpf   Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq 3’mRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. Zebrafish Danio rerio were raised according to standard protocols 28°C water temperature 14/10 hr light/dark cycle. TLAB fish generated by crossing zebrafish AB and the natural variant TL Tupfel Long fin stocks served as wild type zebrafish for all experiments. zPCF11 null and zPCF11 ∆PAS1 mutant zebrafish were generated by Cas9 mediated mutagenesis Gagnon et al. 2014 molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11null +/+ GSM3536450 GSM3536450: 3 primemRNAseq zPCF11null wt e2; Danio rerio; RNA Seq GSM3536450   1 Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq three primemRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. GEO Accession:GSM3536450 RNA-Seq TRANSCRIPTOMIC cDNA SINGLE ILLUMINA Illumina HiSeq 2500   SRP175015     bam4__sel_TTGGTAATC.bam.fastq_trimmed.fastq.gz fastq 496496816.0 5671581.0 GSM3536450 r1 0:87.54 A:167742127;C:88470896;G:98690606;T:141591736;N:1451 87       167742127 88470896 98690606 141591736 1451 SRX5193206 SRS4199206 SRA828489 GEO Sir William Dunn School of Pathology, University of Oxford 1 0.81921   0.15966   0.8071   0.53472   89   B   usable mapping rate illumina hiseq_era 3prime poly_a lexogen bulk unknown unknown   United Kingdom 2019-01-02 Multi-stage Embryo Head Nervous System
51006 51006 SRR8383263 SRX5193206 SRS4199206 SRP175015 PRJNA512489 Selective roles of vertebrate PCF11 in premature and full length transcript termination zebrafish three prime mRNA seq GSE124555 Other The pervasive nature of RNA polymerase II Pol II transcription requires efficient termination. A key player in this process is the cleavage and polyadenylation CPA factor PCF11 which directly binds to the Pol II C terminal domain and dismantles elongating Pol II from DNA in vitro. We demonstrate that PCF11 mediated termination is essential for vertebrate development. A range of genomic analyses including: mNET seq three prime mRNA seq chromatin RNA seq and ChIP seq reveals that PCF11 enhances transcription termination and stimulates early polyadenylation genome wide. PCF11 binds preferentially between closely spaced genes where it prevents transcriptional interference and downstream gene silencing. Notably PCF11 is sub stoichiometric to the CPA complex. Low levels of PCF11 are maintained by an auto regulatory mechanism involving premature termination of its own transcript and are important for normal development. Both in human cell culture and during zebrafish development PCF11 selectively attenuates the expression of other transcriptional regulators by premature CPA and termination. Overall design: three prime mRNA seq in individual zebrafish embryo heads. Two types of mutants: zPCF11 null and zPCF11 with deletion of PAS1. Wild type wt +/+ heterozygous het +/ and homozygous mutant hom / embryos were analyzed. Wild type and heterozygous animals were phenotypically indistinguishable. parent bioproject:PRJNA507553 pubmed:30819644   3 primemRNAseq zPCF11null wt e2 GSM3536450   tissue:single embryo head 19hpf|molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11null +/+ 3 primemRNAseq zPCF11null wt e2 3’ mRNA seq data were mapped according to the guidelines of QuantSeq library kit manufacturer Lexogen. Unaligned bam files from HiSeq2500 were converted to FASTQ files with bam2fastx TopHat 2 component Kim et al. 2013. post overview with FastQC reads were trimmed with BBtools sourceforge.net/projects/bbmap/ script bbduk using following settings: k=13 ktrim=r useshortkmers=t mink=5 qtrim=r trimq=10 minlength=20 . post trimming control with FastQC curated reads were mapped with STAR2.5b Dobin et al. 2013 aligner. STAR index was generated with the GRCz10/danRer10 was used as the reference zebrafish genome together with the corresponding ENSEMBL annotation. Aligned 3’ mRNA seq reads were filtered to remove false positives due to internal priming of the QuantSeq assay on genome encoded polyA stretches. To do this first a crude genomic mask was generated that contained all loci harbouring 6 or more consecutive A bases as well as any 10 nucleotide windows containing more than 6 A bases. For genes expressed from the reverse strand an analogous T rich mask was generated. Those crude masks were then corrected to allow for detection of genuine PAS falling in A/T rich regions by strand specifically unmasking 20 nucleotide intervals centred at annotated 3’ gene ends. 3’ mRNA seq reads falling into those refined strand specific masks were then removed and the filtered reads reduced to the most distal nucleotide 3’ end nucleotide. Genome build: GRCz10/danRer10 Supplementary files format and content: bigWig files: genome coverage files mean from replicates single embryo head 19hpf   Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq 3’mRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. Zebrafish Danio rerio were raised according to standard protocols 28°C water temperature 14/10 hr light/dark cycle. TLAB fish generated by crossing zebrafish AB and the natural variant TL Tupfel Long fin stocks served as wild type zebrafish for all experiments. zPCF11 null and zPCF11 ∆PAS1 mutant zebrafish were generated by Cas9 mediated mutagenesis Gagnon et al. 2014 molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11null +/+ GSM3536450 GSM3536450: 3 primemRNAseq zPCF11null wt e2; Danio rerio; RNA Seq GSM3536450   1 Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq three primemRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. GEO Accession:GSM3536450 RNA-Seq TRANSCRIPTOMIC cDNA SINGLE ILLUMINA Illumina HiSeq 2500   SRP175015     bam5__sel_TTGGTAATC.bam.fastq_trimmed.fastq.gz fastq 510651572.0 5835386.0 GSM3536450 r2 0:87.51 A:172754168;C:90870653;G:101360243;T:145665418;N:1090 87       172754168 90870653 101360243 145665418 1090 SRX5193206 SRS4199206 SRA828489 GEO Sir William Dunn School of Pathology, University of Oxford 1 0.81817   0.15907   0.8071   0.54188   89   B   usable mapping rate illumina hiseq_era 3prime poly_a lexogen bulk unknown unknown   United Kingdom 2019-01-02 Multi-stage Embryo Head Nervous System
51007 51007 SRR8383260 SRX5193205 SRS4199205 SRP175015 PRJNA512489 Selective roles of vertebrate PCF11 in premature and full length transcript termination zebrafish three prime mRNA seq GSE124555 Other The pervasive nature of RNA polymerase II Pol II transcription requires efficient termination. A key player in this process is the cleavage and polyadenylation CPA factor PCF11 which directly binds to the Pol II C terminal domain and dismantles elongating Pol II from DNA in vitro. We demonstrate that PCF11 mediated termination is essential for vertebrate development. A range of genomic analyses including: mNET seq three prime mRNA seq chromatin RNA seq and ChIP seq reveals that PCF11 enhances transcription termination and stimulates early polyadenylation genome wide. PCF11 binds preferentially between closely spaced genes where it prevents transcriptional interference and downstream gene silencing. Notably PCF11 is sub stoichiometric to the CPA complex. Low levels of PCF11 are maintained by an auto regulatory mechanism involving premature termination of its own transcript and are important for normal development. Both in human cell culture and during zebrafish development PCF11 selectively attenuates the expression of other transcriptional regulators by premature CPA and termination. Overall design: three prime mRNA seq in individual zebrafish embryo heads. Two types of mutants: zPCF11 null and zPCF11 with deletion of PAS1. Wild type wt +/+ heterozygous het +/ and homozygous mutant hom / embryos were analyzed. Wild type and heterozygous animals were phenotypically indistinguishable. parent bioproject:PRJNA507553 pubmed:30819644   3 primemRNAseq zPCF11null wt e1 GSM3536449   tissue:single embryo head 19hpf|molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11null +/+ 3 primemRNAseq zPCF11null wt e1 3’ mRNA seq data were mapped according to the guidelines of QuantSeq library kit manufacturer Lexogen. Unaligned bam files from HiSeq2500 were converted to FASTQ files with bam2fastx TopHat 2 component Kim et al. 2013. post overview with FastQC reads were trimmed with BBtools sourceforge.net/projects/bbmap/ script bbduk using following settings: k=13 ktrim=r useshortkmers=t mink=5 qtrim=r trimq=10 minlength=20 . post trimming control with FastQC curated reads were mapped with STAR2.5b Dobin et al. 2013 aligner. STAR index was generated with the GRCz10/danRer10 was used as the reference zebrafish genome together with the corresponding ENSEMBL annotation. Aligned 3’ mRNA seq reads were filtered to remove false positives due to internal priming of the QuantSeq assay on genome encoded polyA stretches. To do this first a crude genomic mask was generated that contained all loci harbouring 6 or more consecutive A bases as well as any 10 nucleotide windows containing more than 6 A bases. For genes expressed from the reverse strand an analogous T rich mask was generated. Those crude masks were then corrected to allow for detection of genuine PAS falling in A/T rich regions by strand specifically unmasking 20 nucleotide intervals centred at annotated 3’ gene ends. 3’ mRNA seq reads falling into those refined strand specific masks were then removed and the filtered reads reduced to the most distal nucleotide 3’ end nucleotide. Genome build: GRCz10/danRer10 Supplementary files format and content: bigWig files: genome coverage files mean from replicates single embryo head 19hpf   Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq 3’mRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. Zebrafish Danio rerio were raised according to standard protocols 28°C water temperature 14/10 hr light/dark cycle. TLAB fish generated by crossing zebrafish AB and the natural variant TL Tupfel Long fin stocks served as wild type zebrafish for all experiments. zPCF11 null and zPCF11 ∆PAS1 mutant zebrafish were generated by Cas9 mediated mutagenesis Gagnon et al. 2014 molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11null +/+ GSM3536449 GSM3536449: 3 primemRNAseq zPCF11null wt e1; Danio rerio; RNA Seq GSM3536449   1 Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq three primemRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. GEO Accession:GSM3536449 RNA-Seq TRANSCRIPTOMIC cDNA SINGLE ILLUMINA Illumina HiSeq 2500   SRP175015     bam4__sel_AGATAGATC.bam.fastq_trimmed.fastq.gz fastq 765801085.0 8788307.0 GSM3536449 r1 0:87.14 A:248760821;C:142857406;G:160149415;T:214031144;N:2299 87       248760821 142857406 160149415 214031144 2299 SRX5193205 SRS4199205 SRA828489 GEO Sir William Dunn School of Pathology, University of Oxford 1 0.83838   0.2174   0.81341   0.56038   89   B   usable mapping rate illumina hiseq_era 3prime poly_a lexogen bulk unknown unknown   United Kingdom 2019-01-02 Multi-stage Embryo Head Nervous System
51008 51008 SRR8383261 SRX5193205 SRS4199205 SRP175015 PRJNA512489 Selective roles of vertebrate PCF11 in premature and full length transcript termination zebrafish three prime mRNA seq GSE124555 Other The pervasive nature of RNA polymerase II Pol II transcription requires efficient termination. A key player in this process is the cleavage and polyadenylation CPA factor PCF11 which directly binds to the Pol II C terminal domain and dismantles elongating Pol II from DNA in vitro. We demonstrate that PCF11 mediated termination is essential for vertebrate development. A range of genomic analyses including: mNET seq three prime mRNA seq chromatin RNA seq and ChIP seq reveals that PCF11 enhances transcription termination and stimulates early polyadenylation genome wide. PCF11 binds preferentially between closely spaced genes where it prevents transcriptional interference and downstream gene silencing. Notably PCF11 is sub stoichiometric to the CPA complex. Low levels of PCF11 are maintained by an auto regulatory mechanism involving premature termination of its own transcript and are important for normal development. Both in human cell culture and during zebrafish development PCF11 selectively attenuates the expression of other transcriptional regulators by premature CPA and termination. Overall design: three prime mRNA seq in individual zebrafish embryo heads. Two types of mutants: zPCF11 null and zPCF11 with deletion of PAS1. Wild type wt +/+ heterozygous het +/ and homozygous mutant hom / embryos were analyzed. Wild type and heterozygous animals were phenotypically indistinguishable. parent bioproject:PRJNA507553 pubmed:30819644   3 primemRNAseq zPCF11null wt e1 GSM3536449   tissue:single embryo head 19hpf|molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11null +/+ 3 primemRNAseq zPCF11null wt e1 3’ mRNA seq data were mapped according to the guidelines of QuantSeq library kit manufacturer Lexogen. Unaligned bam files from HiSeq2500 were converted to FASTQ files with bam2fastx TopHat 2 component Kim et al. 2013. post overview with FastQC reads were trimmed with BBtools sourceforge.net/projects/bbmap/ script bbduk using following settings: k=13 ktrim=r useshortkmers=t mink=5 qtrim=r trimq=10 minlength=20 . post trimming control with FastQC curated reads were mapped with STAR2.5b Dobin et al. 2013 aligner. STAR index was generated with the GRCz10/danRer10 was used as the reference zebrafish genome together with the corresponding ENSEMBL annotation. Aligned 3’ mRNA seq reads were filtered to remove false positives due to internal priming of the QuantSeq assay on genome encoded polyA stretches. To do this first a crude genomic mask was generated that contained all loci harbouring 6 or more consecutive A bases as well as any 10 nucleotide windows containing more than 6 A bases. For genes expressed from the reverse strand an analogous T rich mask was generated. Those crude masks were then corrected to allow for detection of genuine PAS falling in A/T rich regions by strand specifically unmasking 20 nucleotide intervals centred at annotated 3’ gene ends. 3’ mRNA seq reads falling into those refined strand specific masks were then removed and the filtered reads reduced to the most distal nucleotide 3’ end nucleotide. Genome build: GRCz10/danRer10 Supplementary files format and content: bigWig files: genome coverage files mean from replicates single embryo head 19hpf   Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq 3’mRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. Zebrafish Danio rerio were raised according to standard protocols 28°C water temperature 14/10 hr light/dark cycle. TLAB fish generated by crossing zebrafish AB and the natural variant TL Tupfel Long fin stocks served as wild type zebrafish for all experiments. zPCF11 null and zPCF11 ∆PAS1 mutant zebrafish were generated by Cas9 mediated mutagenesis Gagnon et al. 2014 molecule subtype:3 prime end of mRNA|genotype/treatment:zPCF11null +/+ GSM3536449 GSM3536449: 3 primemRNAseq zPCF11null wt e1; Danio rerio; RNA Seq GSM3536449   1 Dechorionated embryos of heterozygous Pcf11 mutant incrosses were cut in half with a razor blade at 19 hpf zPCF11null or 32 hpf zPCF11deltaPAS1 and and each head and tail was collected individually in PCR tubes. The anterior halves heads were lysed in 10 µl of TCL buffer with 1% beta mercaptoethanol and flash frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves tails were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Libraries were prepared according to the protocols of QuantSeq three primemRNA Seq library prep kit FW for Illumina LEXOGEN Cat# SKU 015.96 3 4 wild type 4 5 heterozygous and six homozygous samples of both zPCF11 null and zPCF11 delta PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads A63987 Beckman Coulter. Strand specific libraries were generated using the QuantSeq three prime mRNA Library Prep Kit FW Lexogen and used for 100 bp single end sequencing on the Illumina HiSeq 2500. GEO Accession:GSM3536449 RNA-Seq TRANSCRIPTOMIC cDNA SINGLE ILLUMINA Illumina HiSeq 2500   SRP175015     bam5__sel_AGATAGATC.bam.fastq_trimmed.fastq.gz fastq 786372640.0 9027751.0 GSM3536449 r2 0:87.11 A:255738065;C:146505167;G:164268319;T:219859234;N:1855 87       255738065 146505167 164268319 219859234 1855 SRX5193205 SRS4199205 SRA828489 GEO Sir William Dunn School of Pathology, University of Oxford 1 0.83666   0.21536   0.81428   0.5556   89   B   usable mapping rate illumina hiseq_era 3prime poly_a lexogen bulk unknown unknown   United Kingdom 2019-01-02 Multi-stage Embryo Head Nervous System
55541 55541 SRR10550579 SRX7232580 SRS5733210 SRP233586 PRJNA592212 Transcriptional analysis of the effects of early life exposure to fluoxetine on telencephalon and hypothalamus in SR4G transgenic zebrafish larvae and adults GSE141144 Transcriptome Analysis Our previous study suggested persistent transgenerational effects of fluoxetine FLX exposure in both environmental and therapeutic concentrations on stress response and behaviour up to 3 generation withdrawn from exposure in larvae and adult zebrafish. To lay the foundations of the mechanistic of such transgenerational effects we studied the effect of FLX on stress response in brain tissues telencephalon and hypothalamus of larvae and adult zebrafish using differentially expressed genes at the transcriptome level. To monitor the stress response we used a transgenic zebrafish line SR4G which are expressing green fluorescence protein GFP in response to endogenous and exogenous glucocorticoids. The transcriptome analysis of GFP RNA as an internal control provided us with an invaluable tool to clearly visualize and differentiate between stressed and unstressed groups as per treatments. To stimulate stress in both larvae and adult zebrafish we used a modified net stressing technique consisting of consecutive sequence of air exposure and incubation intervals. A pool of 22 larvae heads and a pool of 5 telencephalon and 5 hypothalamus dissected tissues were used as the target tissues for RNA extraction. All experiments were replicated in n=6 per 4 different groups including Control Unstressed Cnt U Control Stressed Cnt S Fluoxetine Unstressed FLX U and Fluoxetine Stressed FLX S using pools of larvae heads telencephalon and hypothalamus. post total RNA extraction using Illumina technology all samples were sequence and the list of differentially expressed genes DEGs were produced using DSEQ pipeline in R. Overall design: Three different target tissue two from 6 mpf adults Telencephalon and Hypothalamus and one from 6 dpf larvae dissected heads. One treatment Fluoxetine One control ethanol as vehicle two condition Stressed Unstressed       Lrv FLX Str whole 6 GSM4196113   source name:Lrv FLX Str whole|genotype/variation:SR4G transgenic|stress status:Str|drug status:FLX|tissue:larvae head Lrv FLX Str whole 6 Illumina bcl2fastq v2.17.1.14 software used for basecalling. Sequenced reads were trimmed for adaptor sequence with CutAdapt then aligned with STAR v 2.5 with parameter clip5pNbases 10 and quantMode TranscriptomeSAM GeneCounts. Tables of gene counts generated by STAR were imported into R and differential gene expression analysis was performed using DESeq2 to compare exposure groups. Genome build: GRCz11v92 Supplementary files format and content: tab delimited text files include raw count values for each sample. Lrv FLX Str whole Exposing to Fluoxetine 54ug/L as treatment or Ethanol 0.0053% as vehicle from 0 dpf to 6 dpf in larvae Qiagene miliamp total RNA extraction column based Illumina TruSeq Stranded mRNA libraries were built following the manufacturer’s protocol using 250ng of starting material.   genotype/variation:SR4G transgenic|stress status:Str|drug status:FLX|tissue:larvae head GSM4196113 GSM4196113: Lrv FLX Str whole 6; Danio rerio; RNA Seq GSM4196113   1 Qiagene miliamp total RNA extraction column based Illumina TruSeq Stranded mRNA libraries were built following the manufacturer's protocol using 250ng of starting material. GEO Accession:GSM4196113 RNA-Seq TRANSCRIPTOMIC cDNA SINGLE ILLUMINA NextSeq 500   SRP233586     Lrv_FLX_Str_6_S55_R1_001.trimmed.fastq.gz fastq 1667637008.0 22201140.0 GSM4196113 r1 0:75.11 1:0 A:438989346;C:397147842;G:367576938;T:463905385;N:17497 75 0     438989346 397147842 367576938 463905385 17497 SRX7232580 SRS5733210 SRA1003923 GEO Mechanistic Studies Division Genomics Laboratory, Health Canada, Government of Canada 1 0.94774   0.13955   0.69842   0.47883   74   B   usable mapping rate illumina nextseq unknown cdna_unspecified trueseq bulk unknown unknown   Canada 2019-11-27 Multi-stage Multi-stage Head Nervous System
55542 55542 SRR10550578 SRX7232579 SRS5733211 SRP233586 PRJNA592212 Transcriptional analysis of the effects of early life exposure to fluoxetine on telencephalon and hypothalamus in SR4G transgenic zebrafish larvae and adults GSE141144 Transcriptome Analysis Our previous study suggested persistent transgenerational effects of fluoxetine FLX exposure in both environmental and therapeutic concentrations on stress response and behaviour up to 3 generation withdrawn from exposure in larvae and adult zebrafish. To lay the foundations of the mechanistic of such transgenerational effects we studied the effect of FLX on stress response in brain tissues telencephalon and hypothalamus of larvae and adult zebrafish using differentially expressed genes at the transcriptome level. To monitor the stress response we used a transgenic zebrafish line SR4G which are expressing green fluorescence protein GFP in response to endogenous and exogenous glucocorticoids. The transcriptome analysis of GFP RNA as an internal control provided us with an invaluable tool to clearly visualize and differentiate between stressed and unstressed groups as per treatments. To stimulate stress in both larvae and adult zebrafish we used a modified net stressing technique consisting of consecutive sequence of air exposure and incubation intervals. A pool of 22 larvae heads and a pool of 5 telencephalon and 5 hypothalamus dissected tissues were used as the target tissues for RNA extraction. All experiments were replicated in n=6 per 4 different groups including Control Unstressed Cnt U Control Stressed Cnt S Fluoxetine Unstressed FLX U and Fluoxetine Stressed FLX S using pools of larvae heads telencephalon and hypothalamus. post total RNA extraction using Illumina technology all samples were sequence and the list of differentially expressed genes DEGs were produced using DSEQ pipeline in R. Overall design: Three different target tissue two from 6 mpf adults Telencephalon and Hypothalamus and one from 6 dpf larvae dissected heads. One treatment Fluoxetine One control ethanol as vehicle two condition Stressed Unstressed       Lrv FLX Str whole 5 GSM4196112   source name:Lrv FLX Str whole|genotype/variation:SR4G transgenic|stress status:Str|drug status:FLX|tissue:larvae head Lrv FLX Str whole 5 Illumina bcl2fastq v2.17.1.14 software used for basecalling. Sequenced reads were trimmed for adaptor sequence with CutAdapt then aligned with STAR v 2.5 with parameter clip5pNbases 10 and quantMode TranscriptomeSAM GeneCounts. Tables of gene counts generated by STAR were imported into R and differential gene expression analysis was performed using DESeq2 to compare exposure groups. Genome build: GRCz11v92 Supplementary files format and content: tab delimited text files include raw count values for each sample. Lrv FLX Str whole Exposing to Fluoxetine 54ug/L as treatment or Ethanol 0.0053% as vehicle from 0 dpf to 6 dpf in larvae Qiagene miliamp total RNA extraction column based Illumina TruSeq Stranded mRNA libraries were built following the manufacturer’s protocol using 250ng of starting material.   genotype/variation:SR4G transgenic|stress status:Str|drug status:FLX|tissue:larvae head GSM4196112 GSM4196112: Lrv FLX Str whole 5; Danio rerio; RNA Seq GSM4196112   1 Qiagene miliamp total RNA extraction column based Illumina TruSeq Stranded mRNA libraries were built following the manufacturer's protocol using 250ng of starting material. GEO Accession:GSM4196112 RNA-Seq TRANSCRIPTOMIC cDNA SINGLE ILLUMINA NextSeq 500   SRP233586     Lrv_FLX_Str_5_S59_R1_001.trimmed.fastq.gz fastq 1682694124.0 22385497.0 GSM4196112 r1 0:75.17 1:0 A:404424623;C:427018922;G:386032112;T:465200972;N:17495 75 0     404424623 427018922 386032112 465200972 17495 SRX7232579 SRS5733211 SRA1003923 GEO Mechanistic Studies Division Genomics Laboratory, Health Canada, Government of Canada 1 0.95782   0.11464   0.69404   0.47678   76   B   usable mapping rate illumina nextseq unknown cdna_unspecified trueseq bulk unknown unknown   Canada 2019-11-27 Multi-stage Multi-stage Head Nervous System
55543 55543 SRR10550577 SRX7232578 SRS5733209 SRP233586 PRJNA592212 Transcriptional analysis of the effects of early life exposure to fluoxetine on telencephalon and hypothalamus in SR4G transgenic zebrafish larvae and adults GSE141144 Transcriptome Analysis Our previous study suggested persistent transgenerational effects of fluoxetine FLX exposure in both environmental and therapeutic concentrations on stress response and behaviour up to 3 generation withdrawn from exposure in larvae and adult zebrafish. To lay the foundations of the mechanistic of such transgenerational effects we studied the effect of FLX on stress response in brain tissues telencephalon and hypothalamus of larvae and adult zebrafish using differentially expressed genes at the transcriptome level. To monitor the stress response we used a transgenic zebrafish line SR4G which are expressing green fluorescence protein GFP in response to endogenous and exogenous glucocorticoids. The transcriptome analysis of GFP RNA as an internal control provided us with an invaluable tool to clearly visualize and differentiate between stressed and unstressed groups as per treatments. To stimulate stress in both larvae and adult zebrafish we used a modified net stressing technique consisting of consecutive sequence of air exposure and incubation intervals. A pool of 22 larvae heads and a pool of 5 telencephalon and 5 hypothalamus dissected tissues were used as the target tissues for RNA extraction. All experiments were replicated in n=6 per 4 different groups including Control Unstressed Cnt U Control Stressed Cnt S Fluoxetine Unstressed FLX U and Fluoxetine Stressed FLX S using pools of larvae heads telencephalon and hypothalamus. post total RNA extraction using Illumina technology all samples were sequence and the list of differentially expressed genes DEGs were produced using DSEQ pipeline in R. Overall design: Three different target tissue two from 6 mpf adults Telencephalon and Hypothalamus and one from 6 dpf larvae dissected heads. One treatment Fluoxetine One control ethanol as vehicle two condition Stressed Unstressed       Lrv FLX Str whole 4 GSM4196111   source name:Lrv FLX Str whole|genotype/variation:SR4G transgenic|stress status:Str|drug status:FLX|tissue:larvae head Lrv FLX Str whole 4 Illumina bcl2fastq v2.17.1.14 software used for basecalling. Sequenced reads were trimmed for adaptor sequence with CutAdapt then aligned with STAR v 2.5 with parameter clip5pNbases 10 and quantMode TranscriptomeSAM GeneCounts. Tables of gene counts generated by STAR were imported into R and differential gene expression analysis was performed using DESeq2 to compare exposure groups. Genome build: GRCz11v92 Supplementary files format and content: tab delimited text files include raw count values for each sample. Lrv FLX Str whole Exposing to Fluoxetine 54ug/L as treatment or Ethanol 0.0053% as vehicle from 0 dpf to 6 dpf in larvae Qiagene miliamp total RNA extraction column based Illumina TruSeq Stranded mRNA libraries were built following the manufacturer’s protocol using 250ng of starting material.   genotype/variation:SR4G transgenic|stress status:Str|drug status:FLX|tissue:larvae head GSM4196111 GSM4196111: Lrv FLX Str whole 4; Danio rerio; RNA Seq GSM4196111   1 Qiagene miliamp total RNA extraction column based Illumina TruSeq Stranded mRNA libraries were built following the manufacturer's protocol using 250ng of starting material. GEO Accession:GSM4196111 RNA-Seq TRANSCRIPTOMIC cDNA SINGLE ILLUMINA NextSeq 500   SRP233586     Lrv_FLX_Str_4_S50_R1_001.trimmed.fastq.gz fastq 1087107196.0 14458795.0 GSM4196111 r1 0:75.19 1:0 A:267171751;C:268993662;G:244811629;T:306118431;N:11723 75 0     267171751 268993662 244811629 306118431 11723 SRX7232578 SRS5733209 SRA1003923 GEO Mechanistic Studies Division Genomics Laboratory, Health Canada, Government of Canada 1 0.95236   0.12138   0.69725   0.46512   76   B   usable mapping rate illumina nextseq unknown cdna_unspecified trueseq bulk unknown unknown   Canada 2019-11-27 Multi-stage Multi-stage Head Nervous System
55544 55544 SRR10550576 SRX7232577 SRS5733208 SRP233586 PRJNA592212 Transcriptional analysis of the effects of early life exposure to fluoxetine on telencephalon and hypothalamus in SR4G transgenic zebrafish larvae and adults GSE141144 Transcriptome Analysis Our previous study suggested persistent transgenerational effects of fluoxetine FLX exposure in both environmental and therapeutic concentrations on stress response and behaviour up to 3 generation withdrawn from exposure in larvae and adult zebrafish. To lay the foundations of the mechanistic of such transgenerational effects we studied the effect of FLX on stress response in brain tissues telencephalon and hypothalamus of larvae and adult zebrafish using differentially expressed genes at the transcriptome level. To monitor the stress response we used a transgenic zebrafish line SR4G which are expressing green fluorescence protein GFP in response to endogenous and exogenous glucocorticoids. The transcriptome analysis of GFP RNA as an internal control provided us with an invaluable tool to clearly visualize and differentiate between stressed and unstressed groups as per treatments. To stimulate stress in both larvae and adult zebrafish we used a modified net stressing technique consisting of consecutive sequence of air exposure and incubation intervals. A pool of 22 larvae heads and a pool of 5 telencephalon and 5 hypothalamus dissected tissues were used as the target tissues for RNA extraction. All experiments were replicated in n=6 per 4 different groups including Control Unstressed Cnt U Control Stressed Cnt S Fluoxetine Unstressed FLX U and Fluoxetine Stressed FLX S using pools of larvae heads telencephalon and hypothalamus. post total RNA extraction using Illumina technology all samples were sequence and the list of differentially expressed genes DEGs were produced using DSEQ pipeline in R. Overall design: Three different target tissue two from 6 mpf adults Telencephalon and Hypothalamus and one from 6 dpf larvae dissected heads. One treatment Fluoxetine One control ethanol as vehicle two condition Stressed Unstressed       Lrv FLX Str whole 3 GSM4196110   source name:Lrv FLX Str whole|genotype/variation:SR4G transgenic|stress status:Str|drug status:FLX|tissue:larvae head Lrv FLX Str whole 3 Illumina bcl2fastq v2.17.1.14 software used for basecalling. Sequenced reads were trimmed for adaptor sequence with CutAdapt then aligned with STAR v 2.5 with parameter clip5pNbases 10 and quantMode TranscriptomeSAM GeneCounts. Tables of gene counts generated by STAR were imported into R and differential gene expression analysis was performed using DESeq2 to compare exposure groups. Genome build: GRCz11v92 Supplementary files format and content: tab delimited text files include raw count values for each sample. Lrv FLX Str whole Exposing to Fluoxetine 54ug/L as treatment or Ethanol 0.0053% as vehicle from 0 dpf to 6 dpf in larvae Qiagene miliamp total RNA extraction column based Illumina TruSeq Stranded mRNA libraries were built following the manufacturer’s protocol using 250ng of starting material.   genotype/variation:SR4G transgenic|stress status:Str|drug status:FLX|tissue:larvae head GSM4196110 GSM4196110: Lrv FLX Str whole 3; Danio rerio; RNA Seq GSM4196110   1 Qiagene miliamp total RNA extraction column based Illumina TruSeq Stranded mRNA libraries were built following the manufacturer's protocol using 250ng of starting material. GEO Accession:GSM4196110 RNA-Seq TRANSCRIPTOMIC cDNA SINGLE ILLUMINA NextSeq 500   SRP233586     Lrv_FLX_Str_3_S62_R1_001.trimmed.fastq.gz fastq 1652611529.0 21987509.0 GSM4196110 r1 0:75.16 1:0 A:442049125;C:388222499;G:359554341;T:462768582;N:16982 75 0     442049125 388222499 359554341 462768582 16982 SRX7232577 SRS5733208 SRA1003923 GEO Mechanistic Studies Division Genomics Laboratory, Health Canada, Government of Canada 1 0.94763   0.15049   0.69875   0.49665   73   B   usable mapping rate illumina nextseq unknown cdna_unspecified trueseq bulk unknown unknown   Canada 2019-11-27 Multi-stage Multi-stage Head Nervous System
55545 55545 SRR10550575 SRX7232576 SRS5733207 SRP233586 PRJNA592212 Transcriptional analysis of the effects of early life exposure to fluoxetine on telencephalon and hypothalamus in SR4G transgenic zebrafish larvae and adults GSE141144 Transcriptome Analysis Our previous study suggested persistent transgenerational effects of fluoxetine FLX exposure in both environmental and therapeutic concentrations on stress response and behaviour up to 3 generation withdrawn from exposure in larvae and adult zebrafish. To lay the foundations of the mechanistic of such transgenerational effects we studied the effect of FLX on stress response in brain tissues telencephalon and hypothalamus of larvae and adult zebrafish using differentially expressed genes at the transcriptome level. To monitor the stress response we used a transgenic zebrafish line SR4G which are expressing green fluorescence protein GFP in response to endogenous and exogenous glucocorticoids. The transcriptome analysis of GFP RNA as an internal control provided us with an invaluable tool to clearly visualize and differentiate between stressed and unstressed groups as per treatments. To stimulate stress in both larvae and adult zebrafish we used a modified net stressing technique consisting of consecutive sequence of air exposure and incubation intervals. A pool of 22 larvae heads and a pool of 5 telencephalon and 5 hypothalamus dissected tissues were used as the target tissues for RNA extraction. All experiments were replicated in n=6 per 4 different groups including Control Unstressed Cnt U Control Stressed Cnt S Fluoxetine Unstressed FLX U and Fluoxetine Stressed FLX S using pools of larvae heads telencephalon and hypothalamus. post total RNA extraction using Illumina technology all samples were sequence and the list of differentially expressed genes DEGs were produced using DSEQ pipeline in R. Overall design: Three different target tissue two from 6 mpf adults Telencephalon and Hypothalamus and one from 6 dpf larvae dissected heads. One treatment Fluoxetine One control ethanol as vehicle two condition Stressed Unstressed       Lrv FLX Str whole 2 GSM4196109   source name:Lrv FLX Str whole|genotype/variation:SR4G transgenic|stress status:Str|drug status:FLX|tissue:larvae head Lrv FLX Str whole 2 Illumina bcl2fastq v2.17.1.14 software used for basecalling. Sequenced reads were trimmed for adaptor sequence with CutAdapt then aligned with STAR v 2.5 with parameter clip5pNbases 10 and quantMode TranscriptomeSAM GeneCounts. Tables of gene counts generated by STAR were imported into R and differential gene expression analysis was performed using DESeq2 to compare exposure groups. Genome build: GRCz11v92 Supplementary files format and content: tab delimited text files include raw count values for each sample. Lrv FLX Str whole Exposing to Fluoxetine 54ug/L as treatment or Ethanol 0.0053% as vehicle from 0 dpf to 6 dpf in larvae Qiagene miliamp total RNA extraction column based Illumina TruSeq Stranded mRNA libraries were built following the manufacturer’s protocol using 250ng of starting material.   genotype/variation:SR4G transgenic|stress status:Str|drug status:FLX|tissue:larvae head GSM4196109 GSM4196109: Lrv FLX Str whole 2; Danio rerio; RNA Seq GSM4196109   1 Qiagene miliamp total RNA extraction column based Illumina TruSeq Stranded mRNA libraries were built following the manufacturer's protocol using 250ng of starting material. GEO Accession:GSM4196109 RNA-Seq TRANSCRIPTOMIC cDNA SINGLE ILLUMINA NextSeq 500   SRP233586     Lrv_FLX_Str_2_S70_R1_001.trimmed.fastq.gz fastq 1525352669.0 20291623.0 GSM4196109 r1 0:75.17 1:0 A:415549321;C:352941285;G:326282134;T:430564543;N:15386 75 0     415549321 352941285 326282134 430564543 15386 SRX7232576 SRS5733207 SRA1003923 GEO Mechanistic Studies Division Genomics Laboratory, Health Canada, Government of Canada 1 0.94391   0.16406   0.69613   0.48758   76   B   usable mapping rate illumina nextseq unknown cdna_unspecified trueseq bulk unknown unknown   Canada 2019-11-27 Multi-stage Multi-stage Head Nervous System
55546 55546 SRR10550574 SRX7232575 SRS5733206 SRP233586 PRJNA592212 Transcriptional analysis of the effects of early life exposure to fluoxetine on telencephalon and hypothalamus in SR4G transgenic zebrafish larvae and adults GSE141144 Transcriptome Analysis Our previous study suggested persistent transgenerational effects of fluoxetine FLX exposure in both environmental and therapeutic concentrations on stress response and behaviour up to 3 generation withdrawn from exposure in larvae and adult zebrafish. To lay the foundations of the mechanistic of such transgenerational effects we studied the effect of FLX on stress response in brain tissues telencephalon and hypothalamus of larvae and adult zebrafish using differentially expressed genes at the transcriptome level. To monitor the stress response we used a transgenic zebrafish line SR4G which are expressing green fluorescence protein GFP in response to endogenous and exogenous glucocorticoids. The transcriptome analysis of GFP RNA as an internal control provided us with an invaluable tool to clearly visualize and differentiate between stressed and unstressed groups as per treatments. To stimulate stress in both larvae and adult zebrafish we used a modified net stressing technique consisting of consecutive sequence of air exposure and incubation intervals. A pool of 22 larvae heads and a pool of 5 telencephalon and 5 hypothalamus dissected tissues were used as the target tissues for RNA extraction. All experiments were replicated in n=6 per 4 different groups including Control Unstressed Cnt U Control Stressed Cnt S Fluoxetine Unstressed FLX U and Fluoxetine Stressed FLX S using pools of larvae heads telencephalon and hypothalamus. post total RNA extraction using Illumina technology all samples were sequence and the list of differentially expressed genes DEGs were produced using DSEQ pipeline in R. Overall design: Three different target tissue two from 6 mpf adults Telencephalon and Hypothalamus and one from 6 dpf larvae dissected heads. One treatment Fluoxetine One control ethanol as vehicle two condition Stressed Unstressed       Lrv FLX Str whole 1 GSM4196108   source name:Lrv FLX Str whole|genotype/variation:SR4G transgenic|stress status:Str|drug status:FLX|tissue:larvae head Lrv FLX Str whole 1 Illumina bcl2fastq v2.17.1.14 software used for basecalling. Sequenced reads were trimmed for adaptor sequence with CutAdapt then aligned with STAR v 2.5 with parameter clip5pNbases 10 and quantMode TranscriptomeSAM GeneCounts. Tables of gene counts generated by STAR were imported into R and differential gene expression analysis was performed using DESeq2 to compare exposure groups. Genome build: GRCz11v92 Supplementary files format and content: tab delimited text files include raw count values for each sample. Lrv FLX Str whole Exposing to Fluoxetine 54ug/L as treatment or Ethanol 0.0053% as vehicle from 0 dpf to 6 dpf in larvae Qiagene miliamp total RNA extraction column based Illumina TruSeq Stranded mRNA libraries were built following the manufacturer’s protocol using 250ng of starting material.   genotype/variation:SR4G transgenic|stress status:Str|drug status:FLX|tissue:larvae head GSM4196108 GSM4196108: Lrv FLX Str whole 1; Danio rerio; RNA Seq GSM4196108   1 Qiagene miliamp total RNA extraction column based Illumina TruSeq Stranded mRNA libraries were built following the manufacturer's protocol using 250ng of starting material. GEO Accession:GSM4196108 RNA-Seq TRANSCRIPTOMIC cDNA SINGLE ILLUMINA NextSeq 500   SRP233586     Lrv_FLX_Str_1_S57_R1_001.trimmed.fastq.gz fastq 1572443197.0 20910507.0 GSM4196108 r1 0:75.20 1:0 A:391749194;C:388029990;G:350196422;T:442451463;N:16128 75 0     391749194 388029990 350196422 442451463 16128 SRX7232575 SRS5733206 SRA1003923 GEO Mechanistic Studies Division Genomics Laboratory, Health Canada, Government of Canada 1 0.95111   0.1268   0.69587   0.47885   76   B   usable mapping rate illumina nextseq unknown cdna_unspecified trueseq bulk unknown unknown   Canada 2019-11-27 Multi-stage Multi-stage Head Nervous System
55547 55547 SRR10550573 SRX7232574 SRS5733205 SRP233586 PRJNA592212 Transcriptional analysis of the effects of early life exposure to fluoxetine on telencephalon and hypothalamus in SR4G transgenic zebrafish larvae and adults GSE141144 Transcriptome Analysis Our previous study suggested persistent transgenerational effects of fluoxetine FLX exposure in both environmental and therapeutic concentrations on stress response and behaviour up to 3 generation withdrawn from exposure in larvae and adult zebrafish. To lay the foundations of the mechanistic of such transgenerational effects we studied the effect of FLX on stress response in brain tissues telencephalon and hypothalamus of larvae and adult zebrafish using differentially expressed genes at the transcriptome level. To monitor the stress response we used a transgenic zebrafish line SR4G which are expressing green fluorescence protein GFP in response to endogenous and exogenous glucocorticoids. The transcriptome analysis of GFP RNA as an internal control provided us with an invaluable tool to clearly visualize and differentiate between stressed and unstressed groups as per treatments. To stimulate stress in both larvae and adult zebrafish we used a modified net stressing technique consisting of consecutive sequence of air exposure and incubation intervals. A pool of 22 larvae heads and a pool of 5 telencephalon and 5 hypothalamus dissected tissues were used as the target tissues for RNA extraction. All experiments were replicated in n=6 per 4 different groups including Control Unstressed Cnt U Control Stressed Cnt S Fluoxetine Unstressed FLX U and Fluoxetine Stressed FLX S using pools of larvae heads telencephalon and hypothalamus. post total RNA extraction using Illumina technology all samples were sequence and the list of differentially expressed genes DEGs were produced using DSEQ pipeline in R. Overall design: Three different target tissue two from 6 mpf adults Telencephalon and Hypothalamus and one from 6 dpf larvae dissected heads. One treatment Fluoxetine One control ethanol as vehicle two condition Stressed Unstressed       Lrv FLX Bsal whole 6 GSM4196107   source name:Lrv FLX Bsal whole|genotype/variation:SR4G transgenic|stress status:Bsal|drug status:FLX|tissue:larvae head Lrv FLX Bsal whole 6 Illumina bcl2fastq v2.17.1.14 software used for basecalling. Sequenced reads were trimmed for adaptor sequence with CutAdapt then aligned with STAR v 2.5 with parameter clip5pNbases 10 and quantMode TranscriptomeSAM GeneCounts. Tables of gene counts generated by STAR were imported into R and differential gene expression analysis was performed using DESeq2 to compare exposure groups. Genome build: GRCz11v92 Supplementary files format and content: tab delimited text files include raw count values for each sample. Lrv FLX Bsal whole Exposing to Fluoxetine 54ug/L as treatment or Ethanol 0.0053% as vehicle from 0 dpf to 6 dpf in larvae Qiagene miliamp total RNA extraction column based Illumina TruSeq Stranded mRNA libraries were built following the manufacturer’s protocol using 250ng of starting material.   genotype/variation:SR4G transgenic|stress status:Bsal|drug status:FLX|tissue:larvae head GSM4196107 GSM4196107: Lrv FLX Bsal whole 6; Danio rerio; RNA Seq GSM4196107   1 Qiagene miliamp total RNA extraction column based Illumina TruSeq Stranded mRNA libraries were built following the manufacturer's protocol using 250ng of starting material. GEO Accession:GSM4196107 RNA-Seq TRANSCRIPTOMIC cDNA SINGLE ILLUMINA NextSeq 500   SRP233586     Lrv_FLX_Bsal_6_S64_R1_001.trimmed.fastq.gz fastq 1819465903.0 24220618.0 GSM4196107 r1 0:75.12 1:0 A:454377540;C:446747596;G:411720425;T:506601808;N:18534 75 0     454377540 446747596 411720425 506601808 18534 SRX7232574 SRS5733205 SRA1003923 GEO Mechanistic Studies Division Genomics Laboratory, Health Canada, Government of Canada 1 0.95274   0.12081   0.67389   0.47068   75   B   usable mapping rate illumina nextseq unknown cdna_unspecified trueseq bulk unknown unknown   Canada 2019-11-27 Multi-stage Multi-stage Head Nervous System
55548 55548 SRR10550572 SRX7232573 SRS5733204 SRP233586 PRJNA592212 Transcriptional analysis of the effects of early life exposure to fluoxetine on telencephalon and hypothalamus in SR4G transgenic zebrafish larvae and adults GSE141144 Transcriptome Analysis Our previous study suggested persistent transgenerational effects of fluoxetine FLX exposure in both environmental and therapeutic concentrations on stress response and behaviour up to 3 generation withdrawn from exposure in larvae and adult zebrafish. To lay the foundations of the mechanistic of such transgenerational effects we studied the effect of FLX on stress response in brain tissues telencephalon and hypothalamus of larvae and adult zebrafish using differentially expressed genes at the transcriptome level. To monitor the stress response we used a transgenic zebrafish line SR4G which are expressing green fluorescence protein GFP in response to endogenous and exogenous glucocorticoids. The transcriptome analysis of GFP RNA as an internal control provided us with an invaluable tool to clearly visualize and differentiate between stressed and unstressed groups as per treatments. To stimulate stress in both larvae and adult zebrafish we used a modified net stressing technique consisting of consecutive sequence of air exposure and incubation intervals. A pool of 22 larvae heads and a pool of 5 telencephalon and 5 hypothalamus dissected tissues were used as the target tissues for RNA extraction. All experiments were replicated in n=6 per 4 different groups including Control Unstressed Cnt U Control Stressed Cnt S Fluoxetine Unstressed FLX U and Fluoxetine Stressed FLX S using pools of larvae heads telencephalon and hypothalamus. post total RNA extraction using Illumina technology all samples were sequence and the list of differentially expressed genes DEGs were produced using DSEQ pipeline in R. Overall design: Three different target tissue two from 6 mpf adults Telencephalon and Hypothalamus and one from 6 dpf larvae dissected heads. One treatment Fluoxetine One control ethanol as vehicle two condition Stressed Unstressed       Lrv FLX Bsal whole 4 GSM4196106   source name:Lrv FLX Bsal whole|genotype/variation:SR4G transgenic|stress status:Bsal|drug status:FLX|tissue:larvae head Lrv FLX Bsal whole 4 Illumina bcl2fastq v2.17.1.14 software used for basecalling. Sequenced reads were trimmed for adaptor sequence with CutAdapt then aligned with STAR v 2.5 with parameter clip5pNbases 10 and quantMode TranscriptomeSAM GeneCounts. Tables of gene counts generated by STAR were imported into R and differential gene expression analysis was performed using DESeq2 to compare exposure groups. Genome build: GRCz11v92 Supplementary files format and content: tab delimited text files include raw count values for each sample. Lrv FLX Bsal whole Exposing to Fluoxetine 54ug/L as treatment or Ethanol 0.0053% as vehicle from 0 dpf to 6 dpf in larvae Qiagene miliamp total RNA extraction column based Illumina TruSeq Stranded mRNA libraries were built following the manufacturer’s protocol using 250ng of starting material.   genotype/variation:SR4G transgenic|stress status:Bsal|drug status:FLX|tissue:larvae head GSM4196106 GSM4196106: Lrv FLX Bsal whole 4; Danio rerio; RNA Seq GSM4196106   1 Qiagene miliamp total RNA extraction column based Illumina TruSeq Stranded mRNA libraries were built following the manufacturer's protocol using 250ng of starting material. GEO Accession:GSM4196106 RNA-Seq TRANSCRIPTOMIC cDNA SINGLE ILLUMINA NextSeq 500   SRP233586     Lrv_FLX_Bsal_4_S69_R1_001.trimmed.fastq.gz fastq 1802344612.0 23981363.0 GSM4196106 r1 0:75.16 1:0 A:455847939;C:444174899;G:406307504;T:495995239;N:19031 75 0     455847939 444174899 406307504 495995239 19031 SRX7232573 SRS5733204 SRA1003923 GEO Mechanistic Studies Division Genomics Laboratory, Health Canada, Government of Canada 1 0.9537   0.13958   0.6929   0.4996   76   B   usable mapping rate illumina nextseq unknown cdna_unspecified trueseq bulk unknown unknown   Canada 2019-11-27 Multi-stage Multi-stage Head Nervous System
55549 55549 SRR10550571 SRX7232572 SRS5733203 SRP233586 PRJNA592212 Transcriptional analysis of the effects of early life exposure to fluoxetine on telencephalon and hypothalamus in SR4G transgenic zebrafish larvae and adults GSE141144 Transcriptome Analysis Our previous study suggested persistent transgenerational effects of fluoxetine FLX exposure in both environmental and therapeutic concentrations on stress response and behaviour up to 3 generation withdrawn from exposure in larvae and adult zebrafish. To lay the foundations of the mechanistic of such transgenerational effects we studied the effect of FLX on stress response in brain tissues telencephalon and hypothalamus of larvae and adult zebrafish using differentially expressed genes at the transcriptome level. To monitor the stress response we used a transgenic zebrafish line SR4G which are expressing green fluorescence protein GFP in response to endogenous and exogenous glucocorticoids. The transcriptome analysis of GFP RNA as an internal control provided us with an invaluable tool to clearly visualize and differentiate between stressed and unstressed groups as per treatments. To stimulate stress in both larvae and adult zebrafish we used a modified net stressing technique consisting of consecutive sequence of air exposure and incubation intervals. A pool of 22 larvae heads and a pool of 5 telencephalon and 5 hypothalamus dissected tissues were used as the target tissues for RNA extraction. All experiments were replicated in n=6 per 4 different groups including Control Unstressed Cnt U Control Stressed Cnt S Fluoxetine Unstressed FLX U and Fluoxetine Stressed FLX S using pools of larvae heads telencephalon and hypothalamus. post total RNA extraction using Illumina technology all samples were sequence and the list of differentially expressed genes DEGs were produced using DSEQ pipeline in R. Overall design: Three different target tissue two from 6 mpf adults Telencephalon and Hypothalamus and one from 6 dpf larvae dissected heads. One treatment Fluoxetine One control ethanol as vehicle two condition Stressed Unstressed       Lrv FLX Bsal whole 3 GSM4196105   source name:Lrv FLX Bsal whole|genotype/variation:SR4G transgenic|stress status:Bsal|drug status:FLX|tissue:larvae head Lrv FLX Bsal whole 3 Illumina bcl2fastq v2.17.1.14 software used for basecalling. Sequenced reads were trimmed for adaptor sequence with CutAdapt then aligned with STAR v 2.5 with parameter clip5pNbases 10 and quantMode TranscriptomeSAM GeneCounts. Tables of gene counts generated by STAR were imported into R and differential gene expression analysis was performed using DESeq2 to compare exposure groups. Genome build: GRCz11v92 Supplementary files format and content: tab delimited text files include raw count values for each sample. Lrv FLX Bsal whole Exposing to Fluoxetine 54ug/L as treatment or Ethanol 0.0053% as vehicle from 0 dpf to 6 dpf in larvae Qiagene miliamp total RNA extraction column based Illumina TruSeq Stranded mRNA libraries were built following the manufacturer’s protocol using 250ng of starting material.   genotype/variation:SR4G transgenic|stress status:Bsal|drug status:FLX|tissue:larvae head GSM4196105 GSM4196105: Lrv FLX Bsal whole 3; Danio rerio; RNA Seq GSM4196105   1 Qiagene miliamp total RNA extraction column based Illumina TruSeq Stranded mRNA libraries were built following the manufacturer's protocol using 250ng of starting material. GEO Accession:GSM4196105 RNA-Seq TRANSCRIPTOMIC cDNA SINGLE ILLUMINA NextSeq 500   SRP233586     Lrv_FLX_Bsal_3_S53_R1_001.trimmed.fastq.gz fastq 1536979688.0 20489086.0 GSM4196105 r1 0:75.01 1:0 A:379743513;C:383792979;G:353602367;T:419825271;N:15558 75 0     379743513 383792979 353602367 419825271 15558 SRX7232572 SRS5733203 SRA1003923 GEO Mechanistic Studies Division Genomics Laboratory, Health Canada, Government of Canada 1 0.95655   0.12079   0.69666   0.48202   72   B   usable mapping rate illumina nextseq unknown cdna_unspecified trueseq bulk unknown unknown   Canada 2019-11-27 Multi-stage Multi-stage Head Nervous System
55550 55550 SRR10550570 SRX7232571 SRS5733202 SRP233586 PRJNA592212 Transcriptional analysis of the effects of early life exposure to fluoxetine on telencephalon and hypothalamus in SR4G transgenic zebrafish larvae and adults GSE141144 Transcriptome Analysis Our previous study suggested persistent transgenerational effects of fluoxetine FLX exposure in both environmental and therapeutic concentrations on stress response and behaviour up to 3 generation withdrawn from exposure in larvae and adult zebrafish. To lay the foundations of the mechanistic of such transgenerational effects we studied the effect of FLX on stress response in brain tissues telencephalon and hypothalamus of larvae and adult zebrafish using differentially expressed genes at the transcriptome level. To monitor the stress response we used a transgenic zebrafish line SR4G which are expressing green fluorescence protein GFP in response to endogenous and exogenous glucocorticoids. The transcriptome analysis of GFP RNA as an internal control provided us with an invaluable tool to clearly visualize and differentiate between stressed and unstressed groups as per treatments. To stimulate stress in both larvae and adult zebrafish we used a modified net stressing technique consisting of consecutive sequence of air exposure and incubation intervals. A pool of 22 larvae heads and a pool of 5 telencephalon and 5 hypothalamus dissected tissues were used as the target tissues for RNA extraction. All experiments were replicated in n=6 per 4 different groups including Control Unstressed Cnt U Control Stressed Cnt S Fluoxetine Unstressed FLX U and Fluoxetine Stressed FLX S using pools of larvae heads telencephalon and hypothalamus. post total RNA extraction using Illumina technology all samples were sequence and the list of differentially expressed genes DEGs were produced using DSEQ pipeline in R. Overall design: Three different target tissue two from 6 mpf adults Telencephalon and Hypothalamus and one from 6 dpf larvae dissected heads. One treatment Fluoxetine One control ethanol as vehicle two condition Stressed Unstressed       Lrv FLX Bsal whole 2 GSM4196104   source name:Lrv FLX Bsal whole|genotype/variation:SR4G transgenic|stress status:Bsal|drug status:FLX|tissue:larvae head Lrv FLX Bsal whole 2 Illumina bcl2fastq v2.17.1.14 software used for basecalling. Sequenced reads were trimmed for adaptor sequence with CutAdapt then aligned with STAR v 2.5 with parameter clip5pNbases 10 and quantMode TranscriptomeSAM GeneCounts. Tables of gene counts generated by STAR were imported into R and differential gene expression analysis was performed using DESeq2 to compare exposure groups. Genome build: GRCz11v92 Supplementary files format and content: tab delimited text files include raw count values for each sample. Lrv FLX Bsal whole Exposing to Fluoxetine 54ug/L as treatment or Ethanol 0.0053% as vehicle from 0 dpf to 6 dpf in larvae Qiagene miliamp total RNA extraction column based Illumina TruSeq Stranded mRNA libraries were built following the manufacturer’s protocol using 250ng of starting material.   genotype/variation:SR4G transgenic|stress status:Bsal|drug status:FLX|tissue:larvae head GSM4196104 GSM4196104: Lrv FLX Bsal whole 2; Danio rerio; RNA Seq GSM4196104   1 Qiagene miliamp total RNA extraction column based Illumina TruSeq Stranded mRNA libraries were built following the manufacturer's protocol using 250ng of starting material. GEO Accession:GSM4196104 RNA-Seq TRANSCRIPTOMIC cDNA SINGLE ILLUMINA NextSeq 500   SRP233586     Lrv_FLX_Bsal_2_S51_R1_001.trimmed.fastq.gz fastq 1461206417.0 19438024.0 GSM4196104 r1 0:75.17 1:0 A:356069261;C:366658372;G:336019907;T:402443848;N:15029 75 0     356069261 366658372 336019907 402443848 15029 SRX7232571 SRS5733202 SRA1003923 GEO Mechanistic Studies Division Genomics Laboratory, Health Canada, Government of Canada 1 0.95627   0.11745   0.697   0.47178   76   B   usable mapping rate illumina nextseq unknown cdna_unspecified trueseq bulk unknown unknown   Canada 2019-11-27 Multi-stage Multi-stage Head Nervous System
55551 55551 SRR10550569 SRX7232570 SRS5733201 SRP233586 PRJNA592212 Transcriptional analysis of the effects of early life exposure to fluoxetine on telencephalon and hypothalamus in SR4G transgenic zebrafish larvae and adults GSE141144 Transcriptome Analysis Our previous study suggested persistent transgenerational effects of fluoxetine FLX exposure in both environmental and therapeutic concentrations on stress response and behaviour up to 3 generation withdrawn from exposure in larvae and adult zebrafish. To lay the foundations of the mechanistic of such transgenerational effects we studied the effect of FLX on stress response in brain tissues telencephalon and hypothalamus of larvae and adult zebrafish using differentially expressed genes at the transcriptome level. To monitor the stress response we used a transgenic zebrafish line SR4G which are expressing green fluorescence protein GFP in response to endogenous and exogenous glucocorticoids. The transcriptome analysis of GFP RNA as an internal control provided us with an invaluable tool to clearly visualize and differentiate between stressed and unstressed groups as per treatments. To stimulate stress in both larvae and adult zebrafish we used a modified net stressing technique consisting of consecutive sequence of air exposure and incubation intervals. A pool of 22 larvae heads and a pool of 5 telencephalon and 5 hypothalamus dissected tissues were used as the target tissues for RNA extraction. All experiments were replicated in n=6 per 4 different groups including Control Unstressed Cnt U Control Stressed Cnt S Fluoxetine Unstressed FLX U and Fluoxetine Stressed FLX S using pools of larvae heads telencephalon and hypothalamus. post total RNA extraction using Illumina technology all samples were sequence and the list of differentially expressed genes DEGs were produced using DSEQ pipeline in R. Overall design: Three different target tissue two from 6 mpf adults Telencephalon and Hypothalamus and one from 6 dpf larvae dissected heads. One treatment Fluoxetine One control ethanol as vehicle two condition Stressed Unstressed       Lrv FLX Bsal whole 1 GSM4196103   source name:Lrv FLX Bsal whole|genotype/variation:SR4G transgenic|stress status:Bsal|drug status:FLX|tissue:larvae head Lrv FLX Bsal whole 1 Illumina bcl2fastq v2.17.1.14 software used for basecalling. Sequenced reads were trimmed for adaptor sequence with CutAdapt then aligned with STAR v 2.5 with parameter clip5pNbases 10 and quantMode TranscriptomeSAM GeneCounts. Tables of gene counts generated by STAR were imported into R and differential gene expression analysis was performed using DESeq2 to compare exposure groups. Genome build: GRCz11v92 Supplementary files format and content: tab delimited text files include raw count values for each sample. Lrv FLX Bsal whole Exposing to Fluoxetine 54ug/L as treatment or Ethanol 0.0053% as vehicle from 0 dpf to 6 dpf in larvae Qiagene miliamp total RNA extraction column based Illumina TruSeq Stranded mRNA libraries were built following the manufacturer’s protocol using 250ng of starting material.   genotype/variation:SR4G transgenic|stress status:Bsal|drug status:FLX|tissue:larvae head GSM4196103 GSM4196103: Lrv FLX Bsal whole 1; Danio rerio; RNA Seq GSM4196103   1 Qiagene miliamp total RNA extraction column based Illumina TruSeq Stranded mRNA libraries were built following the manufacturer's protocol using 250ng of starting material. GEO Accession:GSM4196103 RNA-Seq TRANSCRIPTOMIC cDNA SINGLE ILLUMINA NextSeq 500   SRP233586     Lrv_FLX_Bsal_1_S58_R1_001.trimmed.fastq.gz fastq 1274881809.0 16955491.0 GSM4196103 r1 0:75.19 1:0 A:313269199;C:315319719;G:289025501;T:357254917;N:12473 75 0     313269199 315319719 289025501 357254917 12473 SRX7232570 SRS5733201 SRA1003923 GEO Mechanistic Studies Division Genomics Laboratory, Health Canada, Government of Canada 1 0.95224   0.12115   0.69749   0.45972   75   B   usable mapping rate illumina nextseq unknown cdna_unspecified trueseq bulk unknown unknown   Canada 2019-11-27 Multi-stage Multi-stage Head Nervous System
55552 55552 SRR10550568 SRX7232569 SRS5733200 SRP233586 PRJNA592212 Transcriptional analysis of the effects of early life exposure to fluoxetine on telencephalon and hypothalamus in SR4G transgenic zebrafish larvae and adults GSE141144 Transcriptome Analysis Our previous study suggested persistent transgenerational effects of fluoxetine FLX exposure in both environmental and therapeutic concentrations on stress response and behaviour up to 3 generation withdrawn from exposure in larvae and adult zebrafish. To lay the foundations of the mechanistic of such transgenerational effects we studied the effect of FLX on stress response in brain tissues telencephalon and hypothalamus of larvae and adult zebrafish using differentially expressed genes at the transcriptome level. To monitor the stress response we used a transgenic zebrafish line SR4G which are expressing green fluorescence protein GFP in response to endogenous and exogenous glucocorticoids. The transcriptome analysis of GFP RNA as an internal control provided us with an invaluable tool to clearly visualize and differentiate between stressed and unstressed groups as per treatments. To stimulate stress in both larvae and adult zebrafish we used a modified net stressing technique consisting of consecutive sequence of air exposure and incubation intervals. A pool of 22 larvae heads and a pool of 5 telencephalon and 5 hypothalamus dissected tissues were used as the target tissues for RNA extraction. All experiments were replicated in n=6 per 4 different groups including Control Unstressed Cnt U Control Stressed Cnt S Fluoxetine Unstressed FLX U and Fluoxetine Stressed FLX S using pools of larvae heads telencephalon and hypothalamus. post total RNA extraction using Illumina technology all samples were sequence and the list of differentially expressed genes DEGs were produced using DSEQ pipeline in R. Overall design: Three different target tissue two from 6 mpf adults Telencephalon and Hypothalamus and one from 6 dpf larvae dissected heads. One treatment Fluoxetine One control ethanol as vehicle two condition Stressed Unstressed       Lrv FLX Bsal whole 5 GSM4196102   source name:Lrv FLX Bsal whole|genotype/variation:SR4G transgenic|stress status:Bsal|drug status:FLX|tissue:larvae head Lrv FLX Bsal whole 5 Illumina bcl2fastq v2.17.1.14 software used for basecalling. Sequenced reads were trimmed for adaptor sequence with CutAdapt then aligned with STAR v 2.5 with parameter clip5pNbases 10 and quantMode TranscriptomeSAM GeneCounts. Tables of gene counts generated by STAR were imported into R and differential gene expression analysis was performed using DESeq2 to compare exposure groups. Genome build: GRCz11v92 Supplementary files format and content: tab delimited text files include raw count values for each sample. Lrv FLX Bsal whole Exposing to Fluoxetine 54ug/L as treatment or Ethanol 0.0053% as vehicle from 0 dpf to 6 dpf in larvae Qiagene miliamp total RNA extraction column based Illumina TruSeq Stranded mRNA libraries were built following the manufacturer’s protocol using 250ng of starting material.   genotype/variation:SR4G transgenic|stress status:Bsal|drug status:FLX|tissue:larvae head GSM4196102 GSM4196102: Lrv FLX Bsal whole 5; Danio rerio; RNA Seq GSM4196102   1 Qiagene miliamp total RNA extraction column based Illumina TruSeq Stranded mRNA libraries were built following the manufacturer's protocol using 250ng of starting material. GEO Accession:GSM4196102 RNA-Seq TRANSCRIPTOMIC cDNA SINGLE ILLUMINA NextSeq 500   SRP233586     Lrv_FLX_Bsal__5_S65_R1_001.trimmed.fastq.gz fastq 1857265782.0 24701079.0 GSM4196102 r1 0:75.19 1:0 A:462562803;C:458171000;G:416821299;T:519691519;N:19161 75 0     462562803 458171000 416821299 519691519 19161 SRX7232569 SRS5733200 SRA1003923 GEO Mechanistic Studies Division Genomics Laboratory, Health Canada, Government of Canada 1 0.95278   0.11954   0.6871   0.46728   76   B   usable mapping rate illumina nextseq unknown cdna_unspecified trueseq bulk unknown unknown   Canada 2019-11-27 Multi-stage Multi-stage Head Nervous System
55553 55553 SRR10550567 SRX7232568 SRS5733199 SRP233586 PRJNA592212 Transcriptional analysis of the effects of early life exposure to fluoxetine on telencephalon and hypothalamus in SR4G transgenic zebrafish larvae and adults GSE141144 Transcriptome Analysis Our previous study suggested persistent transgenerational effects of fluoxetine FLX exposure in both environmental and therapeutic concentrations on stress response and behaviour up to 3 generation withdrawn from exposure in larvae and adult zebrafish. To lay the foundations of the mechanistic of such transgenerational effects we studied the effect of FLX on stress response in brain tissues telencephalon and hypothalamus of larvae and adult zebrafish using differentially expressed genes at the transcriptome level. To monitor the stress response we used a transgenic zebrafish line SR4G which are expressing green fluorescence protein GFP in response to endogenous and exogenous glucocorticoids. The transcriptome analysis of GFP RNA as an internal control provided us with an invaluable tool to clearly visualize and differentiate between stressed and unstressed groups as per treatments. To stimulate stress in both larvae and adult zebrafish we used a modified net stressing technique consisting of consecutive sequence of air exposure and incubation intervals. A pool of 22 larvae heads and a pool of 5 telencephalon and 5 hypothalamus dissected tissues were used as the target tissues for RNA extraction. All experiments were replicated in n=6 per 4 different groups including Control Unstressed Cnt U Control Stressed Cnt S Fluoxetine Unstressed FLX U and Fluoxetine Stressed FLX S using pools of larvae heads telencephalon and hypothalamus. post total RNA extraction using Illumina technology all samples were sequence and the list of differentially expressed genes DEGs were produced using DSEQ pipeline in R. Overall design: Three different target tissue two from 6 mpf adults Telencephalon and Hypothalamus and one from 6 dpf larvae dissected heads. One treatment Fluoxetine One control ethanol as vehicle two condition Stressed Unstressed       Lrv Ctl Str whole 6 GSM4196101   source name:Lrv Ctl Str whole|genotype/variation:SR4G transgenic|stress status:Str|drug status:Ctl|tissue:larvae head Lrv Ctl Str whole 6 Illumina bcl2fastq v2.17.1.14 software used for basecalling. Sequenced reads were trimmed for adaptor sequence with CutAdapt then aligned with STAR v 2.5 with parameter clip5pNbases 10 and quantMode TranscriptomeSAM GeneCounts. Tables of gene counts generated by STAR were imported into R and differential gene expression analysis was performed using DESeq2 to compare exposure groups. Genome build: GRCz11v92 Supplementary files format and content: tab delimited text files include raw count values for each sample. Lrv Ctl Str whole Exposing to Fluoxetine 54ug/L as treatment or Ethanol 0.0053% as vehicle from 0 dpf to 6 dpf in larvae Qiagene miliamp total RNA extraction column based Illumina TruSeq Stranded mRNA libraries were built following the manufacturer’s protocol using 250ng of starting material.   genotype/variation:SR4G transgenic|stress status:Str|drug status:Ctl|tissue:larvae head GSM4196101 GSM4196101: Lrv Ctl Str whole 6; Danio rerio; RNA Seq GSM4196101   1 Qiagene miliamp total RNA extraction column based Illumina TruSeq Stranded mRNA libraries were built following the manufacturer's protocol using 250ng of starting material. GEO Accession:GSM4196101 RNA-Seq TRANSCRIPTOMIC cDNA SINGLE ILLUMINA NextSeq 500   SRP233586     Lrv_Ctl_Str_6_S71_R1_001.trimmed.fastq.gz fastq 1957288376.0 26046412.0 GSM4196101 r1 0:75.15 1:0 A:514272402;C:461987308;G:431480575;T:549528052;N:20039 75 0     514272402 461987308 431480575 549528052 20039 SRX7232568 SRS5733199 SRA1003923 GEO Mechanistic Studies Division Genomics Laboratory, Health Canada, Government of Canada 1 0.94563   0.13089   0.68682   0.47639   76   B   usable mapping rate illumina nextseq unknown cdna_unspecified trueseq bulk unknown unknown   Canada 2019-11-27 Multi-stage Multi-stage Head Nervous System
55554 55554 SRR10550566 SRX7232567 SRS5733198 SRP233586 PRJNA592212 Transcriptional analysis of the effects of early life exposure to fluoxetine on telencephalon and hypothalamus in SR4G transgenic zebrafish larvae and adults GSE141144 Transcriptome Analysis Our previous study suggested persistent transgenerational effects of fluoxetine FLX exposure in both environmental and therapeutic concentrations on stress response and behaviour up to 3 generation withdrawn from exposure in larvae and adult zebrafish. To lay the foundations of the mechanistic of such transgenerational effects we studied the effect of FLX on stress response in brain tissues telencephalon and hypothalamus of larvae and adult zebrafish using differentially expressed genes at the transcriptome level. To monitor the stress response we used a transgenic zebrafish line SR4G which are expressing green fluorescence protein GFP in response to endogenous and exogenous glucocorticoids. The transcriptome analysis of GFP RNA as an internal control provided us with an invaluable tool to clearly visualize and differentiate between stressed and unstressed groups as per treatments. To stimulate stress in both larvae and adult zebrafish we used a modified net stressing technique consisting of consecutive sequence of air exposure and incubation intervals. A pool of 22 larvae heads and a pool of 5 telencephalon and 5 hypothalamus dissected tissues were used as the target tissues for RNA extraction. All experiments were replicated in n=6 per 4 different groups including Control Unstressed Cnt U Control Stressed Cnt S Fluoxetine Unstressed FLX U and Fluoxetine Stressed FLX S using pools of larvae heads telencephalon and hypothalamus. post total RNA extraction using Illumina technology all samples were sequence and the list of differentially expressed genes DEGs were produced using DSEQ pipeline in R. Overall design: Three different target tissue two from 6 mpf adults Telencephalon and Hypothalamus and one from 6 dpf larvae dissected heads. One treatment Fluoxetine One control ethanol as vehicle two condition Stressed Unstressed       Lrv Ctl Str whole 5 GSM4196100   source name:Lrv Ctl Str whole|genotype/variation:SR4G transgenic|stress status:Str|drug status:Ctl|tissue:larvae head Lrv Ctl Str whole 5 Illumina bcl2fastq v2.17.1.14 software used for basecalling. Sequenced reads were trimmed for adaptor sequence with CutAdapt then aligned with STAR v 2.5 with parameter clip5pNbases 10 and quantMode TranscriptomeSAM GeneCounts. Tables of gene counts generated by STAR were imported into R and differential gene expression analysis was performed using DESeq2 to compare exposure groups. Genome build: GRCz11v92 Supplementary files format and content: tab delimited text files include raw count values for each sample. Lrv Ctl Str whole Exposing to Fluoxetine 54ug/L as treatment or Ethanol 0.0053% as vehicle from 0 dpf to 6 dpf in larvae Qiagene miliamp total RNA extraction column based Illumina TruSeq Stranded mRNA libraries were built following the manufacturer’s protocol using 250ng of starting material.   genotype/variation:SR4G transgenic|stress status:Str|drug status:Ctl|tissue:larvae head GSM4196100 GSM4196100: Lrv Ctl Str whole 5; Danio rerio; RNA Seq GSM4196100   1 Qiagene miliamp total RNA extraction column based Illumina TruSeq Stranded mRNA libraries were built following the manufacturer's protocol using 250ng of starting material. GEO Accession:GSM4196100 RNA-Seq TRANSCRIPTOMIC cDNA SINGLE ILLUMINA NextSeq 500   SRP233586     Lrv_Ctl_Str_5_S68_R1_001.trimmed.fastq.gz fastq 1939162181.0 25790355.0 GSM4196100 r1 0:75.19 1:0 A:478145766;C:489708772;G:441830545;T:529458428;N:18670 75 0     478145766 489708772 441830545 529458428 18670 SRX7232567 SRS5733198 SRA1003923 GEO Mechanistic Studies Division Genomics Laboratory, Health Canada, Government of Canada 1 0.95788   0.13698   0.69564   0.50856   76   B   usable mapping rate illumina nextseq unknown cdna_unspecified trueseq bulk unknown unknown   Canada 2019-11-27 Multi-stage Multi-stage Head Nervous System
55555 55555 SRR10550565 SRX7232566 SRS5733197 SRP233586 PRJNA592212 Transcriptional analysis of the effects of early life exposure to fluoxetine on telencephalon and hypothalamus in SR4G transgenic zebrafish larvae and adults GSE141144 Transcriptome Analysis Our previous study suggested persistent transgenerational effects of fluoxetine FLX exposure in both environmental and therapeutic concentrations on stress response and behaviour up to 3 generation withdrawn from exposure in larvae and adult zebrafish. To lay the foundations of the mechanistic of such transgenerational effects we studied the effect of FLX on stress response in brain tissues telencephalon and hypothalamus of larvae and adult zebrafish using differentially expressed genes at the transcriptome level. To monitor the stress response we used a transgenic zebrafish line SR4G which are expressing green fluorescence protein GFP in response to endogenous and exogenous glucocorticoids. The transcriptome analysis of GFP RNA as an internal control provided us with an invaluable tool to clearly visualize and differentiate between stressed and unstressed groups as per treatments. To stimulate stress in both larvae and adult zebrafish we used a modified net stressing technique consisting of consecutive sequence of air exposure and incubation intervals. A pool of 22 larvae heads and a pool of 5 telencephalon and 5 hypothalamus dissected tissues were used as the target tissues for RNA extraction. All experiments were replicated in n=6 per 4 different groups including Control Unstressed Cnt U Control Stressed Cnt S Fluoxetine Unstressed FLX U and Fluoxetine Stressed FLX S using pools of larvae heads telencephalon and hypothalamus. post total RNA extraction using Illumina technology all samples were sequence and the list of differentially expressed genes DEGs were produced using DSEQ pipeline in R. Overall design: Three different target tissue two from 6 mpf adults Telencephalon and Hypothalamus and one from 6 dpf larvae dissected heads. One treatment Fluoxetine One control ethanol as vehicle two condition Stressed Unstressed       Lrv Ctl Str whole 4 GSM4196099   source name:Lrv Ctl Str whole|genotype/variation:SR4G transgenic|stress status:Str|drug status:Ctl|tissue:larvae head Lrv Ctl Str whole 4 Illumina bcl2fastq v2.17.1.14 software used for basecalling. Sequenced reads were trimmed for adaptor sequence with CutAdapt then aligned with STAR v 2.5 with parameter clip5pNbases 10 and quantMode TranscriptomeSAM GeneCounts. Tables of gene counts generated by STAR were imported into R and differential gene expression analysis was performed using DESeq2 to compare exposure groups. Genome build: GRCz11v92 Supplementary files format and content: tab delimited text files include raw count values for each sample. Lrv Ctl Str whole Exposing to Fluoxetine 54ug/L as treatment or Ethanol 0.0053% as vehicle from 0 dpf to 6 dpf in larvae Qiagene miliamp total RNA extraction column based Illumina TruSeq Stranded mRNA libraries were built following the manufacturer’s protocol using 250ng of starting material.   genotype/variation:SR4G transgenic|stress status:Str|drug status:Ctl|tissue:larvae head GSM4196099 GSM4196099: Lrv Ctl Str whole 4; Danio rerio; RNA Seq GSM4196099   1 Qiagene miliamp total RNA extraction column based Illumina TruSeq Stranded mRNA libraries were built following the manufacturer's protocol using 250ng of starting material. GEO Accession:GSM4196099 RNA-Seq TRANSCRIPTOMIC cDNA SINGLE ILLUMINA NextSeq 500   SRP233586     Lrv_Ctl_Str_4_S63_R1_001.trimmed.fastq.gz fastq 1661252715.0 22112240.0 GSM4196099 r1 0:75.13 1:0 A:426247799;C:401717650;G:370519308;T:462751124;N:16834 75 0     426247799 401717650 370519308 462751124 16834 SRX7232566 SRS5733197 SRA1003923 GEO Mechanistic Studies Division Genomics Laboratory, Health Canada, Government of Canada 1 0.95211   0.11993   0.68905   0.47345   76   B   usable mapping rate illumina nextseq unknown cdna_unspecified trueseq bulk unknown unknown   Canada 2019-11-27 Multi-stage Multi-stage Head Nervous System
55556 55556 SRR10550564 SRX7232565 SRS5733196 SRP233586 PRJNA592212 Transcriptional analysis of the effects of early life exposure to fluoxetine on telencephalon and hypothalamus in SR4G transgenic zebrafish larvae and adults GSE141144 Transcriptome Analysis Our previous study suggested persistent transgenerational effects of fluoxetine FLX exposure in both environmental and therapeutic concentrations on stress response and behaviour up to 3 generation withdrawn from exposure in larvae and adult zebrafish. To lay the foundations of the mechanistic of such transgenerational effects we studied the effect of FLX on stress response in brain tissues telencephalon and hypothalamus of larvae and adult zebrafish using differentially expressed genes at the transcriptome level. To monitor the stress response we used a transgenic zebrafish line SR4G which are expressing green fluorescence protein GFP in response to endogenous and exogenous glucocorticoids. The transcriptome analysis of GFP RNA as an internal control provided us with an invaluable tool to clearly visualize and differentiate between stressed and unstressed groups as per treatments. To stimulate stress in both larvae and adult zebrafish we used a modified net stressing technique consisting of consecutive sequence of air exposure and incubation intervals. A pool of 22 larvae heads and a pool of 5 telencephalon and 5 hypothalamus dissected tissues were used as the target tissues for RNA extraction. All experiments were replicated in n=6 per 4 different groups including Control Unstressed Cnt U Control Stressed Cnt S Fluoxetine Unstressed FLX U and Fluoxetine Stressed FLX S using pools of larvae heads telencephalon and hypothalamus. post total RNA extraction using Illumina technology all samples were sequence and the list of differentially expressed genes DEGs were produced using DSEQ pipeline in R. Overall design: Three different target tissue two from 6 mpf adults Telencephalon and Hypothalamus and one from 6 dpf larvae dissected heads. One treatment Fluoxetine One control ethanol as vehicle two condition Stressed Unstressed       Lrv Ctl Str whole 3 GSM4196098   source name:Lrv Ctl Str whole|genotype/variation:SR4G transgenic|stress status:Str|drug status:Ctl|tissue:larvae head Lrv Ctl Str whole 3 Illumina bcl2fastq v2.17.1.14 software used for basecalling. Sequenced reads were trimmed for adaptor sequence with CutAdapt then aligned with STAR v 2.5 with parameter clip5pNbases 10 and quantMode TranscriptomeSAM GeneCounts. Tables of gene counts generated by STAR were imported into R and differential gene expression analysis was performed using DESeq2 to compare exposure groups. Genome build: GRCz11v92 Supplementary files format and content: tab delimited text files include raw count values for each sample. Lrv Ctl Str whole Exposing to Fluoxetine 54ug/L as treatment or Ethanol 0.0053% as vehicle from 0 dpf to 6 dpf in larvae Qiagene miliamp total RNA extraction column based Illumina TruSeq Stranded mRNA libraries were built following the manufacturer’s protocol using 250ng of starting material.   genotype/variation:SR4G transgenic|stress status:Str|drug status:Ctl|tissue:larvae head GSM4196098 GSM4196098: Lrv Ctl Str whole 3; Danio rerio; RNA Seq GSM4196098   1 Qiagene miliamp total RNA extraction column based Illumina TruSeq Stranded mRNA libraries were built following the manufacturer's protocol using 250ng of starting material. GEO Accession:GSM4196098 RNA-Seq TRANSCRIPTOMIC cDNA SINGLE ILLUMINA NextSeq 500   SRP233586     Lrv_Ctl_Str_3_S61_R1_001.trimmed.fastq.gz fastq 2128433399.0 28315796.0 GSM4196098 r1 0:75.17 1:0 A:527968641;C:529216046;G:482537279;T:588689313;N:22120 75 0     527968641 529216046 482537279 588689313 22120 SRX7232565 SRS5733196 SRA1003923 GEO Mechanistic Studies Division Genomics Laboratory, Health Canada, Government of Canada 1 0.95484   0.12615   0.69899   0.48613   76   B   usable mapping rate illumina nextseq unknown cdna_unspecified trueseq bulk unknown unknown   Canada 2019-11-27 Multi-stage Multi-stage Head Nervous System
55557 55557 SRR10550563 SRX7232564 SRS5733195 SRP233586 PRJNA592212 Transcriptional analysis of the effects of early life exposure to fluoxetine on telencephalon and hypothalamus in SR4G transgenic zebrafish larvae and adults GSE141144 Transcriptome Analysis Our previous study suggested persistent transgenerational effects of fluoxetine FLX exposure in both environmental and therapeutic concentrations on stress response and behaviour up to 3 generation withdrawn from exposure in larvae and adult zebrafish. To lay the foundations of the mechanistic of such transgenerational effects we studied the effect of FLX on stress response in brain tissues telencephalon and hypothalamus of larvae and adult zebrafish using differentially expressed genes at the transcriptome level. To monitor the stress response we used a transgenic zebrafish line SR4G which are expressing green fluorescence protein GFP in response to endogenous and exogenous glucocorticoids. The transcriptome analysis of GFP RNA as an internal control provided us with an invaluable tool to clearly visualize and differentiate between stressed and unstressed groups as per treatments. To stimulate stress in both larvae and adult zebrafish we used a modified net stressing technique consisting of consecutive sequence of air exposure and incubation intervals. A pool of 22 larvae heads and a pool of 5 telencephalon and 5 hypothalamus dissected tissues were used as the target tissues for RNA extraction. All experiments were replicated in n=6 per 4 different groups including Control Unstressed Cnt U Control Stressed Cnt S Fluoxetine Unstressed FLX U and Fluoxetine Stressed FLX S using pools of larvae heads telencephalon and hypothalamus. post total RNA extraction using Illumina technology all samples were sequence and the list of differentially expressed genes DEGs were produced using DSEQ pipeline in R. Overall design: Three different target tissue two from 6 mpf adults Telencephalon and Hypothalamus and one from 6 dpf larvae dissected heads. One treatment Fluoxetine One control ethanol as vehicle two condition Stressed Unstressed       Lrv Ctl Str whole 2 GSM4196097   source name:Lrv Ctl Str whole|genotype/variation:SR4G transgenic|stress status:Str|drug status:Ctl|tissue:larvae head Lrv Ctl Str whole 2 Illumina bcl2fastq v2.17.1.14 software used for basecalling. Sequenced reads were trimmed for adaptor sequence with CutAdapt then aligned with STAR v 2.5 with parameter clip5pNbases 10 and quantMode TranscriptomeSAM GeneCounts. Tables of gene counts generated by STAR were imported into R and differential gene expression analysis was performed using DESeq2 to compare exposure groups. Genome build: GRCz11v92 Supplementary files format and content: tab delimited text files include raw count values for each sample. Lrv Ctl Str whole Exposing to Fluoxetine 54ug/L as treatment or Ethanol 0.0053% as vehicle from 0 dpf to 6 dpf in larvae Qiagene miliamp total RNA extraction column based Illumina TruSeq Stranded mRNA libraries were built following the manufacturer’s protocol using 250ng of starting material.   genotype/variation:SR4G transgenic|stress status:Str|drug status:Ctl|tissue:larvae head GSM4196097 GSM4196097: Lrv Ctl Str whole 2; Danio rerio; RNA Seq GSM4196097   1 Qiagene miliamp total RNA extraction column based Illumina TruSeq Stranded mRNA libraries were built following the manufacturer's protocol using 250ng of starting material. GEO Accession:GSM4196097 RNA-Seq TRANSCRIPTOMIC cDNA SINGLE ILLUMINA NextSeq 500   SRP233586     Lrv_Ctl_Str_2_S49_R1_001.trimmed.fastq.gz fastq 1308174499.0 17399094.0 GSM4196097 r1 0:75.19 1:0 A:324097251;C:321883043;G:297347816;T:364832719;N:13670 75 0     324097251 321883043 297347816 364832719 13670 SRX7232564 SRS5733195 SRA1003923 GEO Mechanistic Studies Division Genomics Laboratory, Health Canada, Government of Canada 1 0.95352   0.12304   0.70305   0.47639   76   B   usable mapping rate illumina nextseq unknown cdna_unspecified trueseq bulk unknown unknown   Canada 2019-11-27 Multi-stage Multi-stage Head Nervous System
55558 55558 SRR10550562 SRX7232563 SRS5733194 SRP233586 PRJNA592212 Transcriptional analysis of the effects of early life exposure to fluoxetine on telencephalon and hypothalamus in SR4G transgenic zebrafish larvae and adults GSE141144 Transcriptome Analysis Our previous study suggested persistent transgenerational effects of fluoxetine FLX exposure in both environmental and therapeutic concentrations on stress response and behaviour up to 3 generation withdrawn from exposure in larvae and adult zebrafish. To lay the foundations of the mechanistic of such transgenerational effects we studied the effect of FLX on stress response in brain tissues telencephalon and hypothalamus of larvae and adult zebrafish using differentially expressed genes at the transcriptome level. To monitor the stress response we used a transgenic zebrafish line SR4G which are expressing green fluorescence protein GFP in response to endogenous and exogenous glucocorticoids. The transcriptome analysis of GFP RNA as an internal control provided us with an invaluable tool to clearly visualize and differentiate between stressed and unstressed groups as per treatments. To stimulate stress in both larvae and adult zebrafish we used a modified net stressing technique consisting of consecutive sequence of air exposure and incubation intervals. A pool of 22 larvae heads and a pool of 5 telencephalon and 5 hypothalamus dissected tissues were used as the target tissues for RNA extraction. All experiments were replicated in n=6 per 4 different groups including Control Unstressed Cnt U Control Stressed Cnt S Fluoxetine Unstressed FLX U and Fluoxetine Stressed FLX S using pools of larvae heads telencephalon and hypothalamus. post total RNA extraction using Illumina technology all samples were sequence and the list of differentially expressed genes DEGs were produced using DSEQ pipeline in R. Overall design: Three different target tissue two from 6 mpf adults Telencephalon and Hypothalamus and one from 6 dpf larvae dissected heads. One treatment Fluoxetine One control ethanol as vehicle two condition Stressed Unstressed       Lrv Ctl Str whole 1 GSM4196096   source name:Lrv Ctl Str whole|genotype/variation:SR4G transgenic|stress status:Str|drug status:Ctl|tissue:larvae head Lrv Ctl Str whole 1 Illumina bcl2fastq v2.17.1.14 software used for basecalling. Sequenced reads were trimmed for adaptor sequence with CutAdapt then aligned with STAR v 2.5 with parameter clip5pNbases 10 and quantMode TranscriptomeSAM GeneCounts. Tables of gene counts generated by STAR were imported into R and differential gene expression analysis was performed using DESeq2 to compare exposure groups. Genome build: GRCz11v92 Supplementary files format and content: tab delimited text files include raw count values for each sample. Lrv Ctl Str whole Exposing to Fluoxetine 54ug/L as treatment or Ethanol 0.0053% as vehicle from 0 dpf to 6 dpf in larvae Qiagene miliamp total RNA extraction column based Illumina TruSeq Stranded mRNA libraries were built following the manufacturer’s protocol using 250ng of starting material.   genotype/variation:SR4G transgenic|stress status:Str|drug status:Ctl|tissue:larvae head GSM4196096 GSM4196096: Lrv Ctl Str whole 1; Danio rerio; RNA Seq GSM4196096   1 Qiagene miliamp total RNA extraction column based Illumina TruSeq Stranded mRNA libraries were built following the manufacturer's protocol using 250ng of starting material. GEO Accession:GSM4196096 RNA-Seq TRANSCRIPTOMIC cDNA SINGLE ILLUMINA NextSeq 500   SRP233586     Lrv_Ctl_Str_1_S56_R1_001.trimmed.fastq.gz fastq 1578778770.0 21026926.0 GSM4196096 r1 0:75.08 1:0 A:402927986;C:380655464;G:355045267;T:440133814;N:16239 75 0     402927986 380655464 355045267 440133814 16239 SRX7232563 SRS5733194 SRA1003923 GEO Mechanistic Studies Division Genomics Laboratory, Health Canada, Government of Canada 1 0.95088   0.13051   0.69686   0.48234   71   B   usable mapping rate illumina nextseq unknown cdna_unspecified trueseq bulk unknown unknown   Canada 2019-11-27 Multi-stage Multi-stage Head Nervous System
55559 55559 SRR10550561 SRX7232562 SRS5733193 SRP233586 PRJNA592212 Transcriptional analysis of the effects of early life exposure to fluoxetine on telencephalon and hypothalamus in SR4G transgenic zebrafish larvae and adults GSE141144 Transcriptome Analysis Our previous study suggested persistent transgenerational effects of fluoxetine FLX exposure in both environmental and therapeutic concentrations on stress response and behaviour up to 3 generation withdrawn from exposure in larvae and adult zebrafish. To lay the foundations of the mechanistic of such transgenerational effects we studied the effect of FLX on stress response in brain tissues telencephalon and hypothalamus of larvae and adult zebrafish using differentially expressed genes at the transcriptome level. To monitor the stress response we used a transgenic zebrafish line SR4G which are expressing green fluorescence protein GFP in response to endogenous and exogenous glucocorticoids. The transcriptome analysis of GFP RNA as an internal control provided us with an invaluable tool to clearly visualize and differentiate between stressed and unstressed groups as per treatments. To stimulate stress in both larvae and adult zebrafish we used a modified net stressing technique consisting of consecutive sequence of air exposure and incubation intervals. A pool of 22 larvae heads and a pool of 5 telencephalon and 5 hypothalamus dissected tissues were used as the target tissues for RNA extraction. All experiments were replicated in n=6 per 4 different groups including Control Unstressed Cnt U Control Stressed Cnt S Fluoxetine Unstressed FLX U and Fluoxetine Stressed FLX S using pools of larvae heads telencephalon and hypothalamus. post total RNA extraction using Illumina technology all samples were sequence and the list of differentially expressed genes DEGs were produced using DSEQ pipeline in R. Overall design: Three different target tissue two from 6 mpf adults Telencephalon and Hypothalamus and one from 6 dpf larvae dissected heads. One treatment Fluoxetine One control ethanol as vehicle two condition Stressed Unstressed       Lrv Ctl Bsal whole 6 GSM4196095   source name:Lrv Ctl Bsal whole|genotype/variation:SR4G transgenic|stress status:Bsal|drug status:Ctl|tissue:larvae head Lrv Ctl Bsal whole 6 Illumina bcl2fastq v2.17.1.14 software used for basecalling. Sequenced reads were trimmed for adaptor sequence with CutAdapt then aligned with STAR v 2.5 with parameter clip5pNbases 10 and quantMode TranscriptomeSAM GeneCounts. Tables of gene counts generated by STAR were imported into R and differential gene expression analysis was performed using DESeq2 to compare exposure groups. Genome build: GRCz11v92 Supplementary files format and content: tab delimited text files include raw count values for each sample. Lrv Ctl Bsal whole Exposing to Fluoxetine 54ug/L as treatment or Ethanol 0.0053% as vehicle from 0 dpf to 6 dpf in larvae Qiagene miliamp total RNA extraction column based Illumina TruSeq Stranded mRNA libraries were built following the manufacturer’s protocol using 250ng of starting material.   genotype/variation:SR4G transgenic|stress status:Bsal|drug status:Ctl|tissue:larvae head GSM4196095 GSM4196095: Lrv Ctl Bsal whole 6; Danio rerio; RNA Seq GSM4196095   1 Qiagene miliamp total RNA extraction column based Illumina TruSeq Stranded mRNA libraries were built following the manufacturer's protocol using 250ng of starting material. GEO Accession:GSM4196095 RNA-Seq TRANSCRIPTOMIC cDNA SINGLE ILLUMINA NextSeq 500   SRP233586     Lrv_Ctl_Bsal_6_S67_R1_001.trimmed.fastq.gz fastq 1996483532.0 26552956.0 GSM4196095 r1 0:75.19 1:0 A:486227608;C:499380161;G:458068110;T:552786593;N:21060 75 0     486227608 499380161 458068110 552786593 21060 SRX7232562 SRS5733193 SRA1003923 GEO Mechanistic Studies Division Genomics Laboratory, Health Canada, Government of Canada 1 0.95673   0.10298   0.68511   0.46098   73   B   usable mapping rate illumina nextseq unknown cdna_unspecified trueseq bulk unknown unknown   Canada 2019-11-27 Multi-stage Multi-stage Head Nervous System
55560 55560 SRR10550560 SRX7232561 SRS5733192 SRP233586 PRJNA592212 Transcriptional analysis of the effects of early life exposure to fluoxetine on telencephalon and hypothalamus in SR4G transgenic zebrafish larvae and adults GSE141144 Transcriptome Analysis Our previous study suggested persistent transgenerational effects of fluoxetine FLX exposure in both environmental and therapeutic concentrations on stress response and behaviour up to 3 generation withdrawn from exposure in larvae and adult zebrafish. To lay the foundations of the mechanistic of such transgenerational effects we studied the effect of FLX on stress response in brain tissues telencephalon and hypothalamus of larvae and adult zebrafish using differentially expressed genes at the transcriptome level. To monitor the stress response we used a transgenic zebrafish line SR4G which are expressing green fluorescence protein GFP in response to endogenous and exogenous glucocorticoids. The transcriptome analysis of GFP RNA as an internal control provided us with an invaluable tool to clearly visualize and differentiate between stressed and unstressed groups as per treatments. To stimulate stress in both larvae and adult zebrafish we used a modified net stressing technique consisting of consecutive sequence of air exposure and incubation intervals. A pool of 22 larvae heads and a pool of 5 telencephalon and 5 hypothalamus dissected tissues were used as the target tissues for RNA extraction. All experiments were replicated in n=6 per 4 different groups including Control Unstressed Cnt U Control Stressed Cnt S Fluoxetine Unstressed FLX U and Fluoxetine Stressed FLX S using pools of larvae heads telencephalon and hypothalamus. post total RNA extraction using Illumina technology all samples were sequence and the list of differentially expressed genes DEGs were produced using DSEQ pipeline in R. Overall design: Three different target tissue two from 6 mpf adults Telencephalon and Hypothalamus and one from 6 dpf larvae dissected heads. One treatment Fluoxetine One control ethanol as vehicle two condition Stressed Unstressed       Lrv Ctl Bsal whole 4 GSM4196094   source name:Lrv Ctl Bsal whole|genotype/variation:SR4G transgenic|stress status:Bsal|drug status:Ctl|tissue:larvae head Lrv Ctl Bsal whole 4 Illumina bcl2fastq v2.17.1.14 software used for basecalling. Sequenced reads were trimmed for adaptor sequence with CutAdapt then aligned with STAR v 2.5 with parameter clip5pNbases 10 and quantMode TranscriptomeSAM GeneCounts. Tables of gene counts generated by STAR were imported into R and differential gene expression analysis was performed using DESeq2 to compare exposure groups. Genome build: GRCz11v92 Supplementary files format and content: tab delimited text files include raw count values for each sample. Lrv Ctl Bsal whole Exposing to Fluoxetine 54ug/L as treatment or Ethanol 0.0053% as vehicle from 0 dpf to 6 dpf in larvae Qiagene miliamp total RNA extraction column based Illumina TruSeq Stranded mRNA libraries were built following the manufacturer’s protocol using 250ng of starting material.   genotype/variation:SR4G transgenic|stress status:Bsal|drug status:Ctl|tissue:larvae head GSM4196094 GSM4196094: Lrv Ctl Bsal whole 4; Danio rerio; RNA Seq GSM4196094   1 Qiagene miliamp total RNA extraction column based Illumina TruSeq Stranded mRNA libraries were built following the manufacturer's protocol using 250ng of starting material. GEO Accession:GSM4196094 RNA-Seq TRANSCRIPTOMIC cDNA SINGLE ILLUMINA NextSeq 500   SRP233586     Lrv_Ctl_Bsal_4_S54_R1_001.trimmed.fastq.gz fastq 1827456990.0 24323614.0 GSM4196094 r1 0:75.13 1:0 A:482938455;C:430636548;G:401296463;T:512566219;N:19305 75 0     482938455 430636548 401296463 512566219 19305 SRX7232561 SRS5733192 SRA1003923 GEO Mechanistic Studies Division Genomics Laboratory, Health Canada, Government of Canada 1 0.94602   0.14275   0.69777   0.46678   76   B   usable mapping rate illumina nextseq unknown cdna_unspecified trueseq bulk unknown unknown   Canada 2019-11-27 Multi-stage Multi-stage Head Nervous System
55561 55561 SRR10550559 SRX7232560 SRS5733191 SRP233586 PRJNA592212 Transcriptional analysis of the effects of early life exposure to fluoxetine on telencephalon and hypothalamus in SR4G transgenic zebrafish larvae and adults GSE141144 Transcriptome Analysis Our previous study suggested persistent transgenerational effects of fluoxetine FLX exposure in both environmental and therapeutic concentrations on stress response and behaviour up to 3 generation withdrawn from exposure in larvae and adult zebrafish. To lay the foundations of the mechanistic of such transgenerational effects we studied the effect of FLX on stress response in brain tissues telencephalon and hypothalamus of larvae and adult zebrafish using differentially expressed genes at the transcriptome level. To monitor the stress response we used a transgenic zebrafish line SR4G which are expressing green fluorescence protein GFP in response to endogenous and exogenous glucocorticoids. The transcriptome analysis of GFP RNA as an internal control provided us with an invaluable tool to clearly visualize and differentiate between stressed and unstressed groups as per treatments. To stimulate stress in both larvae and adult zebrafish we used a modified net stressing technique consisting of consecutive sequence of air exposure and incubation intervals. A pool of 22 larvae heads and a pool of 5 telencephalon and 5 hypothalamus dissected tissues were used as the target tissues for RNA extraction. All experiments were replicated in n=6 per 4 different groups including Control Unstressed Cnt U Control Stressed Cnt S Fluoxetine Unstressed FLX U and Fluoxetine Stressed FLX S using pools of larvae heads telencephalon and hypothalamus. post total RNA extraction using Illumina technology all samples were sequence and the list of differentially expressed genes DEGs were produced using DSEQ pipeline in R. Overall design: Three different target tissue two from 6 mpf adults Telencephalon and Hypothalamus and one from 6 dpf larvae dissected heads. One treatment Fluoxetine One control ethanol as vehicle two condition Stressed Unstressed       Lrv Ctl Bsal whole 3 GSM4196093   source name:Lrv Ctl Bsal whole|genotype/variation:SR4G transgenic|stress status:Bsal|drug status:Ctl|tissue:larvae head Lrv Ctl Bsal whole 3 Illumina bcl2fastq v2.17.1.14 software used for basecalling. Sequenced reads were trimmed for adaptor sequence with CutAdapt then aligned with STAR v 2.5 with parameter clip5pNbases 10 and quantMode TranscriptomeSAM GeneCounts. Tables of gene counts generated by STAR were imported into R and differential gene expression analysis was performed using DESeq2 to compare exposure groups. Genome build: GRCz11v92 Supplementary files format and content: tab delimited text files include raw count values for each sample. Lrv Ctl Bsal whole Exposing to Fluoxetine 54ug/L as treatment or Ethanol 0.0053% as vehicle from 0 dpf to 6 dpf in larvae Qiagene miliamp total RNA extraction column based Illumina TruSeq Stranded mRNA libraries were built following the manufacturer’s protocol using 250ng of starting material.   genotype/variation:SR4G transgenic|stress status:Bsal|drug status:Ctl|tissue:larvae head GSM4196093 GSM4196093: Lrv Ctl Bsal whole 3; Danio rerio; RNA Seq GSM4196093   1 Qiagene miliamp total RNA extraction column based Illumina TruSeq Stranded mRNA libraries were built following the manufacturer's protocol using 250ng of starting material. GEO Accession:GSM4196093 RNA-Seq TRANSCRIPTOMIC cDNA SINGLE ILLUMINA NextSeq 500   SRP233586     Lrv_Ctl_Bsal_3_S72_R1_001.trimmed.fastq.gz fastq 1920853986.0 25551216.0 GSM4196093 r1 0:75.18 1:0 A:488636639;C:465699839;G:429574280;T:536923951;N:19277 75 0     488636639 465699839 429574280 536923951 19277 SRX7232560 SRS5733191 SRA1003923 GEO Mechanistic Studies Division Genomics Laboratory, Health Canada, Government of Canada 1 0.95469   0.09186   0.66373   0.47662   76   B   usable mapping rate illumina nextseq unknown cdna_unspecified trueseq bulk unknown unknown   Canada 2019-11-27 Multi-stage Multi-stage Head Nervous System
55562 55562 SRR10550558 SRX7232559 SRS5733190 SRP233586 PRJNA592212 Transcriptional analysis of the effects of early life exposure to fluoxetine on telencephalon and hypothalamus in SR4G transgenic zebrafish larvae and adults GSE141144 Transcriptome Analysis Our previous study suggested persistent transgenerational effects of fluoxetine FLX exposure in both environmental and therapeutic concentrations on stress response and behaviour up to 3 generation withdrawn from exposure in larvae and adult zebrafish. To lay the foundations of the mechanistic of such transgenerational effects we studied the effect of FLX on stress response in brain tissues telencephalon and hypothalamus of larvae and adult zebrafish using differentially expressed genes at the transcriptome level. To monitor the stress response we used a transgenic zebrafish line SR4G which are expressing green fluorescence protein GFP in response to endogenous and exogenous glucocorticoids. The transcriptome analysis of GFP RNA as an internal control provided us with an invaluable tool to clearly visualize and differentiate between stressed and unstressed groups as per treatments. To stimulate stress in both larvae and adult zebrafish we used a modified net stressing technique consisting of consecutive sequence of air exposure and incubation intervals. A pool of 22 larvae heads and a pool of 5 telencephalon and 5 hypothalamus dissected tissues were used as the target tissues for RNA extraction. All experiments were replicated in n=6 per 4 different groups including Control Unstressed Cnt U Control Stressed Cnt S Fluoxetine Unstressed FLX U and Fluoxetine Stressed FLX S using pools of larvae heads telencephalon and hypothalamus. post total RNA extraction using Illumina technology all samples were sequence and the list of differentially expressed genes DEGs were produced using DSEQ pipeline in R. Overall design: Three different target tissue two from 6 mpf adults Telencephalon and Hypothalamus and one from 6 dpf larvae dissected heads. One treatment Fluoxetine One control ethanol as vehicle two condition Stressed Unstressed       Lrv Ctl Bsal whole 2 GSM4196092   source name:Lrv Ctl Bsal whole|genotype/variation:SR4G transgenic|stress status:Bsal|drug status:Ctl|tissue:larvae head Lrv Ctl Bsal whole 2 Illumina bcl2fastq v2.17.1.14 software used for basecalling. Sequenced reads were trimmed for adaptor sequence with CutAdapt then aligned with STAR v 2.5 with parameter clip5pNbases 10 and quantMode TranscriptomeSAM GeneCounts. Tables of gene counts generated by STAR were imported into R and differential gene expression analysis was performed using DESeq2 to compare exposure groups. Genome build: GRCz11v92 Supplementary files format and content: tab delimited text files include raw count values for each sample. Lrv Ctl Bsal whole Exposing to Fluoxetine 54ug/L as treatment or Ethanol 0.0053% as vehicle from 0 dpf to 6 dpf in larvae Qiagene miliamp total RNA extraction column based Illumina TruSeq Stranded mRNA libraries were built following the manufacturer’s protocol using 250ng of starting material.   genotype/variation:SR4G transgenic|stress status:Bsal|drug status:Ctl|tissue:larvae head GSM4196092 GSM4196092: Lrv Ctl Bsal whole 2; Danio rerio; RNA Seq GSM4196092   1 Qiagene miliamp total RNA extraction column based Illumina TruSeq Stranded mRNA libraries were built following the manufacturer's protocol using 250ng of starting material. GEO Accession:GSM4196092 RNA-Seq TRANSCRIPTOMIC cDNA SINGLE ILLUMINA NextSeq 500   SRP233586     Lrv_Ctl_Bsal_2_S66_R1_001.trimmed.fastq.gz fastq 1715077991.0 22808137.0 GSM4196092 r1 0:75.20 1:0 A:412795011;C:426393295;G:392381205;T:483491023;N:17457 75 0     412795011 426393295 392381205 483491023 17457 SRX7232559 SRS5733190 SRA1003923 GEO Mechanistic Studies Division Genomics Laboratory, Health Canada, Government of Canada 1 0.95866   0.07909   0.66352   0.47768   76   B   usable mapping rate illumina nextseq unknown cdna_unspecified trueseq bulk unknown unknown   Canada 2019-11-27 Multi-stage Multi-stage Head Nervous System
55563 55563 SRR10550557 SRX7232558 SRS5733189 SRP233586 PRJNA592212 Transcriptional analysis of the effects of early life exposure to fluoxetine on telencephalon and hypothalamus in SR4G transgenic zebrafish larvae and adults GSE141144 Transcriptome Analysis Our previous study suggested persistent transgenerational effects of fluoxetine FLX exposure in both environmental and therapeutic concentrations on stress response and behaviour up to 3 generation withdrawn from exposure in larvae and adult zebrafish. To lay the foundations of the mechanistic of such transgenerational effects we studied the effect of FLX on stress response in brain tissues telencephalon and hypothalamus of larvae and adult zebrafish using differentially expressed genes at the transcriptome level. To monitor the stress response we used a transgenic zebrafish line SR4G which are expressing green fluorescence protein GFP in response to endogenous and exogenous glucocorticoids. The transcriptome analysis of GFP RNA as an internal control provided us with an invaluable tool to clearly visualize and differentiate between stressed and unstressed groups as per treatments. To stimulate stress in both larvae and adult zebrafish we used a modified net stressing technique consisting of consecutive sequence of air exposure and incubation intervals. A pool of 22 larvae heads and a pool of 5 telencephalon and 5 hypothalamus dissected tissues were used as the target tissues for RNA extraction. All experiments were replicated in n=6 per 4 different groups including Control Unstressed Cnt U Control Stressed Cnt S Fluoxetine Unstressed FLX U and Fluoxetine Stressed FLX S using pools of larvae heads telencephalon and hypothalamus. post total RNA extraction using Illumina technology all samples were sequence and the list of differentially expressed genes DEGs were produced using DSEQ pipeline in R. Overall design: Three different target tissue two from 6 mpf adults Telencephalon and Hypothalamus and one from 6 dpf larvae dissected heads. One treatment Fluoxetine One control ethanol as vehicle two condition Stressed Unstressed       Lrv Ctl Bsal whole 1 GSM4196091   source name:Lrv Ctl Bsal whole|genotype/variation:SR4G transgenic|stress status:Bsal|drug status:Ctl|tissue:larvae head Lrv Ctl Bsal whole 1 Illumina bcl2fastq v2.17.1.14 software used for basecalling. Sequenced reads were trimmed for adaptor sequence with CutAdapt then aligned with STAR v 2.5 with parameter clip5pNbases 10 and quantMode TranscriptomeSAM GeneCounts. Tables of gene counts generated by STAR were imported into R and differential gene expression analysis was performed using DESeq2 to compare exposure groups. Genome build: GRCz11v92 Supplementary files format and content: tab delimited text files include raw count values for each sample. Lrv Ctl Bsal whole Exposing to Fluoxetine 54ug/L as treatment or Ethanol 0.0053% as vehicle from 0 dpf to 6 dpf in larvae Qiagene miliamp total RNA extraction column based Illumina TruSeq Stranded mRNA libraries were built following the manufacturer’s protocol using 250ng of starting material.   genotype/variation:SR4G transgenic|stress status:Bsal|drug status:Ctl|tissue:larvae head GSM4196091 GSM4196091: Lrv Ctl Bsal whole 1; Danio rerio; RNA Seq GSM4196091   1 Qiagene miliamp total RNA extraction column based Illumina TruSeq Stranded mRNA libraries were built following the manufacturer's protocol using 250ng of starting material. GEO Accession:GSM4196091 RNA-Seq TRANSCRIPTOMIC cDNA SINGLE ILLUMINA NextSeq 500   SRP233586     Lrv_Ctl_Bsal_1_S52_R1_001.trimmed.fastq.gz fastq 1771075713.0 23584154.0 GSM4196091 r1 0:75.10 1:0 A:431209517;C:441715839;G:407237232;T:490894491;N:18634 75 0     431209517 441715839 407237232 490894491 18634 SRX7232558 SRS5733189 SRA1003923 GEO Mechanistic Studies Division Genomics Laboratory, Health Canada, Government of Canada 1 0.95568   0.10232   0.65904   0.49156   76   B   usable mapping rate illumina nextseq unknown cdna_unspecified trueseq bulk unknown unknown   Canada 2019-11-27 Multi-stage Multi-stage Head Nervous System
55564 55564 SRR10550556 SRX7232557 SRS5733188 SRP233586 PRJNA592212 Transcriptional analysis of the effects of early life exposure to fluoxetine on telencephalon and hypothalamus in SR4G transgenic zebrafish larvae and adults GSE141144 Transcriptome Analysis Our previous study suggested persistent transgenerational effects of fluoxetine FLX exposure in both environmental and therapeutic concentrations on stress response and behaviour up to 3 generation withdrawn from exposure in larvae and adult zebrafish. To lay the foundations of the mechanistic of such transgenerational effects we studied the effect of FLX on stress response in brain tissues telencephalon and hypothalamus of larvae and adult zebrafish using differentially expressed genes at the transcriptome level. To monitor the stress response we used a transgenic zebrafish line SR4G which are expressing green fluorescence protein GFP in response to endogenous and exogenous glucocorticoids. The transcriptome analysis of GFP RNA as an internal control provided us with an invaluable tool to clearly visualize and differentiate between stressed and unstressed groups as per treatments. To stimulate stress in both larvae and adult zebrafish we used a modified net stressing technique consisting of consecutive sequence of air exposure and incubation intervals. A pool of 22 larvae heads and a pool of 5 telencephalon and 5 hypothalamus dissected tissues were used as the target tissues for RNA extraction. All experiments were replicated in n=6 per 4 different groups including Control Unstressed Cnt U Control Stressed Cnt S Fluoxetine Unstressed FLX U and Fluoxetine Stressed FLX S using pools of larvae heads telencephalon and hypothalamus. post total RNA extraction using Illumina technology all samples were sequence and the list of differentially expressed genes DEGs were produced using DSEQ pipeline in R. Overall design: Three different target tissue two from 6 mpf adults Telencephalon and Hypothalamus and one from 6 dpf larvae dissected heads. One treatment Fluoxetine One control ethanol as vehicle two condition Stressed Unstressed       Lrv Ctl Bsal whole 5 GSM4196090   source name:Lrv Ctl Bsal whole|genotype/variation:SR4G transgenic|stress status:Bsal|drug status:Ctl|tissue:larvae head Lrv Ctl Bsal whole 5 Illumina bcl2fastq v2.17.1.14 software used for basecalling. Sequenced reads were trimmed for adaptor sequence with CutAdapt then aligned with STAR v 2.5 with parameter clip5pNbases 10 and quantMode TranscriptomeSAM GeneCounts. Tables of gene counts generated by STAR were imported into R and differential gene expression analysis was performed using DESeq2 to compare exposure groups. Genome build: GRCz11v92 Supplementary files format and content: tab delimited text files include raw count values for each sample. Lrv Ctl Bsal whole Exposing to Fluoxetine 54ug/L as treatment or Ethanol 0.0053% as vehicle from 0 dpf to 6 dpf in larvae Qiagene miliamp total RNA extraction column based Illumina TruSeq Stranded mRNA libraries were built following the manufacturer’s protocol using 250ng of starting material.   genotype/variation:SR4G transgenic|stress status:Bsal|drug status:Ctl|tissue:larvae head GSM4196090 GSM4196090: Lrv Ctl Bsal whole 5; Danio rerio; RNA Seq GSM4196090   1 Qiagene miliamp total RNA extraction column based Illumina TruSeq Stranded mRNA libraries were built following the manufacturer's protocol using 250ng of starting material. GEO Accession:GSM4196090 RNA-Seq TRANSCRIPTOMIC cDNA SINGLE ILLUMINA NextSeq 500   SRP233586     Lrv_Ctl_Bsal__5_S60_R1_001.trimmed.fastq.gz fastq 2072397830.0 27567241.0 GSM4196090 r1 0:75.18 1:0 A:499993390;C:522808790;G:477072306;T:572501982;N:21362 75 0     499993390 522808790 477072306 572501982 21362 SRX7232557 SRS5733188 SRA1003923 GEO Mechanistic Studies Division Genomics Laboratory, Health Canada, Government of Canada 1 0.95652   0.11456   0.69384   0.4738   76   B   usable mapping rate illumina nextseq unknown cdna_unspecified trueseq bulk unknown unknown   Canada 2019-11-27 Multi-stage Multi-stage Head Nervous System
76963 76963 SRR25396266 SRX21134300 SRS18399045 SRP451003 PRJNA997901 Zebrafish fbln1 mutant ulg075 larvae at 10dpf versus AB wt GSE238059 Transcriptome Analysis We generated a 16 nucleotide deletion mutant in the zebrafish fbln1 gene and we compared the whole larvae transcriptome to wt siblings in 10dpf larvae. Overall design: fbln1+/ parents were crossed wt and fbln1 / homozygous mutants were selected in three independant batches about 20 individuals/sample for extracting RNA and performing RNA Seq. Each mutant batch was compared to its wt siblings.       ABwt rep3 GSM7658116   source name:larvae heads|tissue:larvae heads|strain background:AB|genotype:ABwt|time point:10 dpf|geo loc name:missing|collection date:missing ABwt rep3 Approximatively 20–25 M reads were sequenced per sample. The sequencing reads were processed through the Nf core rnaseq pipeline 3.0 Ewels Peltzer et al. 2020 with default parameters and using the zebrafish reference genome GRCz11 and the annotation set from Ensembl release 103 www.ensembl.org; accessed 1 May 2020 Differential gene expression analysis was performed using the DESeq2 pipeline Love Huber et al. 2014. Assembly: zebrafish reference genome GRCz11 and the annotation set from Ensembl release 103 Supplementary files format and content: Table counts fbln1/wt.csv containing individual counts: Ensembl gene Id gene name fbln1 WT i R1 fbln1 WT ii R1 fbln1 WT iii R1 fbln1 mu i R1 fbln1 mu ii R1 fbln1 mu iii R1 Supplementary files format and content: Table fbln1/wt.csv containing: Ensembl gene ID gene name log2FoldChange lfcSE stat pvalue padj Mu baseMean WT baseMean baseMean larvae heads Homozygous mutants were obtained by crossing heterozygous parents carrying the desired mutation. Resulting larvae were first sacrificed by exposure to MS 222 Ethyl 3 aminobenzoate methane sulfonate; Merck Overijse Belgium 10dpf larvae were stored in RNA later Fisher Scientific Merelbeek Belgium. Individual fish were decapitated the heads anterior of each individual were stored in a 96 well plate for later RNA extraction whereas the body posterior was used for DNA extraction and genotyping using q PCR. Once the genotypes were known the heads were recovered and pooled to constitute wt and fbln1 / batches of 21 individuals. Three independent batches of wt and fbln1 / pooled heads n= 21controls & 21 mutants were prepared and the RNAlater solution was eliminated. RNA was extracted using the RNA mini extraction kit Qiagen Hilden Germany. Samples were lysed in RLT+ buffer with β mercaptoethanol Sigma Aldrich St. Louis MO USA and homogenized at least 10 times with a 26 gauge needle in a 1 mL syringe. An amount of 22 μL of RNAse free water was used to resuspend total RNA. RNA extract was treated with DNAseI Qiagen Hilden Germany to avoid DNA contamination. Quantity ng/μL and quality 260/280 and 260/230 ratios of each extract was assessed by nanodrop spectrophotometer measurements. The pellets were purified by lithium chloride precipitation followed by 2 times pellet washing with 70% ethanol and resuspended in 51 µL of RNAse free water and stored at −80 °C. The integrity of total RNA extracts was assessed using the BioAnalyzer Agilent Santa Clara CA USA. RIN RNA integrity number scores were > 9 for each sample. From the mRNAs extracted from wt and fbln1 mutants the cDNA libraries were generated from 100 to 500 ng of extracted total RNA using the Illumina Truseq mRNA stranded kit Illumina San Diego CA USA according to the manufacturer’s instructions. Zebrafish Danio rerio of the AB strain transgenic line ulg071 Tg were obtained from breeding facilities at the GIGA Institute Liege Belgium. The water characteristics were as follows: pH = 7.4 and temperature = 28°C. The lighting was 14/10 h light/dark. Embryos were used and staged as described [28 29]. Fish were mated and spawning was stimulated by the onset of light. The day before breeding adult male and female zebrafish were set up in several breeding tanks separated by a clear plastic wall. post the light was turned on the next morning walls are removed and eggs are generated by natural mating and then collected from 30 minutes to 2 hours post spawning. post sorting fertilized clean eggs are moved to Petri dishes and incubated at 28°C in E3 medium 5 mM Na Cl 0.17 mM KCl 0.33 mM CaCl2 0.33 mM MgSO4 0.00001% Methylene Blue. The age of the embryos and larvae is indicated as hpf tissue:larvae heads|strain background:AB|genotype:ABwt|time point:10 dpf GSM7658116 GSM7658116: ABwt rep3; Danio rerio; RNA Seq GSM7658116 r1 GSM7658116 1 Three independent batches of wt and fbln1 / pooled heads n= 21controls & 21 mutants were prepared and the RNAlater solution was eliminated. RNA was extracted using the RNA mini extraction kit Qiagen Hilden Germany. Samples were lysed in RLT+ buffer with β mercaptoethanol Sigma Aldrich St. Louis MO USA and homogenized at least 10 times with a 26 gauge needle in a 1 mL syringe. An amount of 22 μL of RNAse free water was used to resuspend total RNA. RNA extract was treated with DNAseI Qiagen Hilden Germany to avoid DNA contamination. Quantity ng/μL and quality 260/280 and 260/230 ratios of each extract was assessed by nanodrop spectrophotometer measurements. The pellets were purified by lithium chloride precipitation followed by 2 times pellet washing with 70% ethanol and resuspended in 51 µL of RNAse free water and stored at −80 °C. The integrity of total RNA extracts was assessed using the BioAnalyzer Agilent Santa Clara CA USA. RIN RNA integrity number scores were > 9 for each sample. From the mRNAs extracted from wt and fbln1 mutants the cDNA libraries were generated from 100 to 500 ng of extracted total RNA using the Illumina Truseq mRNA stranded kit Illumina San Diego CA USA according to the manufacturer's instructions.   RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP451003   loader:fastq load.py fbln1-WT_iii_NGS22-Y626_AH5MKYDSX5_S648_L002_R1_001.fastq.gz fbln1-WT_iii_NGS22-Y626_AH5MKYDSX5_S648_L002_R2_001.fastq.gz fastq fastq 6634706556.0 24788052.0 GSM7658116 r1 0:133.79 1:133.87 A:1794257291;C:1506457485;G:1526166840;T:1807502501;N:322439 133 133     1794257291 1506457485 1526166840 1807502501 322439 SRX21134300 SRS18399045 SRA1678716 Organogenesis and Regeneration, Life Sciences, Université de Liège Organogenesis and Regeneration, Life Sciences, Université de Liège 2 0.88742 0.88401 0.12393 0.12124 0.68306 0.6832 0.47859 0.46588 128 128 B B biological fallback assumption illumina novaseq_era full_length random_priming trueseq bulk unknown unknown   Belgium 2023-07-24 Multi-stage Multi-stage Head Nervous System
76964 76964 SRR25396267 SRX21134299 SRS18399044 SRP451003 PRJNA997901 Zebrafish fbln1 mutant ulg075 larvae at 10dpf versus AB wt GSE238059 Transcriptome Analysis We generated a 16 nucleotide deletion mutant in the zebrafish fbln1 gene and we compared the whole larvae transcriptome to wt siblings in 10dpf larvae. Overall design: fbln1+/ parents were crossed wt and fbln1 / homozygous mutants were selected in three independant batches about 20 individuals/sample for extracting RNA and performing RNA Seq. Each mutant batch was compared to its wt siblings.       ABwt rep2 GSM7658115   source name:larvae heads|tissue:larvae heads|strain background:AB|genotype:ABwt|time point:10 dpf|geo loc name:missing|collection date:missing ABwt rep2 Approximatively 20–25 M reads were sequenced per sample. The sequencing reads were processed through the Nf core rnaseq pipeline 3.0 Ewels Peltzer et al. 2020 with default parameters and using the zebrafish reference genome GRCz11 and the annotation set from Ensembl release 103 www.ensembl.org; accessed 1 May 2020 Differential gene expression analysis was performed using the DESeq2 pipeline Love Huber et al. 2014. Assembly: zebrafish reference genome GRCz11 and the annotation set from Ensembl release 103 Supplementary files format and content: Table counts fbln1/wt.csv containing individual counts: Ensembl gene Id gene name fbln1 WT i R1 fbln1 WT ii R1 fbln1 WT iii R1 fbln1 mu i R1 fbln1 mu ii R1 fbln1 mu iii R1 Supplementary files format and content: Table fbln1/wt.csv containing: Ensembl gene ID gene name log2FoldChange lfcSE stat pvalue padj Mu baseMean WT baseMean baseMean larvae heads Homozygous mutants were obtained by crossing heterozygous parents carrying the desired mutation. Resulting larvae were first sacrificed by exposure to MS 222 Ethyl 3 aminobenzoate methane sulfonate; Merck Overijse Belgium 10dpf larvae were stored in RNA later Fisher Scientific Merelbeek Belgium. Individual fish were decapitated the heads anterior of each individual were stored in a 96 well plate for later RNA extraction whereas the body posterior was used for DNA extraction and genotyping using q PCR. Once the genotypes were known the heads were recovered and pooled to constitute wt and fbln1 / batches of 21 individuals. Three independent batches of wt and fbln1 / pooled heads n= 21controls & 21 mutants were prepared and the RNAlater solution was eliminated. RNA was extracted using the RNA mini extraction kit Qiagen Hilden Germany. Samples were lysed in RLT+ buffer with β mercaptoethanol Sigma Aldrich St. Louis MO USA and homogenized at least 10 times with a 26 gauge needle in a 1 mL syringe. An amount of 22 μL of RNAse free water was used to resuspend total RNA. RNA extract was treated with DNAseI Qiagen Hilden Germany to avoid DNA contamination. Quantity ng/μL and quality 260/280 and 260/230 ratios of each extract was assessed by nanodrop spectrophotometer measurements. The pellets were purified by lithium chloride precipitation followed by 2 times pellet washing with 70% ethanol and resuspended in 51 µL of RNAse free water and stored at −80 °C. The integrity of total RNA extracts was assessed using the BioAnalyzer Agilent Santa Clara CA USA. RIN RNA integrity number scores were > 9 for each sample. From the mRNAs extracted from wt and fbln1 mutants the cDNA libraries were generated from 100 to 500 ng of extracted total RNA using the Illumina Truseq mRNA stranded kit Illumina San Diego CA USA according to the manufacturer’s instructions. Zebrafish Danio rerio of the AB strain transgenic line ulg071 Tg were obtained from breeding facilities at the GIGA Institute Liege Belgium. The water characteristics were as follows: pH = 7.4 and temperature = 28°C. The lighting was 14/10 h light/dark. Embryos were used and staged as described [28 29]. Fish were mated and spawning was stimulated by the onset of light. The day before breeding adult male and female zebrafish were set up in several breeding tanks separated by a clear plastic wall. post the light was turned on the next morning walls are removed and eggs are generated by natural mating and then collected from 30 minutes to 2 hours post spawning. post sorting fertilized clean eggs are moved to Petri dishes and incubated at 28°C in E3 medium 5 mM Na Cl 0.17 mM KCl 0.33 mM CaCl2 0.33 mM MgSO4 0.00001% Methylene Blue. The age of the embryos and larvae is indicated as hpf tissue:larvae heads|strain background:AB|genotype:ABwt|time point:10 dpf GSM7658115 GSM7658115: ABwt rep2; Danio rerio; RNA Seq GSM7658115 r1 GSM7658115 1 Three independent batches of wt and fbln1 / pooled heads n= 21controls & 21 mutants were prepared and the RNAlater solution was eliminated. RNA was extracted using the RNA mini extraction kit Qiagen Hilden Germany. Samples were lysed in RLT+ buffer with β mercaptoethanol Sigma Aldrich St. Louis MO USA and homogenized at least 10 times with a 26 gauge needle in a 1 mL syringe. An amount of 22 μL of RNAse free water was used to resuspend total RNA. RNA extract was treated with DNAseI Qiagen Hilden Germany to avoid DNA contamination. Quantity ng/μL and quality 260/280 and 260/230 ratios of each extract was assessed by nanodrop spectrophotometer measurements. The pellets were purified by lithium chloride precipitation followed by 2 times pellet washing with 70% ethanol and resuspended in 51 µL of RNAse free water and stored at −80 °C. The integrity of total RNA extracts was assessed using the BioAnalyzer Agilent Santa Clara CA USA. RIN RNA integrity number scores were > 9 for each sample. From the mRNAs extracted from wt and fbln1 mutants the cDNA libraries were generated from 100 to 500 ng of extracted total RNA using the Illumina Truseq mRNA stranded kit Illumina San Diego CA USA according to the manufacturer's instructions.   RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP451003   loader:fastq load.py fbln1-WT-ii_NGS22-Y627_AH5MKYDSX5_S649_L002_R1_001.fastq.gz fbln1-WT-ii_NGS22-Y627_AH5MKYDSX5_S649_L002_R2_001.fastq.gz fastq fastq 7071028989.0 26154663.0 GSM7658115 r1 0:135.14 1:135.21 A:1917429043;C:1600107106;G:1615290315;T:1937914414;N:288111 135 135     1917429043 1600107106 1615290315 1937914414 288111 SRX21134299 SRS18399044 SRA1678716 Organogenesis and Regeneration, Life Sciences, Université de Liège Organogenesis and Regeneration, Life Sciences, Université de Liège 2 0.90352 0.90003 0.12911 0.12614 0.67929 0.68016 0.48562 0.48784 103 103 B B biological fallback assumption illumina novaseq_era full_length random_priming trueseq bulk unknown unknown   Belgium 2023-07-24 Multi-stage Multi-stage Head Nervous System
76965 76965 SRR25396268 SRX21134298 SRS18399042 SRP451003 PRJNA997901 Zebrafish fbln1 mutant ulg075 larvae at 10dpf versus AB wt GSE238059 Transcriptome Analysis We generated a 16 nucleotide deletion mutant in the zebrafish fbln1 gene and we compared the whole larvae transcriptome to wt siblings in 10dpf larvae. Overall design: fbln1+/ parents were crossed wt and fbln1 / homozygous mutants were selected in three independant batches about 20 individuals/sample for extracting RNA and performing RNA Seq. Each mutant batch was compared to its wt siblings.       ABwt rep1 GSM7658114   source name:larvae heads|tissue:larvae heads|strain background:AB|genotype:ABwt|time point:10 dpf|geo loc name:missing|collection date:missing ABwt rep1 Approximatively 20–25 M reads were sequenced per sample. The sequencing reads were processed through the Nf core rnaseq pipeline 3.0 Ewels Peltzer et al. 2020 with default parameters and using the zebrafish reference genome GRCz11 and the annotation set from Ensembl release 103 www.ensembl.org; accessed 1 May 2020 Differential gene expression analysis was performed using the DESeq2 pipeline Love Huber et al. 2014. Assembly: zebrafish reference genome GRCz11 and the annotation set from Ensembl release 103 Supplementary files format and content: Table counts fbln1/wt.csv containing individual counts: Ensembl gene Id gene name fbln1 WT i R1 fbln1 WT ii R1 fbln1 WT iii R1 fbln1 mu i R1 fbln1 mu ii R1 fbln1 mu iii R1 Supplementary files format and content: Table fbln1/wt.csv containing: Ensembl gene ID gene name log2FoldChange lfcSE stat pvalue padj Mu baseMean WT baseMean baseMean larvae heads Homozygous mutants were obtained by crossing heterozygous parents carrying the desired mutation. Resulting larvae were first sacrificed by exposure to MS 222 Ethyl 3 aminobenzoate methane sulfonate; Merck Overijse Belgium 10dpf larvae were stored in RNA later Fisher Scientific Merelbeek Belgium. Individual fish were decapitated the heads anterior of each individual were stored in a 96 well plate for later RNA extraction whereas the body posterior was used for DNA extraction and genotyping using q PCR. Once the genotypes were known the heads were recovered and pooled to constitute wt and fbln1 / batches of 21 individuals. Three independent batches of wt and fbln1 / pooled heads n= 21controls & 21 mutants were prepared and the RNAlater solution was eliminated. RNA was extracted using the RNA mini extraction kit Qiagen Hilden Germany. Samples were lysed in RLT+ buffer with β mercaptoethanol Sigma Aldrich St. Louis MO USA and homogenized at least 10 times with a 26 gauge needle in a 1 mL syringe. An amount of 22 μL of RNAse free water was used to resuspend total RNA. RNA extract was treated with DNAseI Qiagen Hilden Germany to avoid DNA contamination. Quantity ng/μL and quality 260/280 and 260/230 ratios of each extract was assessed by nanodrop spectrophotometer measurements. The pellets were purified by lithium chloride precipitation followed by 2 times pellet washing with 70% ethanol and resuspended in 51 µL of RNAse free water and stored at −80 °C. The integrity of total RNA extracts was assessed using the BioAnalyzer Agilent Santa Clara CA USA. RIN RNA integrity number scores were > 9 for each sample. From the mRNAs extracted from wt and fbln1 mutants the cDNA libraries were generated from 100 to 500 ng of extracted total RNA using the Illumina Truseq mRNA stranded kit Illumina San Diego CA USA according to the manufacturer’s instructions. Zebrafish Danio rerio of the AB strain transgenic line ulg071 Tg were obtained from breeding facilities at the GIGA Institute Liege Belgium. The water characteristics were as follows: pH = 7.4 and temperature = 28°C. The lighting was 14/10 h light/dark. Embryos were used and staged as described [28 29]. Fish were mated and spawning was stimulated by the onset of light. The day before breeding adult male and female zebrafish were set up in several breeding tanks separated by a clear plastic wall. post the light was turned on the next morning walls are removed and eggs are generated by natural mating and then collected from 30 minutes to 2 hours post spawning. post sorting fertilized clean eggs are moved to Petri dishes and incubated at 28°C in E3 medium 5 mM Na Cl 0.17 mM KCl 0.33 mM CaCl2 0.33 mM MgSO4 0.00001% Methylene Blue. The age of the embryos and larvae is indicated as hpf tissue:larvae heads|strain background:AB|genotype:ABwt|time point:10 dpf GSM7658114 GSM7658114: ABwt rep1; Danio rerio; RNA Seq GSM7658114 r1 GSM7658114 1 Three independent batches of wt and fbln1 / pooled heads n= 21controls & 21 mutants were prepared and the RNAlater solution was eliminated. RNA was extracted using the RNA mini extraction kit Qiagen Hilden Germany. Samples were lysed in RLT+ buffer with β mercaptoethanol Sigma Aldrich St. Louis MO USA and homogenized at least 10 times with a 26 gauge needle in a 1 mL syringe. An amount of 22 μL of RNAse free water was used to resuspend total RNA. RNA extract was treated with DNAseI Qiagen Hilden Germany to avoid DNA contamination. Quantity ng/μL and quality 260/280 and 260/230 ratios of each extract was assessed by nanodrop spectrophotometer measurements. The pellets were purified by lithium chloride precipitation followed by 2 times pellet washing with 70% ethanol and resuspended in 51 µL of RNAse free water and stored at −80 °C. The integrity of total RNA extracts was assessed using the BioAnalyzer Agilent Santa Clara CA USA. RIN RNA integrity number scores were > 9 for each sample. From the mRNAs extracted from wt and fbln1 mutants the cDNA libraries were generated from 100 to 500 ng of extracted total RNA using the Illumina Truseq mRNA stranded kit Illumina San Diego CA USA according to the manufacturer's instructions.   RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP451003   loader:fastq load.py fbln1-WT-i_NGS22-Y625_AH5MKYDSX5_S647_L002_R1_001.fastq.gz fbln1-WT-i_NGS22-Y625_AH5MKYDSX5_S647_L002_R2_001.fastq.gz fastq fastq 6970434611.0 26052125.0 GSM7658114 r1 0:133.74 1:133.81 A:1884921652;C:1582428412;G:1601771780;T:1900977711;N:335056 133 133     1884921652 1582428412 1601771780 1900977711 335056 SRX21134298 SRS18399042 SRA1678716 Organogenesis and Regeneration, Life Sciences, Université de Liège Organogenesis and Regeneration, Life Sciences, Université de Liège 2 0.89264 0.89039 0.12573 0.12308 0.68734 0.68834 0.48102 0.48212 135 135 B B biological fallback assumption illumina novaseq_era full_length random_priming trueseq bulk unknown unknown   Belgium 2023-07-24 Multi-stage Multi-stage Head Nervous System
76966 76966 SRR25396269 SRX21134297 SRS18399043 SRP451003 PRJNA997901 Zebrafish fbln1 mutant ulg075 larvae at 10dpf versus AB wt GSE238059 Transcriptome Analysis We generated a 16 nucleotide deletion mutant in the zebrafish fbln1 gene and we compared the whole larvae transcriptome to wt siblings in 10dpf larvae. Overall design: fbln1+/ parents were crossed wt and fbln1 / homozygous mutants were selected in three independant batches about 20 individuals/sample for extracting RNA and performing RNA Seq. Each mutant batch was compared to its wt siblings.       fbln rep3 GSM7658113   source name:larvae heads|tissue:larvae heads|strain background:AB|genotype:ulg075|time point:10 dpf|geo loc name:missing|collection date:missing fbln rep3 Approximatively 20–25 M reads were sequenced per sample. The sequencing reads were processed through the Nf core rnaseq pipeline 3.0 Ewels Peltzer et al. 2020 with default parameters and using the zebrafish reference genome GRCz11 and the annotation set from Ensembl release 103 www.ensembl.org; accessed 1 May 2020 Differential gene expression analysis was performed using the DESeq2 pipeline Love Huber et al. 2014. Assembly: zebrafish reference genome GRCz11 and the annotation set from Ensembl release 103 Supplementary files format and content: Table counts fbln1/wt.csv containing individual counts: Ensembl gene Id gene name fbln1 WT i R1 fbln1 WT ii R1 fbln1 WT iii R1 fbln1 mu i R1 fbln1 mu ii R1 fbln1 mu iii R1 Supplementary files format and content: Table fbln1/wt.csv containing: Ensembl gene ID gene name log2FoldChange lfcSE stat pvalue padj Mu baseMean WT baseMean baseMean larvae heads Homozygous mutants were obtained by crossing heterozygous parents carrying the desired mutation. Resulting larvae were first sacrificed by exposure to MS 222 Ethyl 3 aminobenzoate methane sulfonate; Merck Overijse Belgium 10dpf larvae were stored in RNA later Fisher Scientific Merelbeek Belgium. Individual fish were decapitated the heads anterior of each individual were stored in a 96 well plate for later RNA extraction whereas the body posterior was used for DNA extraction and genotyping using q PCR. Once the genotypes were known the heads were recovered and pooled to constitute wt and fbln1 / batches of 21 individuals. Three independent batches of wt and fbln1 / pooled heads n= 21controls & 21 mutants were prepared and the RNAlater solution was eliminated. RNA was extracted using the RNA mini extraction kit Qiagen Hilden Germany. Samples were lysed in RLT+ buffer with β mercaptoethanol Sigma Aldrich St. Louis MO USA and homogenized at least 10 times with a 26 gauge needle in a 1 mL syringe. An amount of 22 μL of RNAse free water was used to resuspend total RNA. RNA extract was treated with DNAseI Qiagen Hilden Germany to avoid DNA contamination. Quantity ng/μL and quality 260/280 and 260/230 ratios of each extract was assessed by nanodrop spectrophotometer measurements. The pellets were purified by lithium chloride precipitation followed by 2 times pellet washing with 70% ethanol and resuspended in 51 µL of RNAse free water and stored at −80 °C. The integrity of total RNA extracts was assessed using the BioAnalyzer Agilent Santa Clara CA USA. RIN RNA integrity number scores were > 9 for each sample. From the mRNAs extracted from wt and fbln1 mutants the cDNA libraries were generated from 100 to 500 ng of extracted total RNA using the Illumina Truseq mRNA stranded kit Illumina San Diego CA USA according to the manufacturer’s instructions. Zebrafish Danio rerio of the AB strain transgenic line ulg071 Tg were obtained from breeding facilities at the GIGA Institute Liege Belgium. The water characteristics were as follows: pH = 7.4 and temperature = 28°C. The lighting was 14/10 h light/dark. Embryos were used and staged as described [28 29]. Fish were mated and spawning was stimulated by the onset of light. The day before breeding adult male and female zebrafish were set up in several breeding tanks separated by a clear plastic wall. post the light was turned on the next morning walls are removed and eggs are generated by natural mating and then collected from 30 minutes to 2 hours post spawning. post sorting fertilized clean eggs are moved to Petri dishes and incubated at 28°C in E3 medium 5 mM Na Cl 0.17 mM KCl 0.33 mM CaCl2 0.33 mM MgSO4 0.00001% Methylene Blue. The age of the embryos and larvae is indicated as hpf tissue:larvae heads|strain background:AB|genotype:ulg075|time point:10 dpf GSM7658113 GSM7658113: fbln rep3; Danio rerio; RNA Seq GSM7658113 r1 GSM7658113 1 Three independent batches of wt and fbln1 / pooled heads n= 21controls & 21 mutants were prepared and the RNAlater solution was eliminated. RNA was extracted using the RNA mini extraction kit Qiagen Hilden Germany. Samples were lysed in RLT+ buffer with β mercaptoethanol Sigma Aldrich St. Louis MO USA and homogenized at least 10 times with a 26 gauge needle in a 1 mL syringe. An amount of 22 μL of RNAse free water was used to resuspend total RNA. RNA extract was treated with DNAseI Qiagen Hilden Germany to avoid DNA contamination. Quantity ng/μL and quality 260/280 and 260/230 ratios of each extract was assessed by nanodrop spectrophotometer measurements. The pellets were purified by lithium chloride precipitation followed by 2 times pellet washing with 70% ethanol and resuspended in 51 µL of RNAse free water and stored at −80 °C. The integrity of total RNA extracts was assessed using the BioAnalyzer Agilent Santa Clara CA USA. RIN RNA integrity number scores were > 9 for each sample. From the mRNAs extracted from wt and fbln1 mutants the cDNA libraries were generated from 100 to 500 ng of extracted total RNA using the Illumina Truseq mRNA stranded kit Illumina San Diego CA USA according to the manufacturer's instructions.   RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP451003   loader:fastq load.py fbln1-mu-iii_NGS22-Y630_AH5MKYDSX5_S652_L002_R1_001.fastq.gz fbln1-mu-iii_NGS22-Y630_AH5MKYDSX5_S652_L002_R2_001.fastq.gz fastq fastq 7897839439.0 29176341.0 GSM7658113 r1 0:135.32 1:135.37 A:2124108601;C:1805125896;G:1825004607;T:2143248362;N:351973 135 135     2124108601 1805125896 1825004607 2143248362 351973 SRX21134297 SRS18399043 SRA1678716 Organogenesis and Regeneration, Life Sciences, Université de Liège Organogenesis and Regeneration, Life Sciences, Université de Liège 2 0.90473 0.90217 0.11614 0.1131 0.66959 0.66996 0.48247 0.48066 150 150 B B biological fallback assumption illumina novaseq_era full_length random_priming trueseq bulk unknown unknown   Belgium 2023-07-24 Multi-stage Multi-stage Head Nervous System
76967 76967 SRR25396270 SRX21134296 SRS18399041 SRP451003 PRJNA997901 Zebrafish fbln1 mutant ulg075 larvae at 10dpf versus AB wt GSE238059 Transcriptome Analysis We generated a 16 nucleotide deletion mutant in the zebrafish fbln1 gene and we compared the whole larvae transcriptome to wt siblings in 10dpf larvae. Overall design: fbln1+/ parents were crossed wt and fbln1 / homozygous mutants were selected in three independant batches about 20 individuals/sample for extracting RNA and performing RNA Seq. Each mutant batch was compared to its wt siblings.       fbln rep2 GSM7658112   source name:larvae heads|tissue:larvae heads|strain background:AB|genotype:ulg075|time point:10 dpf|geo loc name:missing|collection date:missing fbln rep2 Approximatively 20–25 M reads were sequenced per sample. The sequencing reads were processed through the Nf core rnaseq pipeline 3.0 Ewels Peltzer et al. 2020 with default parameters and using the zebrafish reference genome GRCz11 and the annotation set from Ensembl release 103 www.ensembl.org; accessed 1 May 2020 Differential gene expression analysis was performed using the DESeq2 pipeline Love Huber et al. 2014. Assembly: zebrafish reference genome GRCz11 and the annotation set from Ensembl release 103 Supplementary files format and content: Table counts fbln1/wt.csv containing individual counts: Ensembl gene Id gene name fbln1 WT i R1 fbln1 WT ii R1 fbln1 WT iii R1 fbln1 mu i R1 fbln1 mu ii R1 fbln1 mu iii R1 Supplementary files format and content: Table fbln1/wt.csv containing: Ensembl gene ID gene name log2FoldChange lfcSE stat pvalue padj Mu baseMean WT baseMean baseMean larvae heads Homozygous mutants were obtained by crossing heterozygous parents carrying the desired mutation. Resulting larvae were first sacrificed by exposure to MS 222 Ethyl 3 aminobenzoate methane sulfonate; Merck Overijse Belgium 10dpf larvae were stored in RNA later Fisher Scientific Merelbeek Belgium. Individual fish were decapitated the heads anterior of each individual were stored in a 96 well plate for later RNA extraction whereas the body posterior was used for DNA extraction and genotyping using q PCR. Once the genotypes were known the heads were recovered and pooled to constitute wt and fbln1 / batches of 21 individuals. Three independent batches of wt and fbln1 / pooled heads n= 21controls & 21 mutants were prepared and the RNAlater solution was eliminated. RNA was extracted using the RNA mini extraction kit Qiagen Hilden Germany. Samples were lysed in RLT+ buffer with β mercaptoethanol Sigma Aldrich St. Louis MO USA and homogenized at least 10 times with a 26 gauge needle in a 1 mL syringe. An amount of 22 μL of RNAse free water was used to resuspend total RNA. RNA extract was treated with DNAseI Qiagen Hilden Germany to avoid DNA contamination. Quantity ng/μL and quality 260/280 and 260/230 ratios of each extract was assessed by nanodrop spectrophotometer measurements. The pellets were purified by lithium chloride precipitation followed by 2 times pellet washing with 70% ethanol and resuspended in 51 µL of RNAse free water and stored at −80 °C. The integrity of total RNA extracts was assessed using the BioAnalyzer Agilent Santa Clara CA USA. RIN RNA integrity number scores were > 9 for each sample. From the mRNAs extracted from wt and fbln1 mutants the cDNA libraries were generated from 100 to 500 ng of extracted total RNA using the Illumina Truseq mRNA stranded kit Illumina San Diego CA USA according to the manufacturer’s instructions. Zebrafish Danio rerio of the AB strain transgenic line ulg071 Tg were obtained from breeding facilities at the GIGA Institute Liege Belgium. The water characteristics were as follows: pH = 7.4 and temperature = 28°C. The lighting was 14/10 h light/dark. Embryos were used and staged as described [28 29]. Fish were mated and spawning was stimulated by the onset of light. The day before breeding adult male and female zebrafish were set up in several breeding tanks separated by a clear plastic wall. post the light was turned on the next morning walls are removed and eggs are generated by natural mating and then collected from 30 minutes to 2 hours post spawning. post sorting fertilized clean eggs are moved to Petri dishes and incubated at 28°C in E3 medium 5 mM Na Cl 0.17 mM KCl 0.33 mM CaCl2 0.33 mM MgSO4 0.00001% Methylene Blue. The age of the embryos and larvae is indicated as hpf tissue:larvae heads|strain background:AB|genotype:ulg075|time point:10 dpf GSM7658112 GSM7658112: fbln rep2; Danio rerio; RNA Seq GSM7658112 r1 GSM7658112 1 Three independent batches of wt and fbln1 / pooled heads n= 21controls & 21 mutants were prepared and the RNAlater solution was eliminated. RNA was extracted using the RNA mini extraction kit Qiagen Hilden Germany. Samples were lysed in RLT+ buffer with β mercaptoethanol Sigma Aldrich St. Louis MO USA and homogenized at least 10 times with a 26 gauge needle in a 1 mL syringe. An amount of 22 μL of RNAse free water was used to resuspend total RNA. RNA extract was treated with DNAseI Qiagen Hilden Germany to avoid DNA contamination. Quantity ng/μL and quality 260/280 and 260/230 ratios of each extract was assessed by nanodrop spectrophotometer measurements. The pellets were purified by lithium chloride precipitation followed by 2 times pellet washing with 70% ethanol and resuspended in 51 µL of RNAse free water and stored at −80 °C. The integrity of total RNA extracts was assessed using the BioAnalyzer Agilent Santa Clara CA USA. RIN RNA integrity number scores were > 9 for each sample. From the mRNAs extracted from wt and fbln1 mutants the cDNA libraries were generated from 100 to 500 ng of extracted total RNA using the Illumina Truseq mRNA stranded kit Illumina San Diego CA USA according to the manufacturer's instructions.   RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP451003   loader:fastq load.py fbln1-mu-ii_NGS22-Y628_AH5MKYDSX5_S650_L002_R1_001.fastq.gz fbln1-mu-ii_NGS22-Y628_AH5MKYDSX5_S650_L002_R2_001.fastq.gz fastq fastq 6620926405.0 24273768.0 GSM7658112 r1 0:136.35 1:136.41 A:1779196105;C:1513133139;G:1531673859;T:1796617651;N:305651 136 136     1779196105 1513133139 1531673859 1796617651 305651 SRX21134296 SRS18399041 SRA1678716 Organogenesis and Regeneration, Life Sciences, Université de Liège Organogenesis and Regeneration, Life Sciences, Université de Liège 2 0.90799 0.90607 0.10967 0.10744 0.66553 0.66561 0.48504 0.48733 150 150 B B biological fallback assumption illumina novaseq_era full_length random_priming trueseq bulk unknown unknown   Belgium 2023-07-24 Multi-stage Multi-stage Head Nervous System
76968 76968 SRR25396271 SRX21134295 SRS18399040 SRP451003 PRJNA997901 Zebrafish fbln1 mutant ulg075 larvae at 10dpf versus AB wt GSE238059 Transcriptome Analysis We generated a 16 nucleotide deletion mutant in the zebrafish fbln1 gene and we compared the whole larvae transcriptome to wt siblings in 10dpf larvae. Overall design: fbln1+/ parents were crossed wt and fbln1 / homozygous mutants were selected in three independant batches about 20 individuals/sample for extracting RNA and performing RNA Seq. Each mutant batch was compared to its wt siblings.       fbln rep1 GSM7658111   source name:larvae heads|tissue:larvae heads|strain background:AB|genotype:ulg075|time point:10 dpf|geo loc name:missing|collection date:missing fbln rep1 Approximatively 20–25 M reads were sequenced per sample. The sequencing reads were processed through the Nf core rnaseq pipeline 3.0 Ewels Peltzer et al. 2020 with default parameters and using the zebrafish reference genome GRCz11 and the annotation set from Ensembl release 103 www.ensembl.org; accessed 1 May 2020 Differential gene expression analysis was performed using the DESeq2 pipeline Love Huber et al. 2014. Assembly: zebrafish reference genome GRCz11 and the annotation set from Ensembl release 103 Supplementary files format and content: Table counts fbln1/wt.csv containing individual counts: Ensembl gene Id gene name fbln1 WT i R1 fbln1 WT ii R1 fbln1 WT iii R1 fbln1 mu i R1 fbln1 mu ii R1 fbln1 mu iii R1 Supplementary files format and content: Table fbln1/wt.csv containing: Ensembl gene ID gene name log2FoldChange lfcSE stat pvalue padj Mu baseMean WT baseMean baseMean larvae heads Homozygous mutants were obtained by crossing heterozygous parents carrying the desired mutation. Resulting larvae were first sacrificed by exposure to MS 222 Ethyl 3 aminobenzoate methane sulfonate; Merck Overijse Belgium 10dpf larvae were stored in RNA later Fisher Scientific Merelbeek Belgium. Individual fish were decapitated the heads anterior of each individual were stored in a 96 well plate for later RNA extraction whereas the body posterior was used for DNA extraction and genotyping using q PCR. Once the genotypes were known the heads were recovered and pooled to constitute wt and fbln1 / batches of 21 individuals. Three independent batches of wt and fbln1 / pooled heads n= 21controls & 21 mutants were prepared and the RNAlater solution was eliminated. RNA was extracted using the RNA mini extraction kit Qiagen Hilden Germany. Samples were lysed in RLT+ buffer with β mercaptoethanol Sigma Aldrich St. Louis MO USA and homogenized at least 10 times with a 26 gauge needle in a 1 mL syringe. An amount of 22 μL of RNAse free water was used to resuspend total RNA. RNA extract was treated with DNAseI Qiagen Hilden Germany to avoid DNA contamination. Quantity ng/μL and quality 260/280 and 260/230 ratios of each extract was assessed by nanodrop spectrophotometer measurements. The pellets were purified by lithium chloride precipitation followed by 2 times pellet washing with 70% ethanol and resuspended in 51 µL of RNAse free water and stored at −80 °C. The integrity of total RNA extracts was assessed using the BioAnalyzer Agilent Santa Clara CA USA. RIN RNA integrity number scores were > 9 for each sample. From the mRNAs extracted from wt and fbln1 mutants the cDNA libraries were generated from 100 to 500 ng of extracted total RNA using the Illumina Truseq mRNA stranded kit Illumina San Diego CA USA according to the manufacturer’s instructions. Zebrafish Danio rerio of the AB strain transgenic line ulg071 Tg were obtained from breeding facilities at the GIGA Institute Liege Belgium. The water characteristics were as follows: pH = 7.4 and temperature = 28°C. The lighting was 14/10 h light/dark. Embryos were used and staged as described [28 29]. Fish were mated and spawning was stimulated by the onset of light. The day before breeding adult male and female zebrafish were set up in several breeding tanks separated by a clear plastic wall. post the light was turned on the next morning walls are removed and eggs are generated by natural mating and then collected from 30 minutes to 2 hours post spawning. post sorting fertilized clean eggs are moved to Petri dishes and incubated at 28°C in E3 medium 5 mM Na Cl 0.17 mM KCl 0.33 mM CaCl2 0.33 mM MgSO4 0.00001% Methylene Blue. The age of the embryos and larvae is indicated as hpf tissue:larvae heads|strain background:AB|genotype:ulg075|time point:10 dpf GSM7658111 GSM7658111: fbln rep1; Danio rerio; RNA Seq GSM7658111 r1 GSM7658111 1 Three independent batches of wt and fbln1 / pooled heads n= 21controls & 21 mutants were prepared and the RNAlater solution was eliminated. RNA was extracted using the RNA mini extraction kit Qiagen Hilden Germany. Samples were lysed in RLT+ buffer with β mercaptoethanol Sigma Aldrich St. Louis MO USA and homogenized at least 10 times with a 26 gauge needle in a 1 mL syringe. An amount of 22 μL of RNAse free water was used to resuspend total RNA. RNA extract was treated with DNAseI Qiagen Hilden Germany to avoid DNA contamination. Quantity ng/μL and quality 260/280 and 260/230 ratios of each extract was assessed by nanodrop spectrophotometer measurements. The pellets were purified by lithium chloride precipitation followed by 2 times pellet washing with 70% ethanol and resuspended in 51 µL of RNAse free water and stored at −80 °C. The integrity of total RNA extracts was assessed using the BioAnalyzer Agilent Santa Clara CA USA. RIN RNA integrity number scores were > 9 for each sample. From the mRNAs extracted from wt and fbln1 mutants the cDNA libraries were generated from 100 to 500 ng of extracted total RNA using the Illumina Truseq mRNA stranded kit Illumina San Diego CA USA according to the manufacturer's instructions.   RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP451003   loader:fastq load.py fbln1-mu_i_NGS22-Y629_AH5MKYDSX5_S651_L002_R1_001.fastq.gz fbln1-mu_i_NGS22-Y629_AH5MKYDSX5_S651_L002_R2_001.fastq.gz fastq fastq 6961206835.0 25675483.0 GSM7658111 r1 0:135.53 1:135.59 A:1873338006;C:1589509564;G:1608298056;T:1889789695;N:271514 135 135     1873338006 1589509564 1608298056 1889789695 271514 SRX21134295 SRS18399040 SRA1678716 Organogenesis and Regeneration, Life Sciences, Université de Liège Organogenesis and Regeneration, Life Sciences, Université de Liège 2 0.9041 0.90203 0.11033 0.10785 0.66776 0.66906 0.4888 0.47976 150 150 B B biological fallback assumption illumina novaseq_era full_length random_priming trueseq bulk unknown unknown   Belgium 2023-07-24 Multi-stage Multi-stage Head Nervous System

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CREATE TABLE run_metadata("run.accession" VARCHAR, "experiment.accession" VARCHAR, "sample.accession" VARCHAR, "study.accession" VARCHAR, bioproject VARCHAR, "study.title" VARCHAR, "study.alias" VARCHAR, "study.type" VARCHAR, "study.abstract" VARCHAR, "study.attributes" VARCHAR, "study.PMIDs" VARCHAR, "sample.description" VARCHAR, "sample.title" VARCHAR, "sample.alias" VARCHAR, "sample.centername" VARCHAR, "sample.attributes" VARCHAR, "GEOsample.title" VARCHAR, "GEOsample.dataprocessing" VARCHAR, "GEOsample.source" VARCHAR, "GEOsample.treatmentprotocol" VARCHAR, "GEOsample.extractprotocol" VARCHAR, "GEOsample.growthprotocol" VARCHAR, "GEOsample.characteristics" VARCHAR, "GEOsample.accession" VARCHAR, "experiment.title" VARCHAR, "experiment.alias" VARCHAR, "experiment.library_name" VARCHAR, "experiment.design_description" VARCHAR, "experiment.library_construction_protocol" VARCHAR, "experiment.attributes" VARCHAR, "experiment.library_strategy" VARCHAR, "experiment.library_source" VARCHAR, "experiment.library_selection" VARCHAR, "experiment.library_layout" VARCHAR, "experiment.platform" VARCHAR, "experiment.instrument_model" VARCHAR, "experiment.spot_descriptor" VARCHAR, "experiment.study_ref" VARCHAR, "run.title" VARCHAR, "run.attributes" VARCHAR, "run.filename" VARCHAR, "run.semantic_name" VARCHAR, "run.total_bases" DOUBLE, "run.total_spots" DOUBLE, "run.alias" VARCHAR, "run.read_lengths" VARCHAR, "run.base_counts" VARCHAR, "run.r1_length" BIGINT, "run.r2_length" BIGINT, "run.r3_length" BIGINT, "run.r4_length" BIGINT, "run.Acount" BIGINT, "run.Ccount" BIGINT, "run.Gcount" BIGINT, "run.Tcount" BIGINT, "run.Ncount" BIGINT, "run.experiment" VARCHAR, "run.pool_member" VARCHAR, "submission.accession" VARCHAR, "submission.srasource" VARCHAR, "submission.bioprojectsource" VARCHAR, "seqdetective.n_mates" BIGINT, "seqdetective.mapping_rate.mate1" DOUBLE, "seqdetective.mapping_rate.mate2" DOUBLE, "seqdetective.nofeature_rate.mate1" DOUBLE, "seqdetective.nofeature_rate.mate2" DOUBLE, "seqdetective.sparsity.mate1" DOUBLE, "seqdetective.sparsity.mate2" DOUBLE, "seqdetective.pos_strand_rate.mate1" DOUBLE, "seqdetective.pos_strand_rate.mate2" DOUBLE, "seqdetective.readlen.mate1" BIGINT, "seqdetective.readlen.mate2" BIGINT, "seqdetective.judgement.mate1" VARCHAR, "seqdetective.judgement.mate2" VARCHAR, "seqdetective.judgement.reason" VARCHAR, platform_family VARCHAR, instrument_generation VARCHAR, read_bias VARCHAR, selection_class VARCHAR, prep_kit VARCHAR, sc_or_bulk VARCHAR, tech_class VARCHAR, technology VARCHAR, tech_variant VARCHAR, "submission.bioprojectsource.country" VARCHAR, earliest_date DATE, devstage_curation VARCHAR, devstage_curation_coarse VARCHAR, tissue_curation VARCHAR, tissue_curation_coarse VARCHAR);;
CREATE INDEX idx_run_bioproject ON run_metadata(bioproject);;
CREATE INDEX idx_run_run_accession ON run_metadata("run.accession");;