run_metadata: 50979

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