run_metadata: 57247

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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
57247	SRR11293504	SRX7899066	SRS6307322	SRP252498	PRJNA612134	Targeted RNA Knockdown by a Type III CRISPR Cas Complex in Zebrafish	GSE146852	Transcriptome Analysis	RNA interference RNAi is a powerful experimental tool for RNA knockdown but not all organisms are amenable. Here we provide a “proof of principle” demonstration that CRISPR endoribonuclease can be used for programmable mRNA transcript degradation in eukaryotes. Using zebrafish as the animal model and Csm complex as the CRISPR endoribonuclease we targeted EGFP transcript expressed from a variety of promoters. A drastic decrease of fluorescence was achieved in germ cells of the vasa:EGFP line. Weaker effects were also seen in fish lines that express EGFP zygotically. Knockdown was statistically significant in cmcl2:EGFP and fli1:EGFP zebrafish lines at 1 dpf dpf but reduced to background levels at 2 dpf. The nkx2.5:EGFP fish line was least susceptible to Csm mediated EGFP knockdown. We also tested Csm mediated knockdown on the endogenous tdgf1 oep transcript. At optimal Csm dose we observed a penetrance of the characteristic one eyed phenotype at greater than 50% penetrance and hence with similar efficiency to morpholino mediated knockdown. We conclude that Csm mediated knockdown is very efficient for maternal transcripts and can also be used for mixed maternal/early zygotic and early zygotic transcripts in some cases reaching comparable efficiency to morpholino based knockdown with no significant off target effects in our model. Overall design: Zebrafish embryos from the transgenic line Tgddx4:ddx4 EGFP on the background of ABTL were injected with the Csm complex targeted against the EGFP transcript or mock solution StCsm buffer without xxx proteins. The RNA was isolated from the pooled embryos at three time points: 128 cell stage 5 hpf and 24 hpf. Three replicates were obtained from each experimental condition and time point. Pair end sequencing was performed on the Illumina NextSeq platform.		pubmed:32833532		Mock 128 Rep 3	GSM4407863		source name:whole zebrafish embryo\|strain:ABTL Tgddx4:ddx4 EGFP\|tissue:whole embryo\|condition:Mock\|developmental stage:128 cells	Mock 128 Rep 3	Base calling was performed by Illumina RTA 1.18.61. Files were translated into demultiplexed FASTQ files by Illumina bcl2fastq2 2.16.10. The first 12 nucleotides of read 1 containing the UMI sequence were extracted and added to the sequence identifier using UMI tools v. 1.0.0. Low quality sequences and adapter content were trimmed with cutadapt v. 2.4. Reads were aligned to the GRCz11 reference genome supplemented with the EGFP sequence with STAR v. 2.7.2b using the following custom parameters: outFilterType BySJout outFilterMultimapNmax 20 alignSJoverhangMin 8 alignSJDBoverhangMin 1 outFilterMismatchNmax 999 alignIntronMin 20 alignIntronMax 1000000 alignMatesGapMax 1000000 outFilterMismatchNoverLmax 0.6. Alignments were de duplicated based on their Unique Molecular Identifier sequence using UMI tools v. 1.0.0. De duplicated alignments were counted per exon on the gene level with the htseq count script from the HTSeq v. 0.11.2 package using Ensembl GRCz11 version 97 genome annotation. Further analysis was performed in R v. 3.6.1 programming language. Differential expression was analyzed using DESeq2 v. 1.24.0 package. Genome build: GRCz11 Supplementary files format and content: HTSeq counts.tsv files were generated with htseq count script they contain counts for each gene for each sample in tab delimited format; normalized counts files were generated with DESeq2 they contain library size normalized read counts for each gene for every sample from each time point in tab delimited format; egfp.fa file contains the EGFP sequence in FASTA format; egfp.gtf file contains the custom EGFP annotation in GTF format; egfp crrna.fa file contains the crRNA sequence against the EGFP transcript used in the experiment in FASTA format.	whole zebrafish embryo	Zebrafish embryos from three different mating pairs of Tgddx4:ddx4 EGFP females with casper males were pooled during collection. D16A StCsmEGFP was diluted with water for injection 1:1 v/v up to a concentration of 0.7mg/ml. For mock injection Tris storage buffer 10mM Tris HCl pH8.5 300mM Nacl 1mM DTT 0.1mM EDTA 50% glycerol was also diluted in the similar manner. 1nl of either StCsm EGFP solution 0.7ng or buffer alone were injected into the yolk of embryos at the 1 cell stage. In total three independent injections were performed. 25 embryos for both StCsm and mock injected per replica set per time point were frozen in liquid nitrogen stored at xxx℃ and processed together.	25 embryos each for both StCsm and mock injected were pooled at xxx cell stage 5 hpf and 24 hpf from xxx biological replicates. Total RNA was extracted from the whole embryos with Tri Reagent Solution Invitrogen according to the manufacturer’s protocol. RNA was further purified using Zymo RNA Clean and Concentrator 5 kit according to manufacturer's protocol. 1.5 ug of total RNA was subjected to Lexogen polyA RNA Selection kit according to company recommendations. Enriched RNA has been used for sequencing library construction with CORALL Total RNA Seq Library Prep Kit according to the Lexogen manufacturer’s protocol.	Zebrafish embryos were maintained according to standard protocol Westerfield et al. 2000 and staged according to standard morphological criteria Kimmel et al. 1995.	strain:ABTL Tgddx4:ddx4 EGFP\|tissue:whole embryo\|condition:Mock\|developmental stage:128 cells	GSM4407863	GSM4407863: Mock 128 Rep 3; Danio rerio; RNA Seq	GSM4407863		1	25 embryos each for both StCsm and mock injected were pooled at xxx cell stage 5 hpf and 24 hpf from xxx biological replicates. Total RNA was extracted from the whole embryos with Tri Reagent Solution Invitrogen according to the manufacturer's protocol. RNA was further purified using Zymo RNA Clean and Concentrator 5 kit according to manufacturer's protocol. 1.5 ug of total RNA was subjected to Lexogen polyA RNA Selection kit according to company recommendations. Enriched RNA has been used for sequencing library construction with CORALL Total RNA Seq Library Prep Kit according to the Lexogen manufacturer's protocol.	GEO Accession:GSM4407863	RNA-Seq	TRANSCRIPTOMIC	cDNA	PAIRED	ILLUMINA	NextSeq 500		SRP252498			Mock-128-Rep-3_R2.fastq.gz Mock-128-Rep-3_R1.fastq.gz	fastq fastq	3261013600.0	25084720.0	GSM4407863 r1	0:71 1:59	A:900737410;C:688855174;G:770946078;T:895944416;N:4530522	71	59			900737410	688855174	770946078	895944416	4530522	SRX7899066	SRS6307322	SRA1054182	GEO	Laboratory of Zebrafish Developmental Genomics, International Institute of Molecular and Cell Biology in Warsaw	2	0.57905	0.4659	0.03571	0.0302	0.77536	0.79022	0.5161	0.51323	71	59	B	B	biological fallback assumption	illumina	nextseq	unknown	cdna_unspecified	lexogen	bulk	bulk	bulk		Poland	2020-03-12	Blastula	Embryo	Whole Organism	All anatomical structures