run_metadata: 72286

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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
72286	SRR22398736	SRX18368409	SRS15854841	SRP409542	PRJNA904806	Bulk RNA seq of zebrafish 5dpf embryos comparing Klf9 knockout with matched controls at 3 timepoints flanking zeitgeber lights on	GSE218687	Transcriptome Analysis	Krüppel like factor 9 Klf9 is a ubiquitously expressed transcription factor that is a feedforward regulator of multiple stress responsive and endocrine signaling pathways. We previously described how loss of Klf9 function affects the transcriptome of zebrafish larvae sampled at a single time point 5 dpf dpf. However klf9 expression oscillates diurnally and the time point that was sampled corresponded to its expression nadir. To determine if klf9 / function varies with time of day we performed bulk RNA seq on 5 dpf zebrafish embryos sampled at three timepoints encompassing the predawn peak and midmorning nadir of klf9 expression. We found that while the major effects of the klf9 / mutation that we reported previously are robust to time of day the mutation has additional effects that manifest only at the predawn time point. We used a published single cell atlas of zebrafish development to map the effects of the klf9 / mutation onto different cell types and found that the mutation increased representation of genes associated with digestive organs liver pancreas and intestine and decreased representation of genes associated with differentiating neurons blood and somites. Measurements from confocally imaged larvae suggest that overrepresentation of liver genes in klf9 / mutants is due to development of enlarged livers. Overall design: Comparative gene expression profiling analysis of RNA seq data for zebrafish klf9 / mutant larvae vs wild type at three times of day. Klf9 / knockouts were generated through CRISPR creating a 2 bp deletion in the first exon that causes a frameshift and premature stop codon as previously described in Gans et al Scientific Reports 10 11415 2020. https://doi.org/10.1038/s41598 020 68040 z				ko am30 1	GSM6754838		source name:larvae\|tissue:larvae\|developmental stage:5 dpf embryo\|time: 3\|mdibl id:KO 3 1\|conc ng ul:166.574\|a 260s280:1.94\|a 260s230:1.16\|sequence count:50914883	ko am30 1	Quality and adapter trimming was applied to fastq formatted read files with Trim Galore Version 0.6.5 with options: " fastqc args ' noextract'" " illumina" " length 1 35" " length 2 35" " stringency 1" " length 20" and " quality 20". Gene and isoform expression was estimated with RSEM Version 1.3.3 using Ensembl release 101 of the Danio rerio genome for alignment and using command “rsem calculate expression” with options: " keep intermediate files" " star gzipped read file" " p 64" " estimate rspd" " append names" " output genome bam" and " star". This run used 64 threads with the option to estimate the read start position distribution RSPD from the data instead of the default uniform RPSD and the option to output a BAM formatted file of read alignments in genomic coordinates. Counts were formatted into a count matrix with tximport using default options. Due to concerns regarding potential confounding experimental variables this experiment was repeated with improved collection protocols yielding two runs of RNAseq data. PCA of these two runs revealed high similarity between the two so the two were "merged" into one by taking the sum of transcript counts from corresponding samples with respect to time replicate number and genetics from the two runs. The transcripts from this experiment were aligned to Ensembl gene IDs Danio rerio 101. For downstream analysis it is more valuable to refer to genes by their name to estimate expression. There were 581 genes that were associated with more than one ensembl ID so the count matrix collapsed these distinct transcript counts by taking the sum of all transcript counts that correspond to the same gene name effectively removing gene duplicates from the count matrix. Principal Components Analysis PCA was run on the count matrix to identify dominant sources of variance in transcript counts between samples in this experiment. This analysis was conducted with the pipeline developed by Bianca M. Massacci for the MDIBL Bioinformatics Core Massacci 2020. Differential Expression Analysis was performed between wildtype and klf9 / samples in the count matrix with the MDIBL Bioinformatics Core’s DESeq2 R script using R version 4.0.3 and DESeq2 version 1.30.1. In addition to PC1 being used as a covariate time was used as a covariant to isolate the time independent effect of the klf9 knockout. The time dependent effect of klf9 loss was captured by separating samples by time and rerunning the analysis between wildtype and klf9 / samples. Assembly: Zebrafish genome v11 as obtained from Ensembl release 101 and preprocessed with rsem prepare reference using STAR’s genomeGenerate option using STAR v 2.7.5c Supplementary files format and content: tsv and txt files containing raw and transformed/normalized expression data corrected for listed conditions and principal components Supplementary files format and content: jcoffman 008.ZTp2.5.DESeq2 out.tsv; tab separated values DESeq2 output table for comparison of Klf9 / vs Wildtype control WT at the time point 2.5 hours post lights on. The first principal component which was associated with a technical affect of replicates was included as a covariate in the regression. Supplementary files format and content: jcoffman 008.ZTm3.DEseq2 out.tsv; tab separated values DESeq2 output table for comparison of Klf9 / vs Wildtype control WT at the time point 3 hours before lights on. The first principal component which was associated with a technical affect of replicates was included as a covariate in the regression. Supplementary files format and content: jcoffman 008.gene.counts.tsv; tab separated values. RSEM estimated count matrix at the gene level with removal of duplicate genes as described above Supplementary files format and content: jcoffman 008.ZT.DESeq2 out.tsv; tab separated values DESeq2 output table for comparison of Klf9 / vs Wildtype control WT at lights on zeitgeber time. The first principal component which was associated with a technical affect of replicates was included as a covariate in the regression. Supplementary files format and content: jcoffman 008.PC1 time.DESeq2 out.tsv; tab separated values DESeq2 output table for comparison of Klf9 / vs Wildtype control WT across all time points. The first principal component which was associated with a technical affect of replicates was included as a covariate in the regression as was the time of day. Supplementary files format and content: jcoffman 008.rlog.tsv; DESeq normalized count matrix using the rlog regularized log2 function	larvae		At 3 0 and 2.5 hours zeitgeber time onset of the light phase in the incubator on day 5 post fertilization four biological replicates of klf9 / mutant and wildtype larvae were collected as follows. Each biological replicate from each genotype and timepoint consisted of six larvae in a 30mm dish in 4ml of media. Larvae were collected from the dish into Eppendorf tubes media was removed and larvae immediately snap frozen in liquid nitrogen. Collection of the 8 samples at each time point occurred over 10 minutes. Frozen samples were stored at 80 deg C. RNA was prepared using the Qiagen RNeasy Plus mini kit Qiagen 74134. For each set of four biological replicate samples RLT Plus lysis buffer plus beta mercaptoethanol per kit instructions was added to each sample immediately upon its retrieval from the freezer and the sample was then immediately homogenized with a motorized pestle and placed on ice before the next sample was retrieved from the freezer and subjected to the same procedure. Following pestle homogenization replicate samples received simultaneous processing—i.e. passed though Qiashredder columns Qiagen 79654 bound washed and eluted per kit instructions. RNA was quantified and sent to the Oklahoma State Genomics Facility for Illumina library preparation and single end sequencing.	Zebrafish were klf9 / mutants derived from the AB strain and wildtype controls derived from the same grandparents. Husbandry and procedures were as described previously. All animal procedures were approved by the Institutional Animal Care and Use Committee of the MDI Biological Laboratory and all methods were performed in accordance with the relevant guidelines and regulations. Embryo culture and cortisol treatments were performed as previously described. Briefly fertilized eggs were collected in the morning disinfected and at 4 hpf placed in dishes with embryo media. Embryos developed in a 28.5o C incubator with a 14/10 light/dark cycle synchronized with the core fish room. Media was changed daily.	tissue:larvae\|genotype Klf9 / \|developmental stage:5 dpf embryo\|time: 3\|mdibl id:KO 3 1\|conc ng ul:166.574\|a 260s280:1.94\|a 260s230:1.16\|sequence count:50914883	GSM6754838	GSM6754838: ko am30 1; Danio rerio; RNA Seq	GSM6754838 r1	GSM6754838	1	At 3 0 and 2.5 hours zeitgeber time onset of the light phase in the incubator on day 5 post fertilization four biological replicates of klf9 / mutant and wildtype larvae were collected as follows. Each biological replicate from each genotype and timepoint consisted of six larvae in a 30mm dish in 4ml of media. Larvae were collected from the dish into Eppendorf tubes media was removed and larvae immediately snap frozen in liquid nitrogen. Collection of the 8 samples at each time point occurred over 10 minutes. Frozen samples were stored at 80 deg C. RNA was prepared using the Qiagen RNeasy Plus mini kit Qiagen 74134. For each set of four biological replicate samples RLT Plus lysis buffer plus beta mercaptoethanol per kit instructions was added to each sample immediately upon its retrieval from the freezer and the sample was then immediately homogenized with a motorized pestle and placed on ice before the next sample was retrieved from the freezer and subjected to the same procedure. Following pestle homogenization replicate samples received simultaneous processing—i.e. passed though Qiashredder columns Qiagen 79654 bound washed and eluted per kit instructions. RNA was quantified and sent to the Oklahoma State Genomics Facility for Illumina library preparation and single end sequencing.		RNA-Seq	TRANSCRIPTOMIC	cDNA	SINGLE	ILLUMINA	NextSeq 500		SRP409542			ko_am30_1_R1.fastq.gz	fastq	3786315387.0	50914883.0	GSM6754838 r1	0:74.37 1:0	A:965927215;C:875465462;G:846443972;T:1098110795;N:367943	74	0			965927215	875465462	846443972	1098110795	367943	SRX18368409	SRS15854841	SRA1546501	Bioinformatics Core, MDI Biological Laboratory	Bioinformatics Core, MDI Biological Laboratory	1	0.94574		0.11829		0.66574		0.4914		74		B		usable mapping rate	illumina	nextseq	unknown	cdna_unspecified	unknown	bulk	bulk	bulk		United States	2022-11-23	Larval	Larval	Embryo Imprecise	All anatomical structures