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RNA-seq analysis comparing WT, Rad21 MO and CTCF MO zebrafish embryos at stages (2.5, 3.3, 4.5, 5.3, 10 hpf) pre and post ZGA (zygotic genome activation)

ABSTRACT: Methods: Triplicate RNA samples from morphologically stage-matched embryos were sequenced to compare expression profiles over time. Strand-specific libraries were prepared using the TruSeq stranded total RNA-ribozero kit (Illumina) and 100-bp paired-end sequencing was performed to depth of 10 million reads per library on an Illumina HiSeq 2000. Methods: On average, 19 million 100 bp paired-end reads per library were generated. These were then adapter and quality trimmed using cutadapt and SolexaQA. Each sequencing data set was independently mapped to the zebrafish genome with a bowtie2 index generated from Danio_rerio.Zv9.70 (Ensembl) downloaded from Illumina’s iGenomes collection. Zebrafish genome danRer7was used to provide known transcript annotations from Ensembl using TopHat2 (version 2.0.9) with the following options: “tophat2 --GTF genes.gtf --library-type fr-firststrand -p 24 --mate-inner-dist -8 --mate-std-dev 6 zv9” (on average, 75.38% reads mapped uniquely to the genome). Transcriptomes were assembled with Cufflinks (version 2.2.0) using options: ‘cufflinks -p 32 --GTF genes.gtf’ and differential expression analysis between control and knockdown embryos was performed using Cuffdiff. A FDR corrected p-value of 0.05 was applied as the cut off to identify differentially regulated transcripts Results: We could show that MO assisted depletion of Rad21 and CTCF affected the transcriptional profiles of embryos in different ways.

ORGANISM(S): Danio rerio

PROVIDER: GSE84601 | GEO | 2017/12/01

SECONDARY ACCESSION(S): PRJNA330616

REPOSITORIES: GEO

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Project description:Background: Nicotine and alcohol are often co-abused. Adolescence is a vulnerable period for the initiation of both nicotine and alcohol use, which can lead to subsequent neurodevelopmental and behavioral alterations. Aim: The aim was to to determine the effect of nicotine exposure during adolescence on ethanol intake, and the effect of both substances on brain gene expression. Methods: RNA was extracted from a randomly selected subset of mice (16 total; 4 samples from each experimental group). Whole brains (including cerebellum) were dissected. Total RNA was extracted with an RNeasy® Midi Kit (QIAGEN, Valencia, CA). RNA quality was assessed using an Agilent 2100 BioAnalyzer™. An Illumina TruSeq® Stranded mRNA Library Prep Kit (Illumina, San Diego, CA) was used for cDNA library preparation following the manufacturers’ protocol. Libraries were sequenced using an Illumina HiSeq 2500 (Illumina, San Diego, CA). Trimmomatic was used to remove sequencing adapters and low-quality ends. The cleaned dataset was analyzed with the Tuxedo pipeline. Subsequently, readings were mapped to the mouse reference genome (Ensembl GRCm38, mm10) using TopHat2 software (http://tophat.cbcb.umd.edu/). The ‘--library_type’ parameter was set to ‘fr-firststrand’. Default settings were preserved for all other TopHat2 parameters. The resulting alignments files from TopHat2 were used to generate a transcriptome assembly. Gene expression was calculated for each condition using the Cufflinks and Cuffmerge utilities. Cuffdiff2 with default settings was used to identify transcripts that were differentially expressed between each treatment group compared to the water only control group.The significance threshold was set at q < 0.05 (FDR corrected). Finally, a Fisher’s exact test was performed using the GeneOverlap R package to test the significance of DEG overlaps. Gene co-expression networks were identified using the Weighted Gene Co-expression Network Analysis (WGCNA) package. Results: RNA Integrity Number (RIN) was on average 8.23 ± 0.26 for all samples, suggesting high RNA integrity and quality. On average, 43 million, 150 base pair single end reads were generated for each sample and used in the analysis. The resulting alignments files from TopHat2 had an average mapping rate of 87.4%.

Dataset Information

RNA-seq analysis comparing WT, Rad21 MO and CTCF MO zebrafish embryos at stages (2.5, 3.3, 4.5, 5.3, 10 hpf) pre and post ZGA (zygotic genome activation)

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