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)

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.