Stainier Lab, Max Planck Institute For Heart and Lung Research
RNA from two replicates of mutant and sibling samples were isolated using the miRNeasy micro Kit (Qiagen) and treated with in-column DNase digestion. Quality checks on RNA for library preparation were performed with a BioAnalyzer 2100 (Agilent Technologies) and LabChip Gx Touch 24 (Perkin-Elmer). A total of 300 ng of total RNA was used as input for Truseq Stranded mRNA library preparation. Sequencing was performed on the NextSeq 500 instrument (Illumina) using v1 chemistry, obtaining a minimum of 10–15M reads (75 bp paired-end) per library. Quality checks were performed on raw reads with FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc). Adapter sequences and poor quality base calls were trimmed from the raw reads with Trimmomatic v0.33 (parameters: LEADING:3 TRAILING:3 SLIDINGWINDOW:5:20 CROP:500 MINLEN:15)(Bolger et al., 2014). The processed reads were then aligned to Zv10 version of the zebrafish genome with STAR 2.4.0a (parameters: --outFilterScoreMinOverLread 0 --outFilterMatchNminOverLread 0 --outFilterMatchNmin 30 --outFilterMultimapNmax 999 --alignEndsProtrude 10 ConcordantPair) (Dobin et al., 2013). The resulting bam alignment file was passed through the Cufflinks suite v2.2.1 with standard parameters (Trapnell et al., 2012) to calculate the Fragments Per Kilobase of transcript per Million mapped reads (FPKM) for each gene as well as the statistics on the fold-changes.