Similar protocols

Protocol publication

[…] onal file : Table S1). Additionally, two pools of embryos at 5 days post fertilization (dpf) were used. An overview of tissues collected is depicted in Fig. . Tissue samples were stored in RNAlater (Life Technologies, Carlsbad, USA), after which RNA was isolated using the Qiagen Blood and Tissue DNeasy kit according to manufacturer’s manual (Qiagen, Hilden, Germany).Fig. 1, A library was made with the TruSeq Stranded total RNA library prep kit according to the manufacturer’s description (Illumina inc, San Diego, USA). Both paired end and single libraries were sequenced using an Illumina HiSeq 2500 according to the manufacturer’s description. Illumina software (HCS) was used for basecalling. Tophat version 2.0.5 [] was used to align the reads to the reference genome. For each read pair, secondary alignments (which meet alignment criteria but are less likely to be correct) were filtered out using samtools version 0.1.18 []. For each predicted gene, read counts were obtained from the alignment file using HTSeq-count version 0.5.3.p9 [] using the ‘intersection-strict’ setting to ignore reads not aligning to annotated exons., Data quality was assessed using the statistical package R []. Most importantly, raw RNA-Seq counts need to be normalized to correct for sequencing depth and (optionally) transcript length. Dividing counts by simple sequencing depth (the total number of reads) can lead to inaccurate results [], as it is strongly influenced by the most abundantly expressed genes. For example, extreme expression of a single gene (e.g. hormone-encoding genes []) will strongly affect the expression measures of all other genes if naïve CPM (counts per million) or RPKM (reads per million per kil […]

Pipeline specifications

Software tools TopHat, SAMtools, HTSeq