Truseq mrna kit v 2

Wild-type embryos were microinjected with 200 ng of Pcdh18a mRNA or 0.5 mM Pcdh18a MO. At 24 hpf, pools of 50 embryos from the wild-type and the injected clutches were collected. Total RNA extraction was performed with TRIzol (Invitrogen) according to the manufacturer’s protocol. The extracted total RNA samples were tested on RNA nanochips (Bioanalyzer 2100, Agilent) for degradation. Sequencing libraries were generated with the TruSeq mRNA kit v.2 (Illumina). The size and concentration of the sequencing libraries were determined with DNA-chip (Bioanalyzer 2100, Agilent). Multiplexed samples were loaded on a total of six sequencing lanes. Paired end reads (2 × 50 nucleotides) were obtained on a Hiseq1000 using SBS v3 kits (Illumina). The sequencing resulted in 300 million pairs of 50-nucleotide-long reads. The reads were mapped against the zebrafish genome (Zv9) using TopHat version 1.4.1. Gene expression was determined with HTSeq version 0.5.3p3.

RNA quality was assessed using the Experion RNA StdSens Analysis Kit. RNA-seq libraries were prepared from total RNA using the TruSeq Stranded mRNA LT kit according to the manufacturer's instructions. Quality of sequencing libraries was controlled on a Bioanalyzer 2100 using the Agilent High Sensitivity DNA Kit. Pooled sequencing libraries were quantified with digital PCR (QuantStudio 3D) and sequenced on the HiSeq 1500 Illumina platform in Rapid-Run mode with 50 base single reads. RNAseq was performed from RNA isolated using the RNAeasy mini kit with the Illumina Truseq mRNA kit v2 on an Illumina Hiseq 1500 according to the manufacturer's instructions.