Truseq pcr primers

Total RNA was isolated using the RNeasy Micro Kit (QIAGEN, Hilden, Germany). The quality and the integrity of the RNA were assayed using an Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, California). Subsequently, ribosomal RNA (rRNA) was depleted using the RNA Clean XP Kit (Beckman Coulter, Inc., Brea, California) and RNA was fragmented using RNase-Free DNase (QIAGEN). Immediately afterwards, first-strand cDNA was synthesized using the SuperScript II kit (Invitrogen, Carlsbad, California) while adding a hexamer random primer. Double-strand DNA fragments were ligated with a TruSeq adapter (Illumina, San Diego, California) and amplified with TruSeq PCR primers (Illumina) for sequencing.
Reads that were shorter than 25 nt and those that mapped to rRNA were discarded. The remaining clean reads were genome-mapped using spliced mapping in the Hisat2 package (Anaconda, Inc., Austin, Texas). Fragments per kilobase of transcript per million mapped reads (FPKM) of EdgeR (Anaconda, Inc.) were used to screen differentially expressed genes (DEGs). The p-value was corrected by multiple hypothesis testing, and the p-value threshold was determined by controlling false discovery rate (FDR). Corrected p-values are denoted as q-values. Transcripts with a q-value < 0.05 and a fold change (FC) > 2 were categorized as DEGs (Fig. 1).

Genotyping was performed using the SureSelect XT Custom Capture (Agilent). Multiplexing was achieved using TruSeq adapters (Illumina); samples were pooled prior to the capture and amplified post-capture using TruSeq PCR primers (Illumina). Blocking reagents were replaced with water to avoid cross-reactivity. Sequencing was performed using 50-bp paired-end reads (Illumina HiSeq 2000). Reads were aligned to the reference human genome (hg19) using BWA (Li and Durbin 2009 (link)). Genes were sequenced to an average coverage of 200× with 85% of the target sequenced to least at 50×. Genotypes were determined using the Genome Analysis Toolkit (GATK) (McKenna et al. 2010 (link)) “Better” protocol. Only high-confidence (quality score >100) variants predicted to have high or moderate severity and not reported in dbSNP (v129) were considered.