Nextseq 500 desktop next generation sequencing system

Cells were sorted into RLT buffer (Qiagen RNAeasy Plus Micro kit) and RNA was purified according to the manufacturer’s instructions. RNA quality was validated using a Pico Bioanalyzer. RNA sequencing libraries were generated using SMARTer Stranded Total RNA-Seq kit – Pico Input Mammalian (Clonetech). Library fragment size was measured using D1000 Screen Tape (Agilent) and library concentration was measured using Qubit RNA assay kit (Life Technologies). Libraries were sequenced using the Illumina NextSeq 500 desktop Next Generation Sequencing (NGS) system. The quality of RNA-seq raw reads was checked using FastQC 0.11.2. Raw reads were quality-trimmed using Trimmomatic 0.32 and mapped to the mouse genome, GRCm38, using TopHat 2.0.12 with Bowtie2 2.2.3. Mapped reads were quality-filtered using SAMtools 0.1.19, and quantified using HTSeq 0.6.1. Differential expression was assessed using DESeq2 1.8.2 and RUVSeq 1.2.0 with R 3.2.1. A fold change heatmap was generated using Cluster 3.0. Computational analysis was performed using the BioHPC high-performance computing cluster at UTSW.