RNA-seq mus musculus: WT macrophages-2
BMDMs were lysed in Buffer RLT (Qiagen, Germantown, MD) plus ?-mercaptoethanol. RNA was extracted using the Qiagen AllPrep DNA/RNA kit (Qiagen, Germantown, MD) and processed using standard manual protocol. RNA was DNase-treated and purified using the RNA Clean and Concentrator-5 kit (Zymo Research, Irvine, CA) and assessed for RNA quality using an Agilent 2100 Bioanalyzer and the RNA 6000 Nano Kit (Agilent Technologies, Santa Clara, CA). Total RNA was used to prepare cDNA libraries using the Illumina TruSeq Stranded Total RNA with Ribo-Zero Gold Preparation kit (Illumina Inc., San Diego, CA). Total RNA was rRNA depleted followed by enzymatic fragmentation, reverse-transcription and double-stranded cDNA purification using AMPure XP magnetic beads. The cDNA was end repaired, 3? adenylated, with Illumina sequencing adaptors ligated onto the fragment ends, and the stranded libraries were pre-amplified with PCR. The library size distribution was validated and quality inspected using a Fragment Analyzer (Advanced Analytical Solutions, Parkersburg, WV). The quantity of each cDNA library was measured using the Qubit 3.0 (Thermo Fisher Scientific, Walthanm, MA). The libraries were pooled and sequenced to a target read depth of 40M reads per library using single-end 76 cycle sequencing with the High Output 75-cycle kit (Illumina Inc., San Diego, CA) on the Illumina NextSeq 500.