RNA-seq protocol for mouse gene expression

Total RNA was isolated with a combination of RNeasy Plus Kit (Qiagen, Germany) and Trizol (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturers’ instructions. The integrity of the RNA was accessed with TapeStation RNA analysis ScreenTape (Agilent). Libraries were prepared using the NEBNext® Ultra II Directional RNA Library Prep Kit for Illumina (NEB). The starting RNA material was 200 ng. Briefly, polyA+ RNAs were selected with magnetic beads, the RNA was fragmented and cDNA was synthesized. After A-tailing and adaptor ligation, libraries were generated by amplifying the cDNA for 10–12 cycles and purified with Ampure XP beads. Libraries were sequenced on an Illumina HiSeq 2500 or NovaSeq 6000 with pair-end 50-bp reads. RNA-seq data were aligned to the mouse reference genome (mm9) by Hisat2 [88 (link)]. Gene expression was summarized by HTSeq-counts [89 (link)]. The DESeq2 package v1.30.0 [90 ] was used to normalize the raw counts and identify differentially expressed genes (adjusted P-value < 0.05).