RNA-Seq Protocol for Differential Gene Expression

RNA sequencing was performed using the 6G RNA Sequencing Service (150bp paired-end, 40 million reads) (Arraystar Inc., Rockville, MD, USA). In brief, mRNA was enriched using NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs Inc., Ipswich, MA, USA) and RNA sequencing library preparation was performed using a KAPA Stranded RNA-Seq Library Prep Kit (Illumina, San Diego, CA, USA). Sequencing (150 cycles for both ends) was performed on an Illumina Novaseq 6000. Image analysis and base calling was performed using Solexa pipeline v1.8 (Off-Line Base Caller software, v1.8) and sequence quality was examined using FastQC software. Trimmed reads (trimmed 5’, 3’-adaptor bases using cutadaptor) were aligned to the reference genome (GRCm38) using Hisat2 software. The transcript abundances for each sample were estimated using StringTie [45 (link)] and the FPKM, differential gene expression and volcano plots were generated using R package Ballgown [12 (link)]. RNA sequencing data are available at Gene Expression Omnibus (accession no. GSE144796).