RNA Sequencing Analysis Workflow

The RNA isolation was performed using a previously described method [45 (link)]. Briefly, total RNA was isolated using Trizol (Invitrogen, MA, USA), and 5 μg of total RNA was purified using the RNeasy Mini Kit (QIAGEN, Hilden, Germany). Furthermore, 100–200 ng of mRNA was fragmented through hydrolysis and purified (RNAeasy Minelute Kit; QIAGEN, Germany). Library DNAs were prepared according to the Illumina TrueSeq protocol using the Truseq Standard mRNA LT Sample Prep Kit (Illumina, San Diego, CA, USA) and sequenced by Illumina NextSeq 500 (Illumina, CA, USA) using the Nextseq 500/550 High Output v2.5 Kit (Illumina, CA, USA) to obtain single-end 75 bp reads. The resulting reads were aligned to the mouse genome (mm10) using STAR ver.2.6.0a after trimming to remove the adapter sequence and low-quality ends using Trim Galore! v0.5.0 (cutadapt v1.16). The transcript abundance was determined using RSEM v1.3.1. The counts of gene-expression profiles in those selected datasets were further analyzed through RNAseqChef web-based transcriptome analysis [22 (link)]. Pathway Interaction Database was used for enrichment analysis in RNAseqChef. The Z-scored normalized count is shown in the heatmaps (genefilter: methods for filtering genes from high-throughput experiments. R package version 1.72.1.).