mRNA Sequencing Protocol for Differential Expression Analysis

For mRNA sequencing (mRNA-seq), 5 ng high quality RNA (RIN ≥ 7) was used. cDNA was synthesized as previously described [52 (link)], converted to the NGS library using a Nextera XT Library Prep kit (Illumina, San Diego, CA, USA), and sequenced with a NextSeq 500 high-output 75 cycle kit (Illumina, San Diego, CA, USA). A quality check and adapter trimming of FASTQ files were performed as described for the sRNA-seq data. Clean reads were aligned to the GRCh38/hg38 human reference genome by HISAT2 [53 (link)]. Count matrices were created using the featureCounts tool according to the genome annotation and were provided to the DESeq2 package. The DE genes (DEGs) with p < 0.05 and |log2FC| ≥ 1 were considered for further analysis.