Bulk RNA sequencing of human mesenchymal stem cells (hMSC) and hMSC-derived cholangiocytes

The RNA from two biological replicates of hMSCs and hMSC-derived cholangiocytes which were pooled from three samples individually, was extracted and purified using RNeasy Plus Mini Kit (Qiagen). The extracted RNA was sent to Beijing Genomics Institute (BGI) for quality control, sequencing and bioinformatic analysis. The quality of the extracted RNA was assessed using Agilent 2100 Bioanalyzer. All the samples had an RNA Integrity Number (RIN) >9 and a 28S/18S >1. RNA sequencing was performed using the DNBseq platform. The sequencing reads that contained >5% unknown N base or adapters, mapped to rRNA, or with a quality score <15 were filtered and removed. The filtered reads were then mapped to the reference genome using HISAT2. Transcripts were reconstructed using StringTie. Novel transcripts were identified using Cuffcompare, merged with reference transcripts, and mapped with filtered reads using Bowtie2. The expression levels of genes were calculated using RSEM.