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.本研究针对人间充质干细胞（human mesenchymal stem cells, hMSCs）及其来源的胆管上皮细胞的各两个生物学重复样本，每个重复样本均由三份单独的样本混合制备得到，采用RNeasy Plus迷你试剂盒（Qiagen公司）完成RNA的提取与纯化。将提取得到的RNA送往北京基因组研究所（BGI）开展质量控制、测序及生物信息学分析。使用安捷伦2100生物分析仪对提取的RNA质量进行评估，所有样本的RNA完整性数值（RNA Integrity Number, RIN）均大于9，且28S/18S比值大于1。测序工作依托DNBseq平台完成。对包含超过5%未知N碱基、接头序列、比对至核糖体RNA（rRNA），或质量分数低于15的测序读段进行过滤并移除。随后，利用HISAT2将过滤后的读段比对至参考基因组；通过StringTie进行转录本重构；经Cuffcompare鉴定新转录本，将其与参考转录本合并后，使用Bowtie2与过滤后的读段进行比对；最终采用RSEM计算基因的表达水平。