RNA sequencing to compare gene expression in control and PD-L1 knockdown hepatic stellate cells

Primary human hepatic stellate cells (HSCs) isolated from healthy donors were purchased from Sciencell. They were transduced with viruses encoding control or PD-L1 shRNA and stimulated with TGFbeta1 (5 ng/ml) for 24 hours. Cells were collected for RNA isolation and RNA sequencing. The goal of this study is to identify genes transcriptionally regulated by PD-L1. Overall design: There were 4 cell groups: NT shRNA, NT shRNA + TGFbeta1, PD-L1 shRNA, PD-L1 shRNA + TGFbeta1. Each group had 3 repeats. So 12 RNA samples were sent to UMN genomic Center for RNA sequencing. 12 RNA samples were converted to Illumina sequencing libraries by using Illumina's Truseq Stranded mRNA Sample Preparation Kit. Truseq libraries were then subjected to cluster by using Illumina cBot instrument and sequencing with HiSeq2500