GSM5288350: B6_Ctrl_cholang; Mus musculus; RNA-Seq
For flow sorting of individual cell populations from whole liver, tissue was harvested and digested in RPMI containing 0.5 mg/ml collagenase with 100U/ml DNase type I for 30 minutes at 37 C with agitation.  Single cell suspensions were obtained and cells were stained as described above. For isolation of cholangiocytes, we used dolichos biflorus agglutinin (DBA) (RL-1032, Vector Laboratories), a well-established, cell surface marker of bile duct cholangiocytes30. Cells were sorted into individual tubes containing buffer RLT (Catalog #:74104 Qiagen, Hilden, Germany) to lyse the cells. Ultrapure RNA was isolated using the RNeasy kit (Qiagen, Hilden, Germany) following the manufacturer's protocol. The quality of RNA was determined using the Agilent 2100 bioanalyzer and only used if the RNA integrity number was above 7. We combined an equal amount of RNA from 6-8 individual mice per genotype (equal number of males and females) for RNA sequencing studies. Thus, the RNA sequencing data presented is an average of 6-8 biological replicates per group. The NEBNext mRNA library prep kit for Illumina with ribosome reduction was used to generate the cDNA library, which was subsequently sequenced using Illumina NextSeq500 with paired end 75bp sequencing and aligned to the University of California, Santa Cruz (UCSC) GRCm38/mm10 reference genome using the STAR software package31. For each sample, at least 25 million reads were obtained.  Following alignment, HTSeq-count (version 0.9.1) was used to count the number of reads mapping to each gene32.  Fragments per kilobase of transcript per million mapped reads (FPKM) and pairwise differential expression were calculated using the Cufflinks suite (https://github.com/cole-trapnell-lab/cufflinks) and HTSeq-Count/DESeq2 was used to identify the most highly variable genes between groups.