RNAseq analysis between hepatocytes isolated from control livers, β1-integrin mutant livers, P21 over-expressed livers and ductal cells.

RNAseq analysis between cell types. RNA extracted using Qiagen mini total RNA isolation protocols (Qiagen, Germany). Samples were treated with DNase (Ambion), and sample integrity verified on the Agilent Bioanalyser with the RNA Nano chip. Illumina Tru-seq paired end strand specific sequencing (Illumina, USA) was carried out by on a NextSeq-550 sequencer (Edinburgh Clinical Research Facility, Western General Hospital, Edinburgh, Scotland, UK). 500ng of Total RNA underwent ribosomal RNA depletion (rRNA) prior to purification, fragmentation, random hexamer cDNA generation and purification with AMPure XP beads (Beckman-Coulter, USA). Multiple indexing adapters were ligated to ds cDNA with subsequent hybridisation onto flow cells, and DNA fragment enrichment by 15 cycle PCR for sequencing. Completed libraries were quantified by qPCR using the KAPA Illumina Library Quantification Kit (Illumina, USA) before multiplexing in two equimolar pools and running on two flow cells on the Illumina NextSeq 550. The resulting FastQ files were mapped to the reference genome (mm9) using the Tophat alignment tool (V1) on Illumina Basespace software and reads per kilobase per million (RPKM) scores calculated. We carry out paired end, strand specific RNAseq prior to sequencing on NextSeq-550 sequencer to report on the transcriptional landscape control hepatocytes, wild type bile ducts, Biliray derived hepatocytes from Itgb1 fl/fl liver and Biliray derived hepatocytes from AAV8-p21 liver