GSM5444601: Liver PSC_11; Mus musculus; RNA-Seq
Mouse livers were collected at day 35 post-cholangitis induction and stored directly in RNAprotect Tissue Reagent solution (Qiagen) and stored at 4°C. Total RNA extraction and purification were performed using the RNeasy plus mini kit (Qiagen) and the Qiashredder homogenizer columns (Qiagen), according to the supplier's instructions. Lysis and homogenization of the tissues was formerly achieved thanks to the Precellys 24 Tissue Homogenizer (Bertin Instruments) and hard tissue homogenizing CK28-R 2 mL tubes (Bertin Instruments) in the lysis buffer supplied in the RNeasy plus mini kit, at 5 000 rpm for 20 seconds (twice). RNA concentration in samples was then measured using a NanoDropTM 1000 (Thermo Fisher Scientific). The libraries were prepared following the Tru-seq mRNA protocol from Illumina, starting from 1 µg of high-quality total RNA. Paired end (2 × 75) sequencing was performed on an Illumina Nextseq 500 platform. Fluorometric Qubit RNA HS assay (Life Technologies, Grand Island, New York, USA) was applied to measure RNA concentration. RNA quality (RNA integrity number) was assessed on the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). To build libraries, 1 µg of high-quality total RNA sample (RIN >8) was processed using TruSeq stranded mRNA kit (Illumina) according to manufacturer's instructions. Briefly, after purification of poly-A containing mRNA molecules, nucleic acids were fragmented and reverse-transcribed using random primers. Replacement of dTTP by dUTP during the second strand synthesis permitted to achieve strand specificity. Addition of a single A base to the cDNA was followed by ligation of adapters. Libraries were quantified by Qubit DNA HS Assay (Life Technologies, Grand Island, CA, USA) and library profiles were assessed using the DNA High Sensitivity LabChip kit on an Agilent Bioanalyzer. Libraries were sequenced on an Illumina Nextseq 500 instrument using 75 base-length read V2 chemistry in a paired-end mode. After sequencing, a primary analysis based on AOZAN software (ENS, Paris) was applied to demultiplexed and control the quality of the raw data (based of FastQC modules / version 0.11.5) (Perrin et al., 2017).