title: GSM2453293: RNAseq_WT_Rep3; Mus musculus; RNA-Seq
library_construction_protocol: Liver was dissected and the left lateral lobe was prepped for a two-step perfusion adapted from (Li et al 2010 & Goncalves et al 2007): First, the liver was perfused with an EDTA-containing buffer to remove Ca2+ from the tissue in order to weaken the integrity of the desmosomes, which were subsequently digested using a Ca2+ rich buffer containing collagenase. The freed hepatocytes were rinsed through a cell strainer and washed four times with ice-cold Ca2+ rich buffer without collagenase. For each wash, the cells were spun at low centrifugal force (60g for 1min) to enrich intact hepatocytes and reduce non-mesenchymal debris. Part of each sample was fixed with 1% PFA for 10 minutes at room temperature. Fixed and unfixed hepatocytes were aliquoted and frozen in liN2 for later use. Hi-C: Roughly 100 million fixed hepatocytes per sample were processed according to (Kalhor et al, 2012) using HindIII. TCC libraries were PCR-amplified (12 cycles) and size selected. Equimolar pools of libraries were sequenced; ChIP-seq: Fixed aliquots of hepatocytes were hypotonically lysed and sonicated in 1% SDS/TE. An aliquot of each sample was reverse cross-linked in order to determine chromatin concentration and sonication efficiency. 20μg chromatin per sample was diluted in RIPA and incubated with 1.5μg of either αH3k4me3 antibody (C15410003-50, Diagenode) or αH3K27Ac antibody (ab4729, Abcam) at 4°C, overnight. The antibodies were retrieved with Dynabeads (IgA, Invitrogen) and bound chromatin was washed and eluted. After reverse cross-linking, the amount of ChIPped and input DNA was determined with Qubit (Thermo Fisher).  The libraries were prepared with NEBNext® ChIP-Seq Library Prep Kit for Illumina®. After amplification and size selection with E-Gel® SizeSelect™ (Thermo Fisher) their size-distributions were determined with Bioanalyzer. Equimolar pools of libraries were sequenced with Illumina HiSeq2000 (50bp, single end). RNA-seq: RNA integrity was tested with Bioanalyzer (Agilent RNA Nano Kit) and ribosomal RNA was removed using Ribo-Zero rRNA Removal Kit (Illumina) prior to library preparation.  Strand-specific libraries were prepared with NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina®. After amplification and size selection with Agencourt AMPure XP beads (Beckmann Coulter) their size-distributions were determined with Bioanalyzer. Equimolar pools of libraries were sequenced with Illumina HiSeq2000 (50bp, single end).