title: GSM3457021: RNAseq_NCAPH2_Rep2; 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; 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). Tethered Chromosome Capture (TCC) variant of Hi-C; RNA-seq