GSM7015668: m148; Mus musculus; Bisulfite-Seq
Partial hepatectomy was carried out on 12 weeks old mice by removing 2/3 of the liver, including resection of the left lower, upper lobes and the right upper lobe.  Mice were anaesthetized and subjected to liver perfusion. Whole liver was then digested by collagenase (Roche) and hepatocytes removed and isolated by means of a Percoll gradient. Cells were then sorted by FACS according to size. Hepatoblasts were prepared from E11.5 and E16.5 mouse embryos by collecting fetal liver samples from littermate siblings which were then minced and digested for 30 min at 37°C with 0.025% collagenase IV (Sigma-Aldrich, C4-22-1G). After gentle pipetting, the cell suspension was filtered through a 40-mm cell strainer and washed twice with RPMI medium containing 10% FBS. The cells were then stained for 40 minutes at 4°C with moderate agitation with Liv2 (mouse) mAb (MBL, D118-3) for E11.5 hepatoblast, and Dlk1-APC-conjugated mouse Pref-1/DLK1/FA1 antibody (R&D Systems, FAB8634A-100) for E16.5 hepatoblast. Positive hepatoblast cells were then isolated by Flow cytometry. DNA was isolated from purified cells, incubated in lysis buffer (25 mM Tris-HCl (pH  8), 2 mM EDTA, 0.2% SDS, 200 mM NaCl) supplemented with 300 μg/ml proteinase K (Roche) followed by phenol:chloroform extraction and ethanol precipitation. The methylation profile was checked using the RRBS assay [Boyle, 2012]. Briefly, the DNA was digested with the MspI enzyme to yield CpG enriched fragments. Adapter sequences were ligated at both ends, and DNA fragments were then treated with sodium-bisulfite, allowing for easy distinction between methylated and unmethylated bases. The DNA was then amplified by PCR to produce the final libraries.