GSM3358207: Input_P14.hep.sexF.bio1 (ChIP-Seq); Mus musculus; ChIP-Seq
Mice are anesthetized using isoflurane (Butler Animal Health Supply, Cat# 029405) and monitored by paw pinch. A V-shaped incision was used to reveal the abdominal cavity, the intestines moved aside, and a 22 or 24 gauge catheter (Midwest Veterinary Supply, 381423; 22 gauge for M2 animals, 24 gauge for P14 animals) was used to cannulate the venae cavae while liver perfusion media pumps into the circulation, and the portal vein is immediately severed. 37°C liver perfusion media (Invitrogen 17701-038) and then liver digest media (Invitrogen 17703-034) are perfused through the liver (45 mL each for 2-month-old animals, 25 mL each for P14 animals). Livers are dissociated with a cutting motion with cell scrapers in William's E Medium (Sigma W4128) and strained through a 100 μm filter (BD 352360).  Hepatocytes are pelleted by centrifugation at 50g for 5 min at 4°C.  Isolated hepatocytes were fixed in 25 mL 1% formaldehyde in PBS for 10 min at room temperature with gentle rocking and quenched with 2.3 mL 2.5 M glycine for 5min at room temperature with gentle rocking. Fixed hepatocytes were pelleted in a swinging bucket centrifuge for 5 min at 4˚C at 50g. Fixed hepatocytes were resuspended in 10 mL (age: P14) or 20 mL (age: M2) ice-cold RSB buffer (10 mM Tris pH7.4, 10 mM NaCl, 3 mM MgCl2, 0.5% NP40, protease inhibitor cocktail tablet, 0.1 mM PMSF, 1% Triton-X 100) and dounced 25 times in a Wheaton dounce on ice to lyse cytoplasmic membranes. Fixed and dounced hepatocytes were pelleted for 10 min at 4˚C at 100g and resuspended in 2 mL ice-cold AS sonication-lysis buffer (10 mM Tris-HCl, pH8, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% Na-deoxycholate, 0.5% N-lauroylsarcosine, 1 mM DTT, Roche Protease Inhibitor Cocktail (Sigma 11697498001), 0.1 mM PMSF). Hepatocytes in AS-sonication lysis buffer proceeded to sonication. P14 and M2 hepatocyte H3K27me3 (Millipore 07-449) libraries were then generated using the ThruPlex DNA-seq kit Rubicon Genomics catalog #R400428. The M2 hepatocyte H3K27me3 ChIP was previously submitted for another paper (GEO accession: GSE114198, samples: GSM3137721, GSM3137722, GSM3137723, GSM3137724). ThruPlex DNA-seq kit Rubicon Genomics catalog #R40042
GEO
GSM3358207