GSM2424371: TA_rep2; Mus musculus; ATAC-seq
Eight week-old male F1 animals from control (TA) and nicotine-exposed fathers were anaesthetized using ketamine (100 mg/kg BW) and xylazine (10 mg/kg BW). Abdominal cavity was opened with a transversal incision below the rip cage. We dissected the portal vein with blunt forceps and inserted a 26 G catheter needle. After cutting the vena cava inferior cranial of the liver, we perfused the organ firstly with HBBS 1X + 200 mM  EDTA (10ml at 7ml/min) and secondly with 50ml DMEM containing collagenase type I (0.4 mg/ml) at 7 ml/min. Liver was then removed from the abdominal cavity, put in a petri dish containing culture media (DMEM, 20% FBS, 1X ITS, 1X Penicillin/Streptomycin, 0.1 uM Dexamethason, Sigma-Aldrich), and gently dissected to allow release of hepatocytes and supporting cells from connective tissue. After filtration through 70 um nylon cell strainer, cells were washed twice with PBS 1X and once with culture media (centrifugation at 500 rpm for 5 min), and plated on a 0.1% gelatin coated well. Hepatocytes were allowed to adhere to the bottom of the well for three hours. ATAC-Seq was performed as previously descirbed (Buenrostro JD et al. Current Protocols in Molecular Biology 2015), with only slight adjustments. The volume of enzyme mix used for 50,000 cells was adjusted to 3.75 ml due to the fact that approximately 50% of all hepatocytes in culture are binuclear.
GEO Accession
GSM2424371