title: ChIP-seq of CTCF, RAD21, and TOP2B binding sites in three human tissue samples with hepatocellular carcinoma
design: ChIP-seq of CTCF, RAD21, and TOP2B binding sites in three human tissue samples with hepatocellular carcinoma
library_construction_protocol: Three liver hepatocellular carcinoma (HCC) samples were collected from male patients on a background of liver disease due to Hepatitis C, who underwent liver transplantation at the Ajmera Transplant Centre of Toronto General Hospital (ON, Canada) in 2016-2019 Approximately 30 mg of HCC tumor sample was used for each ChIP reaction. Frozen HCC tissues were dounce homogenized into single cell suspensions and cross-linked with 1% formaldehyde in Solution A (50 mM Hepes–KOH, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA) for 20 min at RT. Fixation was quenched by adding glycine to a final concentration of 125 mM. After two washes with ice cold PBS, cells were filtered through 100 μm cell strainer to remove any connective tissue. Cell lysis and nuclei isolation were carried out in 10 mL of LB1 buffer (Diagenode) by rotating 20 min at 4 C, followed by nuclear lysis in 5 mL of LB2 (Diagenode) and rotating 10 min at 4C. For chromatin shearing, the cell pellet was resuspended in Shearing Buffer (Diagenode) supplemented with complete proteinase inhibitors (Roche), and sonicated for 5 cycles (30 s ON, 30 s OFF) with the Bioruptor Pico sonicator (Diagenode). Chromatin was cleared by centrifugation (13,000 rpm for 10 min at 4 C). For ChIP, chromatin lysates were combined with 5 ug of anti-TOP2B, anti-CTCF, or anti-RAD21 antibodies and incubated overnight in IP buffer (Diagenode) rotating at 4 C, and then with 70 μl of pre-blocked (0.5 mg/mL BSA) Dynabeads protein G (ThermoFisher) for 4 hours. Beads were then washed six times with RIPA buffer (50 mM Hepes–KOH, pH 7.5; 500 Mm LiCl; 1 mM EDTA; 1% NP-40 or Igepal CA-630; 0.7% Na– Deoxycholate), and one time with TBS (20 mM Tris–HCl, pH 7.6; 150 mM NaCl), and resuspended in ChIP Elution buffer (50 mM Tris–HCl, pH 8; 10 mM EDTA; 1% SDS). Cross-linking was reversed by overnight incubation at 65 C. Cellular proteins and RNA were digested with Proteinase K (Invitrogen) and RNaseA (Ambion). ChIP DNA was purified with P:C:I method.  ChIP-seq libraries were prepared using NEBNext Ultra II DNA Library preparation Kit following the manufactures instructions (NEB, E7645). Libraries were amplified for 9 cycles using Q5 Hot Start High-Fidelity DNA Polymerase (NEB, M0493L), purified and size-selected with AMPure XP PCR purification beads (Beckman Coulter, A63881), and quantified with 2100 Bioanalyzer (Agilent). Input control DNA extracted from sonicated cell lysates of each sample were processed in parallel.