title: CUT&RUN experiment in colon cancer cells (HCT116, DLD1 and SW620), assessing TBX3 genomic occupancy
design: CUT&RUN experiment in colon cancer cells (HCT116, DLD1 and SW620), assessing TBX3 genomic occupancy
library_construction_protocol: 250,000 cells/sample were harvested using Trypsin-EDTA (Cat. # 25200056, Gibco) for 5 - 10 minutes. Cells were washed in DPBS two times (Cat. #14190094, Thermo Fisher Scientific). HCT116, DLD1, and SW620 human colorectal cancer cells were cultured in a 37 °C incubator in 5% CO2 and 89% humidity. Culture medium used was high glucose Dulbecco's Modified Eagle Medium (Cat. #41965039, Gibco) supplemented with 10 % bovine calf serum (Cat. #1233C, Sigma-Aldrich) and 1X Penicillin-Streptomycin (Cat. #15140148, Gibco).   Nuclear extraction was performed by three washes in Nuclear Extraction (NE) buffer (HEPES-KOH pH-8.2 [20 mM], KCl [10 mM], Spermidine [0.5 mM], IGEPAL [0.05%], Glycerol [20%], Roche Complete Protease Inhibitor EDTA-Free). After extraction, nuclei were resuspended in 40 µl NE per sample and bound to 10 µl Magnetic ConA Agarose beads equilibrated in binding buffer (HEPES pH 7.5 [20 mM], KCl [10 mM], CaCl2 [1 mM], MnCl2 [1 mM]). Bead binding proceeded for 15 min at 4 degrees, then beads were resuspended in 200 µl wash buffer per sample and distributed in PCR tubes. Samples were washed once in 200 µl EDTA wash buffer (wash buffer with EDTA [0.2 mM]), incubating 5 min at room temperature before being resuspended in antibody buffer (wash buffer with antibody 1:100). Incubation happened ON at 4 °C on a rotator. Antibodies used included anti-TBX3 ABIN6265491 and anti-rabbit ABIN101961. The next morning samples were washed 5 times and resuspended in 200 µl of pAG-MNase buffer (wash buffer with pAG-MNase 120ng/sample) and incubated for 45 min at 4 °C on a rotator. Five washes were performed and during the last wash equilibrated 5 min in wet ice. Samples were resuspended in 200 µl ice cold wash buffer with 2 mM CaCl2for digestion of 30 min in wet ice. The digestion buffer was kept and transferred to tubes containing 1.5 µl of 0.5 M EDTA and 1.5 µl of 0.5 M EGTA to inactivate the pAG-MNase. Beads were resuspened in 47 µl of 1X Urea STOP buffer (NaCl [100 mM], EDTA [2 mM], EGTA [2 mM], IGEPAL [0.5%], Urea [8.8 M]) and the DNA was eluted for 1 hr at 4°C for elution. Beads were collected on the magnet and liquid containing DNA was transferred to the PCR tubes and mixed with the digestion buffer. DNA was purified using two successive rounds of bead purifications according to manufacturer's directions, using Mag-Bind TotalPure NGS beads (Cat. #M1327, Omega Bio-Tek) at 2X (200 µl first round and 40 µl second round), final elution was with 20 µl Tris-HCl pH 7.5. Library preparation was performed with the KAPA Hyper Prep Kit for Illumina sequencing (Cat. #KK8504, KAPA Biosystems) according to manufacturer's guidelines with modifications. 0.4X volume reactions were used for End repair and A-tailing steps. The thermocycler conditions were 12 °C for 15 min, 37 °C for 15 min and 58 °C for 25 min. Adapter ligation was also performed in 0.4X volume reactions. KAPA Dual Indexed adapters were used at 0.15 µM. DNA purification was performed after ligation with 1.2X volumes of Mag-Bind TotalPure NGS beads. Library amplification steps were performed in 0.5X volume reactions. The cycling conditions were set to: 45 sec initial denaturation at 98 °C, 15 sec denaturation at 98 °C, 10 sec annealing/elongation at 60 °C, 1 min final extension at 72 °C, hold at 4 °C. Libraries were amplified 13 cycles. DNA purification was performed with 1.2X beads. Libraries were size-selected using the E-Gel EX 2% agarose gel (Cat. # G402022, Invitrogen) and the E-Gel Power Snap Electrophoresis System (Invitrogen), selecting for fragments 150 - 500 bp. DNA from the gel was purified using the QIAquick Gel Extraction Kit (Cat. #28706X4, Qiagen) according to the manufacturer's instructions. Libraries were quantified using Qubit (Thermo Scientific)'s high sensitivity DNA kit (Cat #Q32854, Thermo Scientific).