title: NextSeq 550 paired end sequencing; CUT&RUN LoV-U of H3K4me3, b-catenin, and IgG in HCT116 cells
design: CUT&RUN LoV-U of H3K4me3, b-catenin, and IgG in HCT116 cells
library_construction_protocol: HCT116 human colorectal cancer cells were cultured in a humidified 37 °C incubator in 5% CO2. Culture medium consisted of high glucose Dulbecco's Modified Eagle Medium (Cat. #41965039, Gibco) with 10 % bovine calf serum (Cat. #1233C, Sigma-Aldrich) and 1X Penicillin-Streptomycin (Cat. #15140148, Gibco). 250,000 cells/sample were harvested from culture flasks by incubation with Trypsin-EDTA (Cat. # 25200056, Gibco) for 5 minutes. The trypsin was quenched with culture media and the cells were washed twice with DPBS (Cat. #14190094, Thermo Fisher Scientific).  Nuclear extraction was performed by three washes 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). The nuclei were resuspended after the final wash 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]) as described in (REF Meers 2019). Bead binding proceeded on a rotator for 15 min, after which nuclei and beads were resuspended in 200 µl wash buffer per sample and split into PCR tubes. Beads were collected on the magnet and then resuspended in 200 µl EDTA wash buffer (wash buffer with EDTA [0.2 mM]) and incubated RT for 5 min. Samples were then directly resuspended in antibody buffer (wash buffer with antibody 1:100) and incubated ON at 4 °C on a rotator. Antibodies used included anti-β-catenin ABIN2855042, anti-H3K4me3 ABIN6971977 and anti-rabbit IgG ABIN101961. After ON incubation samples were washed 5 times and resuspended in 200 µl of pAG-MNase buffer (wash buffer with pAG-MNase 0.6 µg/ml) and incubated for 45 min at 4 °C on a rotator. pAG/MNase was a gift from Steven Henikoff (Addgene plasmid #123461; http://n2t.net/addgene:123461; RRID: Addgene_123461) and was expressed and purified in house as described in (Meers 2019). Next, samples were washed five times and during the last wash equilibrated 5 min in wet ice. Samples were resuspended in in 200 µl ice cold wash buffer containing 2 mM CaCl2, and digestion was allowed for proceed for exactly 30 min in wet ice. The digestion buffer was removed and transferred to tubes containing 1.5 µl of 0.5 M EDTA and 1.5 µl of 0.5 M EGTA and mixed well. The reaction was stopped on the beads with 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 samples were incubated 1 hr at 4°C for elution. Beads were collected on the magnet and liquid transferred the PCR tubes containing the digestion buffer. DNA purification was done with two 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), and then finally resuspended in 20 µl Tris-HCl pH 7.5. Library preparation was done using the KAPA Hyper Prep Kit for Illumina sequencing (Cat. #KK8504, KAPA Biosystems) following the manufacturer's guidelines with modifications. 0.4X volume reactions were used for End repair and A-tailing steps, starting with 20 µl CUR&RUN DNA. 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. A DNA cleanup was performed after ligation with Mag-Bind TotalPure NGS beads at 1.2X the sample volume, and DNA was resuspended in 10 mM Tris-HCl pH 7.5. Library amplification steps were performed in 0.5X volume reactions. The cycling conditions were: 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, for a total of 13 cycles. A post-amplification DNA purification was performed with beads at 1.2X sample volume. Libraries were then size-selected using the E-Gel EX 2% agarose gel (Cat. # G402022, Invitrogen) and the E-Gel Power Snap Electrophoresis System (Invitrogen). Run time was set to 10 minutes and DNA between 150 and 500 bp was excised from the gel and purified using the QIAquick Gel Extraction Kit (Cat. #28706X4, Qiagen) according to the manufacturer's instructions. Libraries were quantified using Qubit (Thermo Scientific) and the high sensitivity DNA kit (Cat #Q32854, Thermo Scientific).