title: NextSeq 550 paired end sequencing; CUT&RUN LoV-U of beta-catenin, TCF/LEF transcription factors and histone modifications in HEK293T and mouse embryonic hindlimb tissue at 11.5 days post coitum
design: CUT&RUN LoV-U of beta-catenin, TCF/LEF transcription factors and histone modifications in HEK293T and mouse embryonic hindlimb tissue at 11.5 days post coitum
library_construction_protocol: 500,000 cells were harvested using TrypLE (Cat. #12604013, Gibco). Human embryonic kidney 293T cells (HEK293T) were cultured at 37 °C in a humidified incubator with 5% CO2. Cells were cultured in 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).  HEK293T cells were cultured in media containing 10 µM CHIR99021, a GSK-3 inhibitor, (Cat. #SML1046, Sigma Aldrich) for 24 hr.  Samples were washed twice in wash buffer (HEPES pH 7.5 [20 mM], NaCl [150 mM], Spermidine [0.5 mM], Roche Complete Protease Inhibitor EDTA-Free (Cat. # COEDTAFRO, Roche)) and bound to 20 µl Magnetic ConA Agarose beads (Cat. #ABIN6952467, antibodies-online) equilibrated in binding buffer (HEPES pH 7.5 [20 mM], KCl [10 mM], CaCl2 [1 mM], MnCl2 [1 mM]). Antibody incubation was performed in 150 µl antibody buffer (wash buffer with digitonin [0.01%] and EDTA [2 mM]) with 1.5 µl anti-β-catenin antibody (Cat. #ABIN2855042, antibodies-online) overnight at 4 °C. Thereafter washes were performed with wash buffer containing 0.01% digitonin. After overnight incubation, samples were washed three times and pAG-MN protein was added at 0.6 µg/ml, in total volume of 150 µl. pAG/MNase was a gift from Steven Henikoff (Addgene plasmid #123461; http://n2t.net/addgene:123461; RRID: Addgene_123461), expressed and purified as described in (REF Meers 2019a). Samples were washed three times, followed by digestion for 30 min in wet ice in wash buffer supplemented with 2 mM CaCl2. 2X stop buffer (NaCl [340 mM], EDTA [20 mM], EGTA [4 mM], digitonin [0.05%], RNase A [100 µg/ml], glycogen [50 µl/ml]) was added to stop the reaction, and samples were incubated for 30 min at 37 °C. Beads were collected on the magnet and liquid transferred to a new tube. 2.5 µl proteinase K (Cat. #P8107S, New England BioLabs) and 2 µl 10% SDS were added, and samples incubated for 1 hr at 50 °C.  Library preparation was performed using the KAPA Hyper Prep Kit for Illumina platforms (Cat. #KK8504, KAPA Biosystems) according to the manufacturer's guidelines with the following modifications. End repair and A-tailing was performed with 0.4X volume reactions with 20 µl of purified DNA. The thermocycler conditions were set to 12 °C for 15 min, 37 °C for 15 min and 58 °C for 25 min to prevent thermal degradation of the shortest fragments. Adapter ligation was performed in 0.4X volume reactions. KAPA Dual Indexed adapters were used at 0.15 µM. A post-ligation clean-up was performed with Mag-Bind TotalPure NGS beads at 1.2X the sample volume. Resuspension was done in 10 mM Tris-HCl pH 8.0. Library amplification was performed in 0.5X volume reactions. The cycling conditions were set as follows: 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, with 13 cycles. After amplification, a post-amplification cleanup was performed using NGS beads at 1.2X sample volume. Libraries were then run on an E-Gel EX 2% agarose gel (Cat. # G402022, Invitrogen) for 10 min using the E-Gel Power Snap Electrophoresis System (Invitrogen). Bands of interest between 150 and 500 bp were excised and purified using the GeneJET Gel Extraction Kit (Cat. #K0691, Thermo Scientific) according to manufacturer's instructions.