NextSeq 550 paired end sequencing; CUT&RUN HUDEP1 OE SOX6-Flag
CUT&RUN HUDEP1 OE SOX6-Flag
The cells in each sample are in suspension. Medium with the cells was collected, cells were pelleted and then resuspended as described in the nucleic acid extraction protocol (CUT&RUN) CUT&RUN was performed according to the LoV-U protocol described in (Zambanini et al., 2022). Per each sample 500,000 HUDEP1 (SOX6-Flag expressing) were harvested. Cells were washed three times 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), then resuspended in 40 μl NE per sample and bound to 20 μl Magnetic ConA Agarose beads (ABIN6952467) equilibrated in binding buffer  (HEPES pH-7.5 [20 mM], KCl [10 mM], CaCl2 [1 mM], MnCl2 [1 mM]). After incubation, nuclei and beads were resuspended for 5 minutes in EDTA wash buffer (HEPES pH-7.5 [20 mM], NaCl [150 mM], spermidine [0.5 mM] in Roche Complete Protease Inhibitor EDTA-Free (COEDTAFRO), + EDTA [0.2 mM]). Samples were divided in 200 μl PCR tubes and antibody incubation was performed in 200 μl wash buffer with 2 μl of anti-FLAG antibody (Cat. F7425, Sigma-aldrich) overnight (ON) at 4 °C on a rotator. After ON incubation, samples were washed five times in wash buffer and resuspended in 200 μl of pAG-MN buffer (wash buffer supplemented with pAG-MN [0.6 μg/ml]) for 30 minutes at 4 °C on a rotator.pAG/MNase was a gift from Steven Henikoff (Addgene #123461), expressed and purified as described previously (Meers et al., 2019a). Samples were washed five times, followed by digestion for 30 minutes in wet ice in 50 µl wash buffer with 2 mM CaCl2. After 30 minutes, the digestion buffer was moved to new collection tubes and the reaction on the beads was stopped with 50 μl of 1X Urea STOP buffer (NaCl [100 mM], EDTA [2 mM], EGTA [2 mM], IGEPAL [0.5%], Urea [8.5 M]) and the samples were incubated for 1 hour at 4°C. Beads were collected on the magnetic rack, while the supernatant was mixed in the corresponding collection tube, where it was cleaned up twice using Mag-Bind TotalPure NGS beads (Cat. #M1327, Omega Bio- Tek) at 2X, and then resuspended in 20 μl Tris-HCl pH 7.5. 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 0.4 size 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 done with 0.4 size 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. Resuspension was done in 10 mM Tris-HCl pH 8.0. Library amplification was performed with 0.5 size 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 with 1.2X beads. 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 cut out and purified using the QIAquick Gel Extraction Kit (Cat. #28706, QIAGEN) according to manufacturer's instructions.