GSM6632286: HA-DDX21 epitope, Progenitor, donor 1; Homo sapiens; OTHER
CUT&RUN was performed on 0.5-1 million primary keratinocytes according to the EpiCypher protocol (“CUT&RUN Protocol v2.0”, revised 3/15/2022) and kit (EpiCypher, 14-1048), either using antibody to the endogenous DDX21 (ProteinTech, 10528-1-AP) or infected with virus expressing HA-tagged DDX21 (antibody: CST, #3724). Controls libraries were made for IgG and H3K27me (antibodies: IgG, CST, #2729; anti-H3K27me3, Active Motif, 39055). Nuclei were extracted by resuspending in 100 µL nuclei extraction buffer per 2.5 million cells (20 mM HEPES pH 7.5, 10 mM NaCl, 0.5 mM spermidine, 0.1% BSA, 0.1% NP-40, and 1 tablet Roche protease inhibitor per 10-20 mL) and incubating for 10 minutes on ice. 5-10 volumes of wash buffer were added, cells centrifuged, resuspended in wash buffer, and quantified using a Countess. Nuclei were bound to 10 µL activated Concanavalin A beads (EpiCypher, 21-1401) per sample, stained at 4°C  for 2 hrs to overnight with 1:50 antibody dilutions (anti-HA, CST, #3724; anti-DDX21, ProteinTech, 10528-1-AP; anti-DDX21, Novus Biologicals, NBP1-83310; IgG, CST, #2729; anti-H3K27me3, Active Motif, 39055), washed, and incubated with 2.5 µL pAG-MNase (EpiCypher, 15-1016) per sample for 1 hour at 4°C with rocking, washed, digested for 15-30 minutes, and reactions quenched with EpiCypher STOP buffer for 30 minutes at 37°C according to the manufacturer's protocol. Nuclei and beads were centrifuged for >4,800 rcf at 4°C for 5-15 minutes, placed on a magnet, and supernatant containing released fragments was removed. Cross-links were then reversed by the addition of SDS to ~0.2% and proteinase K to ~0.4 µg/mL (final concentrations), incubating for 55°C in a thermomixer for 4 hours to overnight. DNA was extracted by a Zymo DNA Clean and Concentrator (Zymo Research, D4014), eluting in 27 µL water. Library was prepared using the NEB Ultra II DNA library prep kit (NEB, E7103S) following the manufacturer's protocol with modifications according to published research (Liu et al., 2018).