title: GSM7831330: H9-Ectoderm Day 6, WDR5, rep1; Homo sapiens; OTHER
library_construction_protocol: CUT&RUN was performed using a similar protocol to those previously described (Skene et al., 2018; Kong et al., 2021).  Immediately prior to lifting the cells, magnetic concanavalin A (Con A) beads (Bangs Laboratories, #BP531) were activated by rinsing twice in cold binding buffer (20mM HEPES pH7.5, 10mM KCl, 1mM CaCl2, 1mM MnCl2) and stored on ice in binding buffer equivalent to the original bead volume.  Con A beads were always mixed by gentle pipetting or flick mixing rather than by vortexing.  Cells were rinsed with 1X PBS, lifted with 0.25% Trypsin, and quenched with 2-3 volumes of growth media.  Cell count and viability were determined using a NucleoCounter NC-200, and 3x10^6 cells per sample (ARPE-19) to 6x10^6 cells per sample (neural ectoderm) were pelleted, washed once with 1X PBS, washed once with wash buffer (20mM HEPES pH7.5 and 150mM NaCl supplemented with 1:100 spermidine [Cell Signaling Technology, 27287] and 1:200 protease inhibitor cocktail [Cell Signaling Technology, 7012]), and resuspended in wash buffer of the same composition.  Activated Con A beads were added to the cell suspension, and binding was achieved by end-over-end mixing at room temperature for 15min.  Cells bound to Con A beads were resuspended in antibody buffer (wash buffer supplemented with 2mM EDTA, and 0.01% digitonin [Millipore-Sigma, 300410]) and aliquoted into equal portions.  Antibodies or IgG were added, and incubations were conducted at 4C overnight.  Following antibody binding, beads were washed twice with wash buffer supplemented with 0.01% digitonin and incubated for 10min at room temperature with 1:25 pAG-MNase (EpiCypher CUTANA pAG-MNase for CUT&RUN, 15-1016) diluted in wash buffer of the same composition.  Beads were washed twice with wash buffer containing 0.01% digitonin, and pAG-MNase digestion was performed at 4C for 2h in wash buffer supplemented with 0.01% digitonin and 2mM CaCl2.  Digestion was stopped by addition of 2X stop buffer (340mM NaCl, 20mm EDTA, 4mm EGTA) supplemented with 0.01% digitonin, 1:200 of 10mg/ml RNAse A (Cell Signaling Technology, #7013), and 50pg per sample of S. cerevisiae spike-in normalization DNA (Cell Signaling Technology, 36598S).  Samples were incubated at 37C for 15min with gentle agitation to release DNA, supernatant was separated from magnetic Con A beads into a fresh tube, and samples were diluted with an equal volume of 0.1X TE buffer (1mM Tris-HCl pH7.5, 0.1mM EDTA).  Samples were stored at -20C prior to DNA purification. DNA purification of CUT&RUN samples was performed by Phenol:Chloroform:Isoamyl Alcohol extraction as previously described (Skene et al., 2018).  Briefly, samples were thawed, supplemented with 1:100 of 10% SDS, 1:100 of 20mg/ml proteinase K, and incubated for 10min at 70C.  One sample volume of UltraPure Phenol:Chloroform:Isoamyl Alcohol 25:24:1 v/v (Thermo Fisher Scientific, 15593031) was added and samples were mixed by full-speed vortexing, transferred to phase-lock tubes (Qiagen, 129046), and centrifuged at room temperature for 5min at 16,000g.  One sample volume of chloroform was added, mixed by inversion, and samples were centrifuged at room temperature for 5min at 16,000g.  The aqueous upper phase was removed into a fresh microfuge tube containing 4µg glycogen (Millipore-Sigma, 10901393001), 2.5 volumes of ice cold 100% ethanol were added, and samples were mixed by vortexing followed by incubation on ice for 10min.  Samples were centrifuged at 4C for 10min at 16,000g, supernatant was gently discarded, samples were washed with 5 volumes of ice cold 100% ethanol, centrifuged at 4ºC for 2min at 16,000g, supernatant was gently discarded, and pellets were allowed to air dry for 5-10min.  After excess liquid had evaporated, pellets were resuspended in 0.1X TE buffer, transferred to fresh tubes, and stored at -20C. Libraries were constructed by DNA end repair, A-tailing, Illumina adapter ligation, size selection, PCR amplification, and quantification by qPCR.  Both library construction and 150bp paired-end sequencing on an Illumina NovaSeq 6000 were performed by Novogene Corporation Inc. (Beijing, China).