title: GSM7795458: Stage 8 Pou5f3.3 V5 rep 3; Xenopus laevis; OTHER
library_construction_protocol: CUT&RUN procedure was based on Hainer et al Cell 2019 and Skene et. al. Nature Protocols 2019. 12 embryos per sample were collected, devitellinized in 1mg/mL pronase, dissolved in MR/3, and cells were dissociated in 1 mL of NP2.0 buffer (Briggs et al Science 2018) with gentle agitation. Dissociated cells were lysed in NE buffer (20mM HEPES-KOH, pH 7.9, 10mM KCl, 500uM spermidine, 0.1% Triton X-100, 20% glycerol) with gentle pipetting with a clipped P1000, and the lysate was centrifuged at 600xg in 4C for 3 min. The free nuclei were then bound to 300 uL of activated concanavalin A beads at RT for 10mins. Nuclei were blocked for 5 min at RT then incubated in 1:100 dilution of primary antibody for 2 hr at 4C, washed, incubated in a 1:200 dilution of pA/G MNase for 1 hr at 4C, and washed again. The bound MNase was activated with 2 mM CaCl2 and allowed to digest for 30 mins, then stopped using 2x STOP buffer (200 mM NaCl, 20 mM EDTA, 4 mM EGTA, 50 ug/mL RNase A, 40 g/mL glycogen). Nuclei were incubated at 37C for 20 min followed by centrifuging for 5 min at 16,000xg, drawing off the DNA fragments with the supernatant. The extracted fragments were treated with SDS and proteinase K at 70C for 10 min followed by phenol chloroform extraction. Purified DNA was resuspended in 50 uL of water and verified by Qubit dsDNA high sensitivity and Fragment Analyzer. Sequencing libraries were built using the NEB Next Ultra II library build kit (E7645S) and size selected on an agarose gel to 150-600 bp. Libraries were multiplexed and sequenced paired-end at the Health Sciences Sequencing Core at Children's Hospital of Pittsburgh.