GSM4879209: hTERT RPE1 cells - H3K4me3; Homo sapiens; OTHER
Trypsinized RPE1 cells were bound to Concanavalin A coated magnetic beads (Bangs Laboratories, BP531), permeabilized with 0.025% (w/v) digitonin, then incubated overnight with 5 μg of either anti-H3K4me3 (Active Motif, 39159), anti-H3K27ac (Active Motif, 39133) or anti-H3K27me3 (Active Motif, 39155) at 4 °C. Cells were washed then incubated with protein A-MNase fusion protein (a gift from S. Henikoff, FHCRC) for 15 minutes at room temperature. Cells were washed again and incubated with 2 mM Calcium Chloride to induce MNase cleavage activity for 30 minutes at 0 °C. The reaction was stopped with 2× STOP buffer (340 mM NaCl, 20 mM EDTA, 4 mM EGTA, 100 μg/mL RNase A and 50 μg/mL glycogen) and 0.2 pg of S. cerevisiae spike-in DNA were added to each sample. Cleaved Histone-DNA complexes were isolated by centrifugation and DNA was extracted with a NucleoSpin PCR Clean-up kit (Macherey-Nagel, 740609). Library preparation from each CUT&RUN condition was done with a KAPA Hyper Prep Kit (VWR, 89125-040) with the PCR amplification settings adjusted to have the annealing and extension steps done simultaneously at 60 °C for 10 seconds. Library products between 200-300 base pairs were selected with Agencourt AMPure XP beads (Beckman Coulter, A63880) before being sequenced with an Illumina MiSeq system with paired-end 25 base pair sequencing read length and TruSeq primer standard for approximately 6 million reads per sample.