GSM5706967: MV411-ETOH-H3K27me3; Homo sapiens; OTHER
GSM5706967
Cells were serially washed with PBS and wash buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM spermidine, 1 x protease inhibitor cocktail) at room temperature.  Concanavalin A beads were activated by washing twice with binding buffer (20 mM HEPES pH 7.9, 10 mM KCl, 1 mM CaCl2, 1 mM MnCl2), after which 10 μL of activated beads were added to each sample and incubated for 10 minutes at room temperature.  Bead-bound cells were then resuspended in 150 μL of antibody buffer (wash buffer plus 0.05% digitonin, 2 mM EDTA, 0.1% BSA) with a 1:50 dilution of primary antibody and incubated for 2 hours at room temperature.  The supernatant was then removed and the beads were washed twice with digitonin buffer (wash buffer plus 0.1% digitonin) prior to re-suspension in 50 μL of Dig-300 buffer (20 mM HEPES pH 7.5, 300 mM NaCl, 0.5 mM spermidine, 1 x protease inhibitor cocktail, 0.01% digitonin) with pAG-Tn5 enzyme for 1 hour at room temperature.  The supernatant was again aspirated and the beads washed twice with Dig-300 buffer, followed by re-suspension in 50 μL Tagmentation buffer (Dig-300 buffer plus 10 mM MgCl2) and incubation for 1 hour at 37°C.  An equal volume of 2X stop buffer (Dig-300 buffer plus 40 mM EDTA, 0.2% SDS, 400 μg/mL proteinase K) was then added and the samples were incubated at 55°C for 2 hours, then held at 4°C overnight.  The following day, the supernatant was taken and purified using the MinElute PCR purification kit (Qiagen), with each sample eluted in 25 μL of elution buffer. 21 μL of eluted DNA was mixed with 2 μL of a universal i5 primer and 2 μL of a unique barcoded i7 primer, as well as 25 μL of NEBNext HiFi 2X PCR Master Mix.  Following 14 cycles of PCR amplification, cleanup was performed using 1.1 x volume of AMPure XP beads (Beckman Coulter), analyzed on the Agilent 4200 TapeStation to determine size distribution of libraries and then sequenced on the NextSeq500 using 75 bp paired end chemistry.