GSM5238893: NEUROG3-HA-P2A-Venus - H3K27me3_CR; Homo sapiens; OTHER
We followed the protocol of Hainer and Fazzio (2019) Current Protocols 126 (1):e85 (2019) with minor modifications. Fresh or thawed cells were washed once with 1mL cold PBS and resuspended in nuclear extraction buffer (NE, 20mM HEPES-KOH, pH 7.9, 10mM KCl, 0.5mM Spermidine, 0.1% Triton X-100, 20% glycerol, freshly added protease inhibitors)). After 3 min spinning at 4°C at 600g, cells were resuspended in 600 μL NE buffer. Concanavalin A beads (Polysciences, 25 μL bead slurry/sample) were washed twice with ice-cold Binding buffer (20mM HEPES-KOH, pH 7.9, 10mM KCl, 1mM CaCl2, 1mM MnCl2) and resuspended in 300 μL Binding buffer. Nuclei were added to beads with gentle vortexing and incubated for 10 min at 4°C with gentle rocking. Bead-bound nuclei were blocked with 1 mL cold Blocking buffer (20mM HEPES, pH 7.5, 150mM NaCl, 0.5mM Spermidine, 0.1% BSA, 2mM EDTA, freshly added protease inhibitors) by gentle pipetting, incubated 5 min at RT and washed in 1mL cold Wash buffer (WB, 20mM HEPES, pH 7.5, 150mM NaCl, 0.5mM Spermidine, 0.1% BSA, freshly added protease inhibitors) and resuspended in 250 μL cold WB. 250 μL of primary antibody diluted 1:100 in cold WB were added with gentle vortexing, and samples were incubated overnight with gentle rocking at 4°C. Samples were washed twice in 1 mL cold WB, and resuspended in 250 μL cold WB. When indicated, incubation with a secondary antibody (Donkey anti Sheep IgG, 1:200) was performed for 1h at 4°C in WB under gentle rocking. After 2 washes with 1 ml WB, and resuspension in 250 μl WB, 200 μL of pA-MN (diluted at 1.4 ng/mL in cold WB, in-house production using the pK19pA-MN plasmid, obtained from Ulrich Laemmli (Addgene plasmid # 86973; http://n2t.net/addgene:86973 ; RRID:Addgene_86973) was added with gentle vortexing, and samples were incubated with rotation at 4°C for 1 hour. Samples were washed twice in 1 mL cold WB and resuspended in 150 μL cold WB. Three μL of 100 mM CaCl2 was added upon gentle vortexing to activate the MN. After 30 min of digestion, reactions were stopped by addition of 150 μL 2XSTOP buffer (200mM NaCl, 20mM EDTA, 4mM EGTA, 50ug/mL RNaseA, 40ug/mL glycogen) and DNA fragments were released by passive diffusion during incubation at 37°C for 20 min. After centrifugation for 5 min at 16,000g at +4°C to pellet cells and beads, 3 μL 10% SDS and 2.5 μL Proteinase K 20mg/ml were added to the supernatents, and samples were incubated 10 min at 70°C. DNA purification was done with phenol/chloroform/isoamyl alcohol extraction followed by chloroform extraction using MaxTract tubes (Qiagen). DNA was precipitated with ethanol after addition of 20 µg glycogene, and resuspended in 25µl 0.1XTE. Illumina sequencing libraries were prepared at the Genomeast facility (IGBMC, Illkirch). CUT&RUN samples were purified using Agencourt SPRIselect beads (Beckman-Coulter). Libraries were prepared from 10 ng of double-stranded purified DNA using the MicroPlex Library Preparation kit v2 (Diagenode) following the manufacturer's protocol with some modifications. Illumina compatible indexes were added through a PCR amplification (3 min at 72°C, 2 min at 85°C, 2 min at 98°C; [20 sec at 98°C, 10 sec at 60°C] x 13 cycles). Amplified libraries were purified and size-selected using Agencourt SPRIselect beads (Beckman Coulter) by applying the following ratio: volume of beads / volume of libraries = 1,4 / 1. The libraries were sequenced on Hiseq 4000 as Paired-End 2x100 base reads following Illumina's instructions.