GSM5569916: Primed_H3K27me3_Replicate_2; Drosophila melanogaster; Homo sapiens; OTHER
Concanavalin A conjugated paramagnetic beads were resuspended and washed twice on a magnetic rack in a bead activation buffer composed of: 20 mM HEPES pH 7.9, 10 mM KCl, 1 mM CaCl2 and 1 mM MnCl2. Naive or primed hPSCs and Drosophila S2 cells were dissociated and counted. Cell pellets were then washed twice with a wash buffer composed of: 20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM Spermidine and supplemented with EDTA-free protease inhibitor. 50,000 human cells and 20,000 Drosophila cells were added in wash buffer to Concanavalin A beads and incubated for 10 min at RT with rotation to immobilise cells. Concanavalin A beads were then collected with a magnetic rack, the supernatant discarded and beads were resuspended in antibody buffer, composed of wash buffer supplemented with 0.08% Digitonin and 2 mM EDTA. 1 μg of antibody was added to beads which were then incubated overnight at 4 °C with rotation to allow permeabilisation and target binding. Antibodies used were: rabbit anti-Tri-Methyl-Histone H3 (Lys27) (Cell Signaling Technology, 9733), rabbit anti-IgG (Invitrogen, 31188) and anti-Histone H2Av spike-in antibody (Active Motif, 61686). The following day, beads were washed twice with digitonin buffer, composed of wash buffer supplemented with 0.08% Digitonin. To 50 μl digitonin buffer, 2.5 μl CUTANA™ pA/G MNase (EpiCypher, 15-1016) was added and incubated for 10 min at RT to promote pA/G MNase binding to the antibody constant region. Beads were again washed twice with digitonin buffer and MNase was activated with 2 mM CaCl2 and incubated for 2 h at close to 0 °C to facilitate chromatin fragmentation under sites of antibody binding. MNase activity was terminated by adding 100 μl stop buffer, composed of 340 mM NaCl, 20 mM EDTA, 4 mM EGTA, 50 μg/ml RNase A and 50 μg/ml Glycogen. Cleaved DNA fragments were released from nuclei by incubation for 10 min at 37 °C, spinning for 5 min at 16,000 xg at 4 °C and collecting the supernatant from the beads on a magnetic rack. DNA was then purified by incubation with 1 μl SDS (20 %) and 1.5 μl Proteinase K (20 mg/ml) at 70 °C for 10 min, followed by a 1.8x AMPure XP bead (Beckman Coulter) clean-up into DNA lo-bind tubes (Eppendorf) and eluted in 50 μl 0.1x TE. Libraries were prepared using the NEBNext Ultra II DNA library preparation kit for Illumina (NEB) using the manufacturer's protocol, with libraries indexed using NEBNext Multiplex Oligos for Illumina (Index Primers Set 1 and Set 2) (NEB). Following library preparation, library fragment size and concentration were determined using the Qubit fluorometer double stranded DNA high sensitivity assay kit, using an Agilent Bioanalyzer 2100 and by KAPA Library Quantification Kit (KAPA Biosystems). Samples were sequenced on an Illumina NextSeq500 instrument as HighOutput 75 bp paired-end reads at the Babraham Institute Next Generation Sequencing Facility. CUT&RUN