Instrument: Illumina HiSeq 4000
Strategy: ChIP-Seq
Source: GENOMIC
Selection: ChIP
Layout: SINGLE
Construction protocol: Approximately 0.5-2 x 10^6 FACS purified ESCs were centrifuged at 500 g for 3 min, washed twice with RT PBS and resuspended in 200 µl of culture media. Cells were fixed by the addition of an equal volume of culture media containing 2 % methanol free formaldehyde (Thermo Scientific Pierce PN28906; final concentration of 1 %) and incubated at RT for 10 min. Fixation was stopped by 5 min incubation with 125 mM glycine at room temperature. Cells were washed in PBS prior to short-term storage at -80 °C. All of the following buffers were supplemented with the following just prior to use: 1 mM DTT and 1 x Protease inhibitors (Calbiochem - 539134-1SET). Cell pellets were resuspended in lysis buffer 1 (50 mM Tris-HCl pH 8.1, 10 mM EDTA and 20 % SDS) and incubated for 10 min at 4 °C. Lysates were diluted 1:10 in ChIP dilution buffer (0.1 % Triton X-100, 2mM EDTA, 150 mM NaCl, 20 mM and Tris-HCl pH 8.1) and sonicated with a single 30 s pulse using a soniprep 150 plus (MSE; set to 3.5) followed by a further 7 pulses using a chilled Bioruptor (Diagenode; 1 min cycles of 30 sec on / 30 sec off on ‘high’ setting at 4 °C). The sonicated extract was pre-cleared by centrifugation at 16,000 g for 10 min at 4 °C. The supernatant was transferred to a fresh tube and supplemented with BSA and triton X-100 to a final concentration of 25 mg/ml and 1 % respectively. 10 % of the IP volume of the chromatin was retained as an input reference. Anti-H3K27me3 antibody (Millipore 07-449) was pre-coupled to protein A Dynabeads (Life Technologies; 10001D) at a ratio of 1 µg antibody per 5 µl of dynabeads. Antibody-bound beads were added to the fragmented chromatin at 1 µg antibody per million cell equivalents and incubated for 10 h on a rotating wheel at 4 ºC. Bead-associated immune complexes were washed once with IP buffer, three times with wash buffer 1 (150 mM NaCl, 10 mM TrisHCl pH 8, 2 mM EDTA, 1% NP40 and 1 % sodium deoxycholate) and twice in 1x TE buffer (10mM Tris pH8 and 10mM EDTA). Each wash was performed at 4ºC on a rotating wheel for 10 min. Chromatin was released from the beads by incubation with elution buffer (0.1 M NaHCO3 and 1 % SDS) for 15 min at 37 ºC followed by the addition of RNaseA and Tris pH 6.8 (final concentration of 20 mg/ml and 100 mM respectively) and incubation at 65ºC for 2 hours followed by the addition of 50 µg of proteinase K and incubation at 65ºC for a further 4-6 h to degrade proteins and reverse the cross-links. Dynabeads were removed using a magnetic rack and the chromatin purified using PCR Purification columns (Qiagen) according to manufacturer’s instructions. Libraries were prepared as previously described (Bowman et al., 2013 - PMID: 23837789) with the following modifications. No purification was performed between the A-tailing reaction and the ligation reactions; instead the ligation was supplemented with ligation reagents (400 U of T4 DNA ligase (NEB), 1 x buffer 2 (NEB), 7.5 % PEG-6000, 1 mM ATP and 13.3 nM of annealed Illumina adaptors (AU)) and incubated at 25 °C for 90 min. Size selection purifications following the ligation and amplification PCR steps were performed with 1x and 0.8 x reaction volumes of Agencourt AMPure XP beads (Beckman Coulter - A63880). Library amplification was performed using Illumina index primers and amplified with NEBNext High-Fidelity 2X PCR Master Mix (M0541S). 4 or 5 samples were multiplexed per single Illumina HiSeq 4000 lane. libraries were seqenced as 50bp single end reads on an Illumina HiSeq 4000.