Trypsinized mESCs (20 x 10e6) were washed twice in PBS. Cells were resuspended in 250 µl of PBS and fixed by the addition of an equal volume of PBS 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. Fixed cells were washed in PBS and combined at this stage with 1.3x10e6 formaldehyde fixed S2 cells (Drosophila melanogaster cells; for downstream calibration of ChIP-seq data). All buffers were supplemented with 1 mM DTT and 1x Protease inhibitors (Roche, 11836170001) just prior to use. Cell pellets were resuspended in lysis buffer (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 (1% Triton X-100, 2 mM EDTA, 150 mM NaCl, 2 0mM and Tris-HCl pH8.1) and sonicated, first with a single 30s pulse with a probe sonicator (labtech Soniprep 150) on ice followed by a further 45 cycles using a cooled Bioruptor (Diagenode; 1 min cycles of 30sec on / 30 sec off on 'high' setting at 4 °C). The sonicated extract was pre-cleared by centrifugation at 16000 g for 10 min at 4 °C. The supernatant was transferred to a fresh tube and supplemented with BSA to a final concentration of 25 mg/ml. Anti-H3K27me3 (Cell Signalling C36B11) antibodies were pre-coupled to a protein A Dynabeads (Life Technologies; catalogue no. 10001D) at a ratio of 1 mg antibody per 30 ml of dynabead suspension by rotation at 4 °C for 1 h. 6 x 10e6 cell equivalents of lysate were added to 5 µg of antibody bound beads and incubated for 6 h on a rotating wheel at 4 ºC. Following incubation, bead-associated immune complexes were washed sequentially with ChIP dilution buffer, wash buffer A and wash buffer B, each for 10 min at 4 ºC on a rotating wheel followed by two washes in TE buffer at RT (wash buffer A - 1% Triton X-100, 0.1% Sodium-Deoxycolate, 0.1% SDS, 1mM EDTA, 500 mM NaCl, 50 mM HEPES pH7.9; wash buffer B – 0.5% NP40, 0.5% Sodium-Deoxycolate, 1 mM EDTA, 250 mM LiCl, and 20 mM Tris-HCl pH8.1). Chromatin was released by incubating the beads in 100 µl of elution buffer (0.1 M NaHCO3 and 1 % SDS) for 15 min at 37 ºC, followed by the addition of 50 µg of RNaseA and 6µl of 2 M Tris pH6.8 and incubation at 65 ºC for 2 h and finally by the addition of 50 µg of proteinase K and incubation at 65 ºC for 8 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 constructed using the NEBNext® Ultra™ II DNA Library Prep Kit for Illumina according to manufacturer's instructions (NEB - catalogue no. E7645S). Library PCRs were supplemented with 2x SYBR dye (Sigma – catalogue no. S9430) so that amplification could be monitored by quantitative PCR on a Roche lightcycler 480. To allow for sample multiplexing, PCRs were performed using index primers (NEBNext Multiplex Oligos for Illumina - Set 1. Catalogue no. E7335) and amplified to linear phase. Size selection purifications following the ligation and amplification PCR steps were performed with 1x and 0.9x reaction volumes of Agencourt AMPure XP beads (Beckman Coulter - A63880). Purified libraries were combined as a 12 sample equimolar pool containing the indexes 1-12 and sequenced on an Illumina NextSeq on a single high-output flow cell (single-end 75 bp reads).