Briefly, around 3.5 million cells were used per immunoprecipitation (IP) experiment. A fraction of these cells were always used to quantify levels of Xist induction by RNA FISH. Ten million cells was resuspended and lysed in 90µl of Lysis Buffer (50mM Tris-HCl, pH 7.5; 150mM NaCl; 0.1% sodium deoxycholate; 1% Triton X-100; 5mM CaCl2; Protease Inhibitor Cocktail; 5mM sodium butyrate) for 10min on ice. Lysis Buffer with MNase (62 µl) was then added for chromatin digestion and incubated at 37ºC for exactly 10min. Then, 20mM EGTA was added to stop the reaction, followed by centrifugation (13000rpm) for 5min at 4ºC to sediment undigested debris. Supernatant was then transferred and equal amount of STOP buffer (50mM Tris-HCl, pH 7.5; 150mM NaCl ; 0.1% sodium deoxycholate; 1% Triton X-100; 30mM EGTA; 30mM EDTA; Protease Inhibitor Cocktail; 5mM sodium butyrate) was added to the samples, which were always kept on ice. Lysate (5µl) was mixed with 45µl of ProtK Digestion Buffer (20mM HEPES; 1mM EDTA; 0.5% SDS) and incubated at 56°C for 30min. AMPure XP beads (50µl) were mixed with the digested lysate with 60µl of 20% PEG8000 1.25M NaCl for 15min at RT. Beads were separated on a magnet and washed twice with 80% Ethanol for 30 seconds. DNA was eluted in 12µl of Low-EDTA TE and measured using Qubit DNA High-Sensitivity kit to normalise lysate concentration between samples. DNA isolated in this step was used for the input sample. The volume of each undigested lysate was adjusted for equal concentration to obtain 1ml per IP using a 1:1 mix of Lysis Buffer and STOP Buffer. Protein-A Dynabeads (10µl/IP) were washed twice in Blocking Buffer (0.5% BSA; 0.5% Tween in PBS) before being resuspended in Blocking buffer and coated with H3K27me3 [1µg/IP] (Cell Signalling, Cat#9733S) or H2AK119ub [0.4µg/IP] (Cell Signalling, Cat# 8240S) antibodies for 4 hours at 4°C. Once coated beads were magnet-separated and resuspended in 1ml of concentration-adjusted lysate. Samples were left rotating overnight at 4°C. In the following day beads were magnet-separated and washed quickly with ice-cold washing buffers with Low Salt Buffer (0.1% SDS; 1% TritonX-100; 2mM EDTA; 20mM Tris-HCl, pH 8.1; 150mM NaCl; 0.1% sodium deoxycholate). IPs were then washed four times with Low Salt Buffer, twice with High Salt Buffer (0.1% SDS; 1% TritonX-100; 2mM EDTA; 20mM Tris-HCl, pH 8.1; 360mM NaCl; 0.1% sodium deoxycholate) and twice with LiCl buffer (0.25M LiCl; 1% NP40; 1.1% sodium deoxycholate; 1mM EDTA; 10mM Tris-HCl pH 8.1). Prior to elution all samples were rinsed once in TE. ChIP-DNA was eluted in ProtK-Digestion buffer by incubating at 56°C for 15min. Beads were separated and the supernatant was further digested for more 2 hours at 56°C. DNA was isolated using AMPure XP beads as described for the input sample. DNA concentration was adjusted and used for library preparation using Ovation® Ultralow Library System V2 following suppliers protocol. Amplified libraries were size-selected for dinucleotide fraction (350-600 bp fragments) using agarose gel-separation and MinElute Gel Extraction Kit (Qiagen). Sample quality was inspected using D1000 tapestation. Samples were sequenced with HiSeq2500 using single-end 50bp mode.