25M cells were resuspended in 1XPBS and crosslinked with 1% formaldehyde for 10 min at room temperature. The formaldehyde was quenched with addition of 0.125M glycine, washed three times with PBS-0.05% tween-20, and cell pellets were snap freeze in liquid nitrogen. Cell pellets were thawed on ice with 1ml sucrose buffer (10mM HEPES pH7.5, 0.3M sucrose, 1% Triton X-100, 100mM potassium acetate, 0.1mM EGTA, supplemented with 0.5mM spermidine, 0.15mM spermine, 1mM DTT, 1XProtease Inhibitor Cocktail (11873580001, Sigma), 10U/mL SUPERaseIN RNase Inhibitor (AM2694, Thermo Fisher Scientific) freshly), resuspended and rotated for 10min at 4C. then cells were dounced 20 times with dounce homogenizer (357544, Wheaton) in 3ml sucrose buffer. The cells were layered on a cushion of 7.5ml glycerol buffer (10mM HEPES pH7.5, 25% glycerol, 1mM EDTA, 0.1mM EGTA, 100mM potassium acetate, freshly supplemented with 0.5mM spermidine, 0.15mM spermine, 1mM DTT, 1X PIC, and 5U/mL SUPERaseIN) and centrifugated for 10min in 4C at 1500g. pellets were resuspended in 3ml 1X PBS and 3ml of 6% formaldehyde in 1X PBS (3% formaldehyde final) was added for second fixation, that was carried in rotated for 30 minutes at room temperature. Next, the cells were centrifugated at 1000g for 5min in 4C, and washed 3 times with cold PBS-0.05% tween-20. The pellets were resuspended in 1ml nuclear extraction buffer (50mM HEPES pH 7.5, 250mM NaCl, 0.1mM EGTA, 0.5%N-lauroylsarcosine, 0.1% sodium deoxycholate, 5mM DTT, 10U/mL SUPERaseIN), rotated 10 minutes at 4C, spinned-down at 400g for 5 minutes at 4C and nuclei were resuspended in sonication buffer (50mM HEPES pH 7.5, 75mM NaCl, 0.1mM EGTA, 0.5%N-lauroylsarcosine, 0.1% sodium deoxycholate, 0.1% SDS, 5mM DTT, 10U/mL SUPERaseIN). The lysate was divided to two Covaris microtubes, with 130μl in each and sonication was preformed with Covaris E220 sonicator at power: 140W, duty factor: 10%, cycles per burst: 200 for 5 minutes at 4C. Chromatin was pooled, cleared by centrifugation at full speed for 20 minutes at 4C, supernatant was taken to new tube, and volume was adjusted to 390μl. The lysate was pre-cleared with addition of 640μl 2X hybridization buffer (50mM Tris-HCl pH 7.0, 750mM NaCl, 1% SDS, 1mM EDTA, 15% formamide, 1mM DTT, 1mM PMSF, 1X PIC, and 100U/mL SUPERaseIN) and 60μl of washed Dynabeads™ MyOne™ Streptavidin C1 beads (Invitrogen) in rotation at room temperature for 1 hour. Then, supernatant was divided to two tubes for probe hybridization, each reaction with 520μl. 1% was kept frozen for input. Each tube received 3.6μl from a 10μM mix of 10 Xist probes and hybridization was performed at room temperature in rotation overnight. Next, 120μl of washed beads were added for each reaction and the tubes were incubated in rotation at 37C for 1 hour. Then beads were washed once with 1X hybridization buffer (33% sonication buffer, 67% 2X hybridization buffer) for 10 min, 5 times with 2% SDS buffer (10mM HEPES pH 7.6, 250mM NaCl, 2% SDS, 2mM EDTA, 2mM EGTA, and 1mM DTT) for 5 min and twice with 0.5% NP40 buffer (10mM HEPES pH7.6, 250mM NaCl, 0.5% NP-40, 3mM MgCl2, and 10mM DTT) for 5min, at 37C. Beads were resuspended with 180μl of 0.5% NP40 buffer and 20μl RNase H (5U/μl, NEB) for 20 minutes in room temperature to elute the DNA twice. Input DNA was also resuspended with 0.5% NP40 buffer to 400μl. Eluted DNA was spined down at full speed for 1 min to pellet residual beads, 10μl RNase A (12091-021,Thermo Fisher Scientific) was added to the supernatant, and DNA was incubated for 1h at 37C in rotation. Next, DNA was treated with 10μl of Proteinase K (20mg/ml), SDS to 1% final, and EDTA to 10mM final concentration and the samples were incubated at 55°C for 1 hour. To reverse crosslinking the DNA, NaCl was added to 300mM and the samples were incubated at 65°C overnight. DNA was extracted with phenol-chloroform extraction. Xist pulldown was validated by qPCR with primers around Xist-promoter and compared to primers of Gapdh promoter. To proceed for sequencing, DNA was diluted to 130µl in TE and sonicated in Covaris microtubes at power: 140W, duty factor: 10%, cycles per burst: 200 for 2min at 4C. DNA was cleaned up with 1.8X ampure XP beads (A63881, Beckman Coulter). Sequencing libraries were prepared with NEBNext® Ultra II DNA Library Prep Kit for Illumina (#E7645S)