CHART-seq: Sheared chromatin from 2.5x10^7 cells was prepared as described earlier as in Simon et al., Nature 504, 465-469 (2013). 160 μL chromatin was mixed with 320 μL 2X hybridization buffer (50 mM Tris-HCl pH 7.0, 750 mM NaCl, 1% SDS, 1 mM EDTA, 15% formamide, 1 mM DTT, 200 mM PMSF, 1 cOmplete EDTA-free protease inhibitor cocktail tablet [Roche], and 100 U/mL RNase Inhibitor [Roche]) and pre-cleared with 60 μl MyOne Streptavidin C1 beads blocked with yeast total RNA and bovine serum albumin at room temperature for 1 hr. The pre-cleared chromatin was mixed with 36 pmol of either antisense (Xist-targeting) or sense (control) capture probes. We used a pool of 9 capture probes (biotinylated oligos). Hybridization was carried out at 37°C for 4 hr followed by incubation with 240 μL blocked MyOne Streptavidin C1 beads at 37°C for 1 hr. The beads were washed once with 1X hybridization buffer (1:2 mixture of sonication buffer and 2X hybridization buffer) at 37°C for 10 min, five times with wash buffer (10 mM HEPES pH 7.6, 150 mM NaCl, 2% SDS, 2 mM EDTA, 2 mM EGTA, and 1 mM DTT) at 37°C for 5 min, and twice with elution buffer (10 mM HEPES pH 7.6, 150 mM NaCl, 0.5% NP-40, 3 mM MgCl2, and 10 mM DTT) at 37°C for 5 min. CHART-enriched DNA was eluted by 20 μL RNase H (5 U/μL, New England BioLabs) in 200 μL elution buffer two times at 37°C for 30 min. Eluent was treated with 10 μL RNase A (20 mg/mL) at 37°C for 1 hr and then treated with SDS (final 1%), EDTA (final 10 mM), and 10 μL proteinase K (20 mg/mL) at 55°C for 1 hr. Reversal of crosslinks was performed by supplementing the eluent with NaCl (final 0.3 M) and incubation at 65°C overnight. DNA was extracted by phenol-chloroform and further sheared to below 500-bp fragments. ChIP-seq: Cells were crosslinked with 1% formaldeyde for 10 min, then nuclei were isolated and lysed. Chromatin was sheared by sonication to a size of ~200-400 bp. Cleared lysates were precleared for 1 hour, then 10% saved as input, and lysates were incubated with Dynabeads Protein G pre-bound with the antibody of choice. After stringent washes, ChIP DNA was eluted from beads, and together with input chromtain was reverse-crosslinked with proteinase K. DNA was purified by phenol/chloroform extraction and ethanol precipitation. RNA-seq: Total cell RNA was extracted using TRIzol (Thermo Fisher), from which mRNA was isolated using oligo(dT) beads and RNA-seq libraries prepared using NEBNext Ultra Directional RNA Library Prep Kit for Illumina (New England BioLabs) as per manufacturer's instructions. Libraries were constructed using the NEBNext ChIP-seq Library Preparation Kit with no modifications to the protocol. We used 9-14 cycles of PCR amplification, depending on library concentration measured by qPCR.