GSM7729016: Human Female naive hESC H9 – Input; Homo sapiens; OTHER
After washing with PBS, 5-30 million cells were harvested from confluent cell cultures and incubated with freshly-made 10ml 2mM DSG in 1x PBS at room temperature for 45 minutes. Cells were subsequently crosslinked further at room temperature with 10ml 3% formaldehyde for 10 minutes, and the reaction was stopped by the addition of 2ml 2.5M glycine. Cells were pelleted at 4˚C, aliquoted into vials of 10 million cells, and subjected to lysate preparation as described by Engreitz et al. (Engreitz et al., 2013). Briefly, the cells were passively lysed in Cell Lysis Buffer (10mM HEPES at pH7.5, 20mM KCl, 2mM MgCl2, 1mM EDTA with 0.1% NP-40) for 10min on ice, then lysis was completed either with glass dounce homogenizer (UCLA1, naïve iPSCs and NHDFs) or without (naïve (WT and with XIST KO) and primed H9, and primed iPSCs). Nuclei were lysed in Nuclear Lysis Buffer (20mM HEPES at pH7.5, 50mM KCl, 1.5mM MnCl2, 1% NP-40, 0.4% Sodium deoxycholate, 0.1% N-lauroylsarcosine) for 10min on ice. Chromatin was solubilized by sonication. Briefly, for UCLA1, naïve iPSCs and NHDFs, five cycles of sonication were performed, with each cycle lasting 45 seconds on high power. For naïve and primed H9, and primed iPSCs sonication was done with a Misonix S-400 sonicator with microtip (model number: U1240A0418) at an amplitude of 12, total process time of 2 min with pulse on for 1sec and pulse off for 3sec. After sonication, chromatin was further segmented by incubating it with TURBO DNase digestion at a concentration of 0.1-0.4U/ul for 12-20min at 37C (time was optimized for each sample). The digestion was stop by the addition of DNase Stop Solution (mixture of EDTA and EGTA). XIST RNA pulldown from 5 million cells was done using 1ug (UCLA1, iPSCs, NHDF R1) or 50pmol (primed and naïve H9 and primed iPSCs) of non-overlapping 90nt long biotinylated oligonucleotide probes (Eurofins) (Table S3). To test the effect of higher concentration of the biotinylated oligonucleotides, a higher concentration was added to one replicate of NHDF (R2), using 5ug probes. 1mg (UCLA1, naïve iPSCs and NHDFs) or 0.6mg (naïve and primed H9, and primed iPSCs) of C1 Streptavidin beads were used. As a control, 5% of each sample was taken as an input control and the remaining sample was used for the pulldown. DNA was eluted by Rnase H digestion, and crosslinking was reversed via proteinase K digestion of eluted DNA at 60˚C 10-12 hours. DNA libraries were prepared using NEBNext Ultra End Rpair/dA-Tailing Module (NEB) and TruSeq DNA adapters (Illumina) ligated using Quick Ligase (NEB). Libraries were amplified by KAPA HiFi Polymerase (Roche), pooled, and sequenced on the Illlumina HiSeq platform to generate 50bp single-end reads (UCLA1, naïve iPSCs and NHDFs) or pair-end reads (naïve and primed H9 and primed iPSCs).