Name: GSM7946799
Instrument: Illumina NovaSeq 6000
Strategy: OTHER
Source: GENOMIC
Selection: other
Layout: PAIRED
Construction protocol: Cell pellets were thawed on ice with 1 ml 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, 1X Protease Inhibitor Cocktail, 10U/mL SUPERaseIN RNase Inhibitor. Resuspended samples were then homogenized with dounce homogenizer in a volume of 3 ml sucrose buffer. The mixture was 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 then 3% formaldehyde was added, second fixation was carried out with rotation for 30 min at room temperature. Next, the samples were centrifugated at 1000g for 5min at 4 °C and washed 3 times with ice-cold 1x PBS 0.05% Tween-20. The pellets were resuspended in 1 ml 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 4°C, spinned-down at 400g for 5 minutes at 4C and nuclei were resuspended in 130μl 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). Sonication was performed with Covaris E220 sonicator at power: 140W, duty factor: 10%, cycles per burst: 200 for 5 minutes at 4C. The lysate was pre-cleared with addition of Dynabeads MyOne Streptavidin C1 beads (Invitrogen) in 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) . Pre-cleared mixtures received 3.6μl from a 10μM mix of 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. Eluted DNA was incubated with RNaseA for 1h at 37C in rotation, following by Proteinase K (20mg/ml) treatment at 55°C 1hr. To reverse crosslinking the DNA, 300 mM NaCl was added to the samples and incubated at 65°C overnight. DNA was extracted with phenol-chloroform extraction. CHART libraries were prepared with NEBNext Ultra II DNA Library Prep Kit for Illumina