title: GSM6721803: Patski cells, Xist O-MAP ChIP, Pulldown, Rep2; Mus musculus x Mus spretus; OTHER
library_construction_protocol: For DNA isolation, thawed pellets were gently resuspended by pipetting in 1000 µL of CLB (20mM Tris pH 8.0, 85mM KCl, 0.5% NP40) supplemented with 1X Halt protease inhibitor cocktail (ThermoFisher Scientific) and 10 mM NaN3 for 10 minutes. Lysates were then clarified by centrifugation at 3,000 x g for 5 minutes at 4℃. Supernatant was aspirated, and lysates were subjected to another round of CLB lysis, clarification, and supernatant aspiration. Lysates were then lysed by gentle pipeting in 1000 µL of NLB (10mM Tris-HCl pH 7.5, 1% NP40, 0.5% Na Deoxycholate, 0.1% SDS) for 10 minutes. Samples were then sheared using a Branson sonicator at 10-12 Watts for 18 30 s intervals (0.7 s on +1.3 s off), with 30 s resting steps between intervals. Samples were held in ice-cold metal thermal blocks throughout sonication. Lysates were then clarified by centrifugation at 15,000 x g for 10 min at 4℃ and moved to fresh tubes. For each sample, 10% was removed as 'input'; to the remainder was added 100 µL of streptavidin-coated magnetic bead slurry (ThermoFisher Scientific) that had been equilibrated by two washes in NLB. Samples were incubated for 2 hr at room-temperature with end-over-end agitation. Beads were subsequently washed with the following series of buffers (1 mL each, 5 min per wash, room-temperature, with gentle end-over-end agitation): (1) NLB + 5mM EDTA, 10mM NaN3, 1X Halt protease inhibitor cocktail, 150mM NaCl, (2) NLB + 5mM EDTA, 10mM NaN3, 1X Halt protease inhibitor cocktail, (3+4) 1M KCl, 10mM Tris-HCl pH 7.5, 5mM EDTA, (5+6) 2M Urea, 10mM Tris-HCl pH 7.5, 5mM EDTA, (7) 10mM Tris-HCl, 1% SDS, (8+9) 10mM Tris-HCl pH 7.5, 1mM EDTA. Enriched DNA was released from the beads by proteolysis in 100uL of Elution Buffer (2% N-lauryl sarcoside, 10 mM EDTA, 5 mM DTT, in 1X PBS, supplemented with 200 μg proteinase K (ThermoFisher Scientific)) at 65℃, shaking in a Mixer HC (USA Scientific) 700 rpm for 1 hour. Samples were then placed in a thermocycler overnight at 65℃ for proteinase K treatment. DNA was then extracted using 1:1 phenol:chloroform, and a second chloroform cleanup. Samples were then EtOH precipitated in 300mM NaCl supplemented with glycoblue at -20C overnight. DNA was precipitated by centrifugation at 15,000 x g for 30 minutes at 4℃, washed with 70% EtOH, and resuspended in 20 uL nuclease free water. Each sample then received 1 µL RNaseA (ThermoFisher) and incubated at 37℃ for 1 hour. DNA was then extracted using 1:1 phenol:chloroform with two rounds of chloroform cleanup. Samples were then EtOH precipitated in 300mM NaCl supplemented with glycoblue at -20C overnight, washed in 70% EtOH, and resuspended in 20 µL nuclease free water. Purified DNA samples were quality assessed using agarose electrophoresis to ensure chromatin sheared size was ~200bp. Samples were quantified on a Nanodrop (ThermoFisher) prior to library preparation and 300ng of DNA per sample was used during library preparation. Libraries were prepared using the NebNext Ultra II DNA Library Prep Kit (NEB) according to manufacturer's instructions. Each library was given a unique index during synthesis. Library concentration was measured using an NebNext Library Quant Kit for Illumina (NEB). Quality was confirmed using an Agilent 4200 TapeStation with a 'DNA High Sensitivity' kit.