title: GSM6721809: Patski cells, Xist O-MAP RIP, Pulldown, Rep3; Mus musculus x Mus spretus; OTHER
library_construction_protocol: For RNA isolation, pellets were thawed on ice and resuspended by gentle pipetting in 1000 µL ice cold RIPA Buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% (w/v) SDS, 0.5% (w/v) Sodium Deoxycholate, 1% (v/v) Triton X-100, 5 mM EDTA, 0.5 mM DTT), supplemented with 1x EDTA-Free Halt Protease Inhibitor Cocktail, 0.1 U/µL RNase-OUT, and 10mM Sodium Azide, and rocked end-over-end at 4°C for five minutes. Samples were then sheared using a Branson Digital Sonifier 250 outfitted with a double stepped microtip, at 10–12 Watts for 30 s intervals (0.7 s on; 1.3 s off), with 30 s resting steps between intervals, seven intervals total. 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°C, moved to fresh tubes and diluted with 1 mL Native Lysis Buffer (NLB: 25 mM Tris-HCl pH 7.5, 150 mM KCl, 0.5% (v/v) NP-40, 5 mM EDTA, 0.5 mM DTT), supplemented with 1X Halt Protease Inhibitor Cocktail, 0.1 U/µL RNase-OUT and 10 mM sodium azide. For each sample, 5% was removed as "input," and to the remainder was added 100 µL of Pierce streptavidin magnetic bead slurry (ThermoFisher Scientific) that had been equilibrated by two washes in 1:1 RIPA:NLB. Samples were incubated for 2 hr at room temperature with end-over-end agitation. Beads were then washed with the following series of buffers (1 mL each, 5 min per wash at room-temperature with end-over-end agitation). All buffers were supplemented with 1x EDTA-Free Halt protease inhibitor cocktail, 0.1 U/µL RNase-OUT, and 0.5 mM DTT, unless otherwise noted: (1) RIPA, supplemented with 10 mM Sodium Azide; (2) RIPA alone (3) High Salt Buffer (1 M KCl, 50 mM Tris-HCl pH 7.5, 5 mM EDTA) (4) Urea Buffer (2 M Urea, 50mM Tris-HCl pH 7.5, 5 mM EDTA) (5) 1:1 RIPA:NLB, without protease inhibitors (6) NLB, without protease inhibitors, (7–8) two washes in TE buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA), without protease inhibitors. RNA was isolated from both input and O-MAP-enriched samples by proteolysis in 100 µL Elution Buffer (2% (v/v) N-lauryl sarcoside, 10 mM EDTA, 5 mM DTT, 200 μg proteinase K, in 1x PBS). Reactions were shaken at 700 rpm in a Mixer HC for 1 hour at 42°C, followed by 1 hour at 60°C. RNA was then extracted once with 25:24:1 phenol:chloroform:isoamyl alcohol, and twice thereafter with 24:1 chloroform:isoamyl alcohol. Reactions were supplemented with 15 µg Glycoblue and NaCl to 300 mM, and ethanol precipitated at –20°C overnight. RNA was harvested by centrifugation at 15,000 x g for 30 minutes at 4°C, washed once with 70% ethanol, and resuspended in 84.75 µL nuclease free water. Contaminating DNA was removed by digestion with 5 U RQ1 RNase-free DNase I in 100 µL of the manufacturer's supplied buffer (1x final concentration) at 37°C for 30 min, and this reaction was terminated by addition of EDTA to 15 mM, final. RNA was purified by phenol extraction and ethanol precipitation, as described above, and resuspended in 15 µL nuclease free water. rRNA was removed using a NEBNext rRNA Depletion Kit (NEB; E6310), according to the manufacturer's protocol. Purified RNA samples were quality assessed using an Agilent 4200 TapeStation with a 'RNA High Sensitivity' kit. Samples were quantified on a Nanodrop prior to library preparation and 300ng of RNA per sample was used during library preparation. Libraries were prepared using the NebNext Ultra II Directional RNA 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 'RNA High Sensitivity' kit. Sequencing libraries were prepared from 300 ng RNA using the NEBNext Ultra II Directional RNA Library Prep Kit and NEBNext Multiplex Oligos for Illumina, according to the manufacturer's instructions. Three biological replicates were used for each experimental condition; each library was given a unique index. Libraries were quantified using the NEBNext Library Quant Kit for Illumina, following the manufacturer's instructions, and the quality of these libraries was confirmed using an Agilent 4200 TapeStation with an "DNA High Sensitivity" kit.