GSM9288254: FKBP-SETDB1 G6D2, dTAGd1, NPC d3; Mus musculus; RNA-Seq
Chromatin-RNA isolation and sequencing was as previously described. Briefly, cells were collected in a 15mL tube, washed with PBS and snap frozen immediately after PBS removal. Cells pellets were stored at -80oC until further processing. In order to purify chromatin-RNA, pellets were thawed on ice and resuspended in 800mL of hypotonic buffer HLBN (10mM NaCl, 2.5mM MgCl2, 10mM Tris pH 7.5, 0.1% NP40) and transferred to a 1.5mL tube. This solution was underlaid with a 480mL HLBN containing 24% sucrose to create a sucrose cushion for nuclear isolation by centrifugation at 1000g for 5min at 4oC. For fractionation, the nuclear pellet was then resuspended in 120mL NUN1 (20mM Tris pH8.0, 75mM NaCl, 0.5mM EDTA, 50% glycerol) freshly supplemented with 1mM DTT and protease inhibitors. After resuspension, 1200mL NUN2 (20mM HEPES pH7.6, 7.5mM MgCl2, 0.2mM EDTA, 0.3M NaCl, 1M urea, 1% NP40) freshly supplemented with 1mM DTT and protease inhibitors were added and mixed by gentle vortexing. Sample was incubated on ice for 15 min with occasional vortexing. After centrifugation for 10min at 15000rpm at 4oC, the supernatant containing the nucleoplasmic fraction was discarded and the chromatin pellet was resuspended in 200mL HSB (10mM Tris pH7.5, 500mM NaCl, 10mM MgCl2) supplemented with 2mL TURBO DNAse (Life Technologies) to digest the sample DNA by incubating at 37oC for at least 10min, shaking at 1400rpm. After this incubation, 2μL of 20% SDS and 4μL of proteinase K (20mg/ml) were added and the samples were incubated at 37oC for at least 30min for protein digestion. Chromatin-bound RNA was then purified using Trizol reagent according to manufacturer's recommendations with addition of glycogen to aid precipitation of the RNA at -20oC overnight. Purified chromatin-RNA was subjected to a second round of TURBO DNAse treatment and a second round of Trizol reagent purification as previously described. Chromatin-RNA concentrations were initially measured by Nanodrop to inform further dilution for quantification using Qubit HS RNA assay (Invitrogen) and quality control using RNA Pico 6000 Bioanalyser kit (Agilent). RNA Libraries were prepared for sequencing using the Illumina TruSeq stranded total RNA kit including the rRNA depletion according to the manufacturer's instructions. Libraries were quantified and verified using Qubit High Sensitivity DNA (Invitrogen) and the Bioanalyser High Sensitivity DNA (Agilent) kits before pooling and paired-end sequencing on an Illumina NextSeq 500 machine using NextSeq 500/550 High-Output v2.5 Kit (150 cycles) (Illumina).