GSM1820982
GSM1820982: 2h30_JQ1_797R2; Homo sapiens; RNA-Seq
Nascent RNA purification was based on the Nascent-seq protocol (Khodor et al. 2011; Ferrari et al. 2013). Briefly, cells were harvested and homogenized 3 times with a 25G needle in 1ml buffer CSK buffer (15mM HEPES pH 7.8; 100mM NaCl; 3mM MgCl2; 1mM EGTA; 500mM Sucrose; 0.5% Triton X-100; Complete protease inhibitor (Roche) and 0.5x SUPERaseIN (Life technologies) to isolate the nuclei. Nuclei were collected by centrifugation and resuspended in 80ul of CF buffer (10mM Tris pH 8.0; 100mM NaCl; 25mM EDTA; 1x SUPERaseIN (Life technologies). Immediately afterwards, 1ml of NUN buffer (1.5M UREA; 20mM HEPES pH 7.8; 300mM NaCl; 7.5mM MgCl2; 500mM EDTA; 1% NP40) was added, and the sample was vortexed to lyse the nuclei. The mixture was incubated on ice with occasional agitation for 10 min to allow precipitation of genomic DNA and core histones together with engaged RNA Pol II and nascent transcripts. After spinning the sample at 20000g for 10 min at 4°C, the pellet was washed twice with NUN buffer. Next, the pellet was resuspended in 700 ul of CF buffer supplemented with 1% SDS and 1 mg/ml Proteinase K using a 25G needle and incubated at 55°C for 60 min. The lysate was extracted with Phenol/Chloroform. Nucleic acids were precipitated, dissolved in H2O and treated with Turbo DNaseI (Ambion) for 60 min at 37°C to eliminate DNA contamination. 50 ng of nascent RNA was used to make the RNA library for Illumina following the instructions provided by manufacture for NEBNext Ultra directional RNA library prep kit (New England BioLabs).
GEO
GSM1820982
GEO Accession
GSM1820982