GSM4826634: POINT5_HCT116_+ExT_Rep1; Homo sapiens; OTHER
POINT method was performed as previously described mNET-seq protocol (Nojima et al., 2016) with some alterations.  In brief, crude nuclear fraction was prepared from HeLa or HCT116 cells (1x107 for POINT-seq and POINT-5, 4x107 for POINT-nano) as in mNET protocol. Chromatin pellet was resuspended in NUN1 (20 mM Tris-HCl (pH 7.9), 75 mM NaCl, 0.5 mM EDTA and 50% Glycerol) and treated with modified NUN2 buffer (20 mM HEPES-KOH (pH 7.6), 300 mM NaCl, 0.2 mM EDTA, 7.5 mM MgCl2, 1% NP-40, 1 M Urea, 3% Empigen, 1x proteasome inhibitor Complete (-EDTA) and 1x PhosSTOP). Note Empigen-treated nuclear solution was gently mixed by inverting tube a few times to avoid chromatin aggregation and incubated on ice for 10 min. The chromatin pellet was centrifuged at 400g for 30 sec. The pellet was washed with PBS twice and then digested in DNase solution (10 mM Tris-HCl (pH 7.5), 400 mM NaCl, 100 mM MnCl2, 2 U/mL RiboLock and 0.2 U/mL Turbo DNase) for 15 min. After DNA digestion, soluble digested chromatin was collected by 13,000 rpm centrifugation for 10 min. The supernatant was ten times-diluted in ice-cold NET-2E buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.05 % NP-40 and 3% Empigen BB) and Pol II antibody-conjugated beads were added. 10 or 40 mg of Pol II antibody (200 or 800 mL of Dynabeads anti-mouse IgG) was used for POINT-seq and POINT-5 or POINT-nano methods. Immunoprecipitation was performed at 4oC for 1 hr. The beads were washed with 1 ml of ice-cold NET-2E buffer six times. The isolated nascent RNA was purified using Trizol reagent technology twice with once DNase treatment. Note that polyA tails were added to the isolated RNA by in vitro polyadenylation with E.coli PAP for POINT-nano. Then the pA+RNA was size-selected using SPRISelect reagent (x0.6 volume). Library prep for illumina sequencing (NovaSeq6000) employed NEBNext Ultra II Directional RNA library prep kit and SMARTer Stranded RNA-seq kit for POINT-seq and POINT-5 analyses, respectively.  For ONT sequencing, Direct cDNA library prep kit was used for POINT-nano analysis. Illumina and ONT sequencing (PromethION) were conducted by Novogene UK and the high throughput genomics team of the Wellcome Trust Centre for Human Genetics (WTCHG), Oxford.