GSM3477884: GROseq_SH-EP-NMYCER; Homo sapiens; OTHER
Cells were washed two times with cold 1x PBS and harvested in 1x PBS. Cells were re-suspended in 10 ml swelling buffer (10 mM Tris-HCl pH 7.5, 2 mM MgCl2, 3 mM CaCl2) and incubated for 5 min on ice. After centrifugation (400 xg, 10 min) cells were re-suspended in 10 ml swelling buffer containing 10 % glycerol. 10 ml lysis buffer (10 mM Tris-HCl pH 7.5, 2 mM MgCl2, 3 mM CaCl2, 10 % glycerol, 1 % Igepal) was added with gently swirling and incubated for 5 min on ice. The volume of cell suspension was brought to 45 ml with lysis buffer. Resultant nuclei were washed with 10 ml freezing buffer (40 % glycerol, 50 mM Tris-HCl pH 8.0, 5 mM MgCl2, 0.1 mM EDTA). The pellet was resuspended in 100 µl freezing buffer per 1x107 of nuclei.   For run-on reaction, resuspended nuclei were mixed with the same amount of run-on reaction buffer (10 mM Tris-HCl pH 8.0, 5 mM MgCl2, 300 mM KCl, 1 mM DTT, 500 µM ATP, 500 µM GTP, 500 µM Br-UTP, 2 µM CTP, 200 U/ml Superase In, 1 % N-Laurylsarcosine) and incubated 7 min at 30 °C. The reaction was blocked by adding 600 µl Trifast, thoroughly vortexing and incubating for 5 min. RNA was extracted by adding 160 µl chloroform, precipitated with ethanol and resuspended in 20 µl H2O. RNA was next subjected to DNAse treatment (Turbo DNA-free kit, Thermo fisher) for two times according to manufacturer's protocol. RNA was fragmented using Ambion RNA fragmentation kit and end repaired by T4 PNK (NEB).  Anti-BrdU agarose beads (Santa-Cruz, 50 µl per sample) were twice washed and then blocked for 1 hour with blocking buffer (1 x binding buffer, 0.1 % polyvinylpyrrolidone, 0.1 % BSA). Beads were washed twice with binding buffer (0.25 x SSPE, 0.05 % Tween20, 37.5 mM NaCl, 1mM EDTA) and mixed with RNA in 700 µl binding buffer for 1 h on a rotating wheel. After binding, beads were washed twice with binding buffer, twice with low-salt wash buffer (0.2 x SSPE, 0.05 % Tween20, 1 mM EDTA), one with high-salt wash buffer (0.2 x SSPE, 137.5 mM NaCl, 0.05 % Tween20, 1 mM EDTA) and twice with TE with 0.05 % Tween20. Finally, RNA was eluted four times with 100 µl elution buffer (50 mM Tris pH 7.5, 150 mM NaCl, 0.1 % SDS, 20 mM DTT, 1 mM EDTA). RNA was purified with Trizol and Chloroform as described for mRNA-seq..   RNA was decapped using RppH (NEB) with 10 x NEB Thermopol buffer for 1 hour at 37 °C. Reaction was stopped by adding EDTA and incubation at 65 °C for 5 min. Afterwards RNA was extracted as described for mRNA-seq. Library preparation was done according to manufacturer's instructions using NEBNext Multiplex Small RNA Library Prep Kit for Illumina. Library size was tested with the Biorad Experion system and the amount of library DNA was quantified using a picogreen assay
GEO Accession
GSM3477884