GSM6626514: neuron, vehicle, rep2; Mus musculus; OTHER
Five to 16 million cells were incubated in swelling buffer (10 mM Tris–HCl, 2 mM MgCl2, 3 mM CaCl2 and 2 U/ml SUPERase In RNAse inhibitor) for 5 min on ice. Cells were pelleted for 10 min at 400 × g and resuspended in 500 μl of swelling buffer supplemented with 10% glycerol. Subsequently, 500 μl of swelling buffer supplemented with 10% glycerol and 1% Igepal was added drop by drop to the cells under gentle vortexing. Nuclei were washed twice with lysis buffer (10 ml of swelling buffer supplemented with 0.5% Igepal and 10% glycerol), and once with 1 ml of freezing buffer (50 mM Tris–HCl pH 8.3, 40% glycerol, 5 mM MgCl2 and 0.1 mM ethylenediaminetetraacetic acid). Nuclei were counted, centrifuged at 900 × g for 6 min and suspended to a concentration of 5 million nuclei per 100 μl of freezing buffer, snap-frozen in LN2 and stored −80°C until run-on reactions The nuclear run-on reactions were performed for 5 minutes at 30°C in the presence of Sarkosyl and BrUTP. RNA was extracted with Trizol, DNase-treated, base-hydrolyzed and dephosphorylated with PNK. BrUTP-labeled run-on RNA was immunopurified with anti-BrdUTP-coated agarose beads and precipitated overnight. Poly(A)-tailing was followed by cDNA synthesis using complementary poly(T)-primers. Excess oligo was removed by Exonuclease I and cDNA fragments of were purified. The recovered cDNA was circularized, linearized, amplified for 11-14 cycles. The final product was ran on 10% TBE gel, gel purified and cleaned-up using ChIP DNA clean & Concentrator Kit. The final libraries were sequenced using an Illumina HiSeq 2000