GSM4498075: NURF301BAP170_PROseq_Rep2; Drosophila melanogaster; OTHER
For PRO-seq, we started with 2 × 10e7 cells per condition, and spiked in 2.7 × 10e5 human K562 cells to facilitate spike-in normalization immediately after harvest. Cells were pelleted at 4 °C and washed with ice-cold PBS, then with wash buffer (10 mM Tris-Cl pH 7.5, 10 mM Kcl, 150 mM Sucrose, 5 mM MgCl2, 0.5 mM CaCl2, 0.5 mM DTT, 1X Pierce Protease inhibitors, and SUPERase-In RNase inhibitor). Cells were then incubated for 5 min on ice in permeabilization buffer (10 mM Tris-Cl pH 7.5, 10 mM Kcl, 250 mM Sucrose, 5 mM MgCl2, 1 mM EGTA, 0.5 mM DTT, 0.05% Tween-20, 1X Pierce Protease inhibitors, and SUPERase-In RNase inhibitor) and > 99% permeabilization was observed using trypan blue staining. Nuclei were then washed twice with wash buffer, and snap frozen in freezing buffer (50 mM Tris-Cl pH 8.0, 40% Glycerol, 5 mM MgCl2, 0.1 mM EDTA, 0.5 mM DTT). Run-on and PRO-seq library construction was performed as in Mahat et al. 2016 (PMID: 27442863) with some modification. The run-on reaction was performed with biotin-11-UTP and biotin-11-CTP, and ATP and GTP. We substituted MyOne C1 Streptavidin beads for the M280 beads recommended by the protocol, and used 5' and 3' adapters with 6N UMIs at the ligation junction. Libraries were amplified for 11 PCR cycles.
GEO Accession
GSM4498075