GSM6469487: PROcap_LacZKD_noTAP; Drosophila melanogaster; OTHER
S2 cells (with spike-in mouse embryonic fibroblasts) were permeabilized following the PRO-seq protocol in Mahat et al. 2016 (PMID: 27442863), with all steps performed within a cold room (4˚C), with cells kept on ice between handling, and all centrifugation steps at 4˚C. Cells were first spun and resuspended in 5 mL 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 Protease inhibitor cocktail (Roche cOmplete); 20 units Superase-In RNase inhibitor) before being resuspended in Permeabilization Buffer (10 mM Tris-Cl, pH 7.5; 10 mM KCl; 250 mM sucrose; 5 mM MgCl2, 1 mM EGTA; 0.05% Tween-20; 0.1% Nonidet P-40 substitute; 0.5 mM DTT; 1x Protease inhibitor cocktail (Roche); 20 units Superase-In) and left on ice for 5 minutes. Cell permeability was assessed >99% under a microscope in the presence of 0.2% Trypan Blue. Permeabilized cells were then washed twice in 5 mL Wash Buffer before being resuspended in 220 µL Storage Buffer (50 mM Tris-Cl pH 8.3; 40% glycerol; 5 mM MgCl2; 0.1 mM EDTA; 0.5 mM DTT) which was divided between 2 aliquots (~9 million cells each) and flash frozen in liquid nitrogen before storage at -80˚C.  PRO-cap libraries were prepared as in ﻿Mahat et al. 2016 (PMID: 27442863), but prior to enzymatic removal of the nascent RNA 5' cap, the PRO-cap sample was split into two tubes. One tube received enzymatic treatment with tobacco acid pyrophosphatase (TAP; EpiCentre T19500, now discontinued) to remove the 5' cap structure, while the other received only reaction buffer, lacking TAP, and thus serves as a measurement of remaining background (uncapped) signal (Kruesi et al. 2013, PMID: 23795297). The remainder of the library preparation was performed identically for control and experimental PRO-cap samples.