title: GSM6469474: PROseq_exp2_NELFeKD_DMSO_rep1; Drosophila melanogaster; OTHER
library_construction_protocol: 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-seq was performed as in ﻿Mahat et al. 2016 (PMID: 27442863), but for the time-course experiments, a 3' adapter containing an in-line library-specific barcode (5'-/5Phos/GNNNNNNGAUCGUCGGACUGUAGAACUCUGAAC-/Inverted dT/-3') which allows libraries to be pooled following the 3' adapter ligation to reduce the number of samples being handled for all steps downstream.