alias: GSM2734408
title: GSM2734408: Cdk9as_5min_10uM_3MBPP1_rep1; Schizosaccharomyces pombe; OTHER
library_construction_protocol: Immediately prior to treatments, a fixed amount of spike-in culture (i.e.  S. cerevisaie samples) was added to each sample culture for PRO-seq experiment.  After treatment, cultures were then spun down and washed once in ice-cold water.  Samples were then spun again and resuspended in ice-cold permeabilization buffer (0.05% sarkosyl) and left on ice for 20 minutes.  Permeabilized cells were then spun down at 400XG for 5 minutes.  Permeabilized cell pellets were resuspeneded in 120 uL 2.5X transcription buffer (50 mM Tris-HCl, pH 7.7, 500 mM KCl, 12.5 mM MgCl2).  To the reaction buffer we then added 3.75 uL of each biotin-11-NTP (epicentre), 6 uL .1M DTT, 3 uL SUPERase Inhibitor (invitrogen) and 141 uL DEPC treated water.  The run-on was then performed by adding 15 uL 10% sarkosyl and placing samples at 30°C for 5 minutes.  After the run-on, samples were spun and the reaction buffer was removed.  RNA was then isolated using the hot acid phenol extraction protocol followed by ethanol precipitation. PRO-seq libraries were prepared according to Mahat et al. Nature Protocols (2016).
identifiers.namespace: GEO
identifiers.id: GSM2734408