Assay summary for Ribo-seq_Schier_Lab_0001AS

Assay Type:
Schier Lab, Harvard University
Embryos were lysed by repeated micropipetting in 1.5ml of cold polysome buffer (20 mM Tris-HCl pH 7.4, 250 mM NaCl, 15 mM MgCl2, 1mM dithiothreitol, 100 micrograms/ml cycloheximide) with added 0.5 perc. Triton X-100, 500 micrograms/ml GMP-PNP, 24 U/ml TurboDNase (Ambion AM2238), incubated with agitation for 10 min at 4 C, and clarified by centrifugation at 1300 rcf for 10 min at 4 C. 20microliters RNAseI (Ambion AM2294) was added to the 1.5 ml of supernatant and incubated for 30 min at 37 C, then stopped by chilling on ice and addition of 40 microliters of SuperaseIn (Ambion AM2694). Footprinted samples were pelleted through a sucrose cushion (1M sucrose in polysome buffer with added 100 U/ml SuperaseIn) by centrifugation at 260,000 rcf for 4.5 hours at 4 C, and resuspended in 800 microliters 10mM Tris pH 7.4 with 1 perc. SDS. RNA was purified by hot acid phenol/chloroform extraction and precipitated by standard ethanol precipitation.
Added by User:
Date Added:
May 24, 2016, 5:35 a.m.
Last Edited:
May 24, 2016, 5:35 a.m.