GSM2842830: Scer_RPF; Saccharomyces cerevisiae; OTHER
Lysis proceeded according to McGlincy and Ingolia, 2017, except with no cycloheximide in the lysis buffer (20 mM Tris pH 7.4, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 1% v/v Triton X-1000, 25 U/ml Turbo DNase I). To quantify RNA content of the lysate, total RNA was purified from 200 μL of lysate using the Direct-zol RNA MiniPrep kit (Zymo Research) and the concentration of RNA was measured with a NanoDrop 2000 spectrophotometer (ThermoFisher). Lysate containing 30 μg of total RNA was thawed on ice and diluted to 200 μL with polysome buffer with no cycloheximide (20 mM Tris pH 7.4, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT). 0.1 μl (1 U) of RNase I (Epicentre) was added to the diluted cell lysate and then incubated at room temperature for 45 minutes. Digestion and monosome isolation proceeded according to McGlincy and Ingolia, except with no cycloheximide in the sucrose cushion.  Purified RNA was separated on a 15% TBE/Urea gel, and fragments of 18-34 nt were gel extracted. Size was determined relative to RNA size markers NI-NI-800 and NI-NI-80124 and NEB microRNA size marker (New England Biolabs). Library preparation proceeded according to McGlincy & Ingolia. The library was made with downstream linker NI-NI-811 (/5Phos/NNNNNAGCTAAGATCGGAAGAGCACACGTCTGAA/3ddC/) and a modified RT primer with a preferred CircLigase II substrate (AG) at the 5´ end (oLFL075, 5′-/5Phos/AGATCGGAAGAGCGTCGTGTAGGGAAAGAG/iSp18/GTGACTGGAGTTCAGACGTGTGCTC). Library amplification PCR used primers NI-NI-798 and NI-NI-825799 (Illumina index ATCACG). The resulting library was sequenced as single-end 50 nt reads on an Illumina HiSeq4000 according to the manufacturer's protocol by the Vincent J. Coates Genomics Sequencing Laboratory at the University of California, Berkeley.
GEO Accession
GSM2842830