| description |
GST-4E was recombinantly expressed in E. coli BL21 and crude lysate prepared by sonication was loaded onto glutathione sepharose beads (GE Healthcare). Total RNA was extracted from a P1 mouse (C57BL/6 strain) using Trizol reagent and size-selected after extraction on an 8 M urea-8% polyacrylamide gel to obtain 40- to 100-nt small RNAs. Purified small RNAs were then incubated with recombinant Deinococcus radiodurans polynucleotide phosphorylase (PNPase) and incubated with GST-4E- or GST-4E-W102L-coated glutathione beads in NET-2 buffer. Purified RNAs were ligated to a ³²P-labeled 3′ adaptor, CIP-TAP treated and ligated to the 5′ adaptor. The cDNA library was constructed as described (Cazalla et al., 2011) and sequenced at the Yale Stem Cell Center Genomics Core on an Illumina HiSeq2000 instrument using 50 bp runs. |