GSM2634057: GFP; Mus musculus; RIP-Seq
At complete confluency, cells were suspended in 1000ul of RIP lysis buffer (50mM tris HCl pH 7.4, 150mM NaCl, 1mM EDTA, 1% NP-40), kept in ice for 20min and vortex frequently. Samples were then centrifuged at 10000g for 10min at 4°C and supernatant were saved. Meanwhile, 40µl of agarose-Flag Ab beads (Sigma Aldrich, A2220) were washed in 380µl of TBS and centrifuged at 8000g for 1min at 4°C. Beads were suspended in 40µl with RIP lysis buffer and added to each RIP sample at equal volume. Samples were then incubated through constant rotation for 2 hr at 4°C. After extensive washes, 1/50 of each mixture was saved for IP western blot validation. Finally, beads were centrifuged and suspended in 300µl of pureZOL for subsequent bound RNAs isolation. Total RNA was subsequently quantified on 2100 Bioanalyzer (Agilent). SMART-seq2 protocol adapted from (Picelli et al., 2014b) was used to retrotranscribe 10ng total RNA into full-length cDNA with oligo(dT) primers and LNA-containing template-switching oligo (TSO). Purified cDNA was tagmented with Tn5 transposase and enriched by PCR to create the final library for sequencing on an Illumina HiSeq2000 platform.
GEO Accession
GSM2634057