GSM5516169: GFP-(GR)50_1; Homo sapiens; RIP-Seq
Cells were crosslinked with 100 mJ/cm2 UVC (254 nm). Cells were lysed in 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS with EDTA-free complete mini protease inhibitor cocktail and 1 mM phenylmethylsulfonyl fluoride. Lysates were incubated with 4 U/ml Turbo DNase and 0.04 (library1) or 0.02 (library2) units/ml of RNase I. Immunoprecipitation was performed by incubating the lysates with GFP-Trap Magnetic beads for 1.5 hours at 4°C. 3' linker ligation was performed on beads overnight at 16°C. After labeling with [γ-32P]-ATP, RNPs were resolved on NuPAGE 4-12% Bis-Tris gels. After gel fractionation, RNPs were transferred to nitrocellulose and labeled complexes visualized using a storage phosphor screen. RNA-protein complexes of the expected size were excised from the nitrocellulose membranes, along with the same size region from GFP, GFP-(GP)10, or untransfected control experiments. Associated proteins were removed by digesting with proteinase K and the associated RNA was extracted with phenol chloroform. Extracted 3' linker ligated RNA was used as input for reverse transcription performed using Superscript III. RT primers used were as described in Huppertz et. al. (Methods. 2014 Feb; 65(3): 274–287. doi: 10.1016/j.ymeth.2013.10.011) library 1 GPF - Rt1clip, GFP-(GR)50 - Rt6clip; library 2 untransfected - Rt1clip, GFP-(GP)10 - Rt6clip, GFP-(GR)50 - Rt9clip. cDNA was size selected and circularized with Circligase II. Circularized cDNA was cut with BamHI and amplified using Accuprime Supermix I. The PCR cycle number was optimized to prevent over amplification of the library. Amplified samples were sequenced on the Illumina MiSeq platform in single end read mode with 110 nt reads.