GSM2409784: IGF2BP3-Input; Homo sapiens; RIP-Seq
When reaching a confluency of 70-80%, cells in 10 cm dishes were cross-linked by UV and harvested by trypsinization. Nuclear extraction was isolated and sonicated. One microgram of Flag (Sigma-Aldrich) antibody was conjugated to Protein A/G Magnetic Beads (Thermo Fisher Scientific) by incubation for 4 hours at 4 °C, followed by 3x wash and incubation with pre-cleared nuclear extraction in RIP buffer [150 mM KCl, 25 mM Tris (pH 7.4), 5 mM EDTA, 0.5 mM DTT, 0.5% NP40, 1x protease inhibitor] at 4 °C overnight. After washing with RIP buffer for three times, beads were resuspended in 80 μL PBS, followed by DNA digestion at 37 °C for 15 min and incubation with 50 μg of Proteinase K (Thermo Fisher) at 37 °C for 15 min. Input and co-immunoprecipitated RNAs were recovered by TRIzol (Invitrogen) extraction. NEBNext Ultra Directional RNA Library Prep Kit (New England BioLabs, Ipswich, MA) was used for library preparation, which used dUTP in cDNA synthesis to maintain strand specificity. In short, the immunoprecipitated RNAs were reverse transcribed to 1st strand cDNA, followed by 2nd strand cDNA synthesis labelled with dUTP. The purified cDNA was end repaired and dA tailed, and then ligated to adapter with a stem-loop structure. The dUTP-labelled 2nd strand cDNA was removed by USER enzyme to maintain strand specificity. After indexing via PCR (~12 cycles) enrichment, the amplified libraries together with library preparation negative control were cleaned up by AMPure XP beads for QC analysis. To check the quality and yield of the purified library, one microliter library was analyzed by Bioanalyzer (Agilent, Santa Clara, CA) using DNA high sensitivity chip.
GEO Accession
GSM2409784