GSM9308665: DDX24-FLAG_Input_RIPseq_rep1; Homo sapiens; RNA-Seq
For each immunoprecipitation, HUVECs stably expressing FLAG tagged DDX24-WT or DDX24-dRBD were pelleted; resuspended in an equal volume of ice-cold polysome lysis buffer (10mM HEPES pH 7.0, 100 mM KCl, 5 mM MgCl2, 0.5% NP-40) supplemented with 1 mM DTT, 100 U/mL RNase inhibitor (Thermo Fisher Scientific), 1× protease inhibitor cocktail (Roche) and 0.4 mM RVC (NEB); incubated on ice for 15 minutes; snap frozen in liquid nitrogen. After the lysate was thawed on ice and centrifuged at 15,000 g for 15 minutes, the supernatant was pre-cleared with Dynabeads Protein G (Invitrogen) at 4 °C for 1 hour. 20 μL of the supernatant was saved as input and mixed with 1 mL of RNAzol. Subsequently, other 200 μL of the supernatant extract was diluted with 1 mL of NT2 buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1 mM MgCl2, 0.05% NP-40) supplemented with 1 mM DTT, 100 U/mL RNase inhibitor (Thermo Fisher Scientific), 1× protease inhibitor cocktail (Roche) and 20 mM EDTA. The extracts were mixed with 4 μg of an anti-FLAG antibody (Proteintech) or a normal IgG antibody (EMD Millipore) and incubated overnight at 4 °C with end-over-end rotation. The next day, the RNP/antibody complex was precipitated by incubation with Dynabeads Protein G at 4 °C for 4 hours. The beads were collected and washed five times in NT2 buffer. After the final wash, the beads were resuspended in 1 mL of RNAzol. RNAs of Input and FLAG were extracted for subsequent RIP-seq. The immunoprecipitated RNA was then subjected to mRNA selection using Oligo dT-based Poly(A) mRNA Magnetic Isolation Module (NEB) and prepared for sequencing libraries using Ultra II Directional RNA Library Prep Kit for Illumina (NEB). Libraries were pooled and sequenced on an Illumina Hiseq 3000 PE150 platform.