GSM3244865: F-MDA5 fRIP, replicate1; Homo sapiens; RNA-Seq
BC-3 cells were crosslinked with 1% formaldehyde for 10 min in PBS, and unreacted formaldehyde was neutralized with 0.3 M glycine, pH 7, for 5 min. Cells were washed 2X with PBS, then resuspended in RIPA buffer (50 mM Tris, pH 8, 1% IGEPAL CA 630, 0.5% sodium deoxycholate, 0.05% SDS, 1 mM EDTA, 150 mM NaCl, 1 mM DTT, and RNAse and protease inhibitors) and kept on ice for 10 min. Soluble cell extracts were incubated with anti-FLAG M2 magnetic resin at 4 °C for 2 h. Resin was washed three times for 10 min and then two times for 5 min at room temperature in RIPA buffer containing 0.1% SDS, 1 M NaCl, and 1 M urea. Resin was eluted with 1xFLAG peptide in RIPA buffer for 45 min at 4 °C. Protein–RNA crosslinks were reversed by adding 100 mM Tris, pH 8, 10 mM EDTA, 1% SDS, and 2 mM DTT to eluted samples and heating to 70 °C for 45 min. RNA was recovered by extraction with TRIzol and then again with phenol:chloroform:isoamyl alcohol [25:24:1 (vol/vol)] followed by ethanol precipitation. Paired-end RNA-sequencing libraries were prepared from the recovered RNA using the NEBNext Ultra II Directional RNA Library Prep Kit (NEB) according to the manufacture recommendations. Libraries were then subjected to paired-end sequencing on a HiSeq3000 with 150 cycles at the Vanderbilt Technologies for Advanced Genomics (VANTAGE).
GEO Accession
GSM3244865