GSM7429579: Flp-In HEK293 RBMX iCLIP replicate 3; Homo sapiens; RIP-Seq
Cell pellets were resuspended in lysis buffer and sonicated in the Bioruptor (Diagenode) for 10 cycles with alternating 30 s on/off at low intensity. 1 mg/mL of the protein was treated with Turbo DNase I (Ambion) and high (2.5 U/mL) or low (0.8 U/mL) RNase I (Ambion) Purified RNA was reverse transcribed into cDNA, using Superscript IV with distinct primers irCLIP_ddRT for each replicate. The PCR was amplified using Platinum™ II Hot-Start PCR Master Mix (Thermo Fisher Scientific). The final PCR libraries were purified by agarose gel electrophoresis on a 6% TBE gel, DNA in the range of 150-400nt was gel extracted using Costar SpinX column (Corning Incorporated, 8161) and submitted for sequencing.