title: GSM7031990: HCT116, size-matched input; Homo sapiens; RIP-Seq
library_construction_protocol: Cells were lysed, Turbo DNase/RNase I treated, sonicated, and then subjected to immunoprecipitation with two different anti-DDX6 antibody  precoupled with protein G/A beads. After stringent washes, the RNA/protein complex was dephosphorylated with T4 polynucleotide kinase and FastAP alkaline phosphatase, and then ligated to an RNA adaptor at the 3ʹ end. Next, the resulting sample was eluted through LDS sample buffer. 10% of the eluted sample was used for western gel and the remaining 90% for CLIP gel. The region of the CLIP gel above the 54 kDa protein was isolated followed by proteinase K digestion to release RNA. The purified RNA was reverse transcribed with AffinityScript enzyme and AR17 primer. Then, a DNA adaptor was ligated to the purified cDNA 3ʹ ends. After cleanup, the library was amplified using Q5 DNA polymerase and F-D501 and R-D701 primers, and purified through a 2% low melting temp agarose gel, recycling the library from 175-400bp gels. The final library was used for paired-end Illumina sequencing.