title: GSM5957006: Arpp21-iCLIP1; Mus musculus; RIP-Seq
library_construction_protocol: Cells were washed once with PBS and cross-linked twice with 200 mJ/cm2 UV light (254 nm) in a Stratalinker. After cross-linking, cells were lysed and partial RNA digestion was perfomed by using optimized RNase I dilution (Ambion) together with Turbo DNase (Ambion). After centrifugation, cell lysates were loaded onto a Proteus Clarification Mini Spin Column (Generon) and spun. Protein lysates were incubated with the antibody-coupled beads. After stringent washing, RNA 3' ends were dephosphorylated with T4 PNK and subsequentlys the 3' adaptor was ligated. The samples were radioactively end-labled and were subjected to NuPAGE gel electrophoresis and tranferred onto Nitrocellulose Membrane. The RNA was isolated from the membrane by treatment with proteinase K. After phonol/chloroform extraction and precipitation, the RNA was reversed transcribed and cDNA was purified with Dynabeads MyOne Silane (Invitrogen). The 5' adaptor was ligated. First-strand cDNA was synthesized with 6 cycles and size-selected using ProNex Size-Selective Purification System (Promega). Libraries were PCR amplified for 8–13 cycles and sequencing was performed on a Illumina HiSeq1500 sequencer.