title: GSM9204044: QKI_D8_IP3; Homo sapiens; OTHER
library_construction_protocol: eCLIP was performed as described in Van Nostrand et al. Nature Methods 2016 (PMID 27018577). Briefly, cells were lysed in iCLIP lysis buffer, followed by limited digestion with RNase I (Ambion), immunoprecipitation of RBP-RNA complexes with a specific antibody of interest as indicated and Protein G sheep anti-rabbit Dynabeads, and stringent washes. After dephosphorylation with FastAP (Thermo Fisher) and T4 PNK (NEB), a barcoded RNA adapter was ligated to the 3' end (T4 RNA Ligase, NEB). Samples were then run on SDS-PAGE gels and transferred to nitrocellulose membranes, and a region from 40-115 kDa (75 kDa = 225 nt of RNA above the protein size) was isolated and proteinase K (NEB) treated to isolate RNA. RNA was reverse transcribed and treated with ExoSAP-IT (Affymetrix) to remove excess oligonucleotides. A second DNA adapter (containing a random-mer of 10 (N10) random bases at the 5' end) was then ligated to the cDNA fragment 3' end (T4 RNA Ligase, NEB). After cleanup (Dynabeads MyOne Silane, ThermoFisher), an aliquot of each sample was first subjected to qPCR (to identify the proper number of PCR cycles), and then the remainder was PCR amplified (Q5, NEB) and size selected via agarose gel electrophoresis.