title: GSM5542618: PTBP iCLIP2 1-500; Homo sapiens; RIP-Seq
library_construction_protocol: Cells were harvested by scraping and collected by centrifugation at 1000x g for 1 min at 4 °C. The supernatant was subsequently removed and the cell pellets were either snap-frozen and stored or immediately subjected to immunoprecipitation. Protein-RNA complexes were immunoprecipitated by using a monoclonal mouse anti-PTBP1(hnRNP I) antibody from SCBT (sc-56701). Then, RNase digestion was performed by diluted RNase I (Invitrogene) to each sample. Further processing of the sample including RNA extraction was performed according to a previously published protocol (Buchbender, Andreas, et al. Methods 178 (2020): 33-48). cDNA synthesis was carried out using the Superscript III (Invitrogene) reverse transcriptase according to the manufacturer's protocol. Subsequently, the cDNA was gel-purified and circularized according to a previously published protocol (Buchbender, Andreas, et al. Methods 178 (2020): 33-48). Next, the cDNA was PCR-amplified and the products were purified and size-selected using the ProNex® chemistry.