title: GSM9118554: iCLIP, MCF7, HNRNPH immunoprecipitation, rep1; Homo sapiens; OTHER
library_construction_protocol: The RNA was extracted from the cells using immunoprecipitation of HNRNPH. MCF7 cells were UV-crosslinked at 254 nm with 150 mJ/cm2 and lysed. After RNA digestion using RNase I, the protein-RNA complexes were immunoprecipitated using Protein G Dynabeads with 5 µg HNRNPH antibody (Abcam #10374) per replicate. For iCLIP2, the protocol from a previous publication was used (PMID: 31610236). Briefly, after protein-RNA complex extraction using the HNRNPH antibody, the RNA was 3' end dephosphorylated and subsequently ligated with a pre-adenylated L3-App linker. Then, the 5' end was radioactively labelled and the protein-RNA complexes were separated using an SDS-PAGE. After transfer to a nitrocellulose membrane and exposure to a Fuji film, the protein-RNA complexes were isolated. RNA was eluted using proteinase K treatment, phenol-chloroform extraction and ethanol precipitation. Using an RT primer against the L3-App linker, the RNA was reversely transcribed to cDNA. After MyONE Silane bead clean up to purify the cDNA, the 5' linker was ligated to the cDNA. The cDNA was pre-amplified using a 6-cycle PCR and size-selected with ProNex beads. For the final library preparation, the samples were PCR-amplified using 10 cycles, which was followed by a second size selection using ProNex beads. The samples were quality checked and profiled on a High Sensitivity D1000 ScreenTape using TapeStation 2200 (Agilent). After quantification with the Qubit dsDNA HS Assay Kit (Life technologies) the samples were pooled in an equimolar ratio, Sequencing was performed on a NextSeq 500 High Output Flowcell.