GSM9155831: in vitro iCLIP, RNA oligo library, 7-deaza-GTP RNA, rep3; Homo sapiens; OTHER
Recombinant MBP-HNRNPH1 was incubated with the radioactively labelled RNA libraries and UV-crosslinked at 254 nm with 250 mJ/cm2. The protein-RNA complexes were separated using an SDS-PAGE and transferred to a nitrocellulose membrane. The RNA was extracted from the protein-RNA complexes using proteinase K treatment, phenol-chloroform extraction and ethanol precipitation. The protocol for the RNA extraction from the nitrocellulose membrane was taken from the iCLIP2 protocol (PMID: 31610236). The library preparation followed the iCLIP2 protocol (PMID: 31610236). In brief, the RNA was reversely transcribed to cDNA using an RT primer against the L3-App linker that was included at the 3' end of each oligo on the sequence level. After MyONE Silane bead clean up to purify the cDNA, the 5' linker was ligated to the cDNA. The cDNA was amplified using a 6-cycle PCR and size-selected with 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 Mid Output Flowcell using paired-end sequencing with 150 cycles (Read 1: 141nt, Read 2: 25nt).