title: GSM8733935: SK-OV-3(V5-RRN3)_IP (PAR-CLIP); Homo sapiens; OTHER
library_construction_protocol: V5-RRN3-expressing cells were grown in its complete medium supplemented with 100 μM 4-thiouridine (4-SU) for 14 h before crosslinking. After washing the cells with ice-cold phosphate-buffered saline (PBS), cells were cross-linked by exposure to 365-nm UV light for receiving a total energy of 0.2 J/cm2. The cells were then suspended in 3 volumes of NP40 lysis buffer [50 mM HEPES-KOH, pH 7.5, 150 mM KCl, 2 mM EDTA, 1 mM NaF, 0.5% (v/v) Nonidet P-40 (NP-40), 0.5 mM DTT, protease inhibitor cocktail (Roche, Swiss)] and incubate on ice for 10 min. The cell lysates were then treated with 10 μg/mL RNase A and 4 U/mL DNase I (Thermo-Fisher Scientific). Ten percent of cleared cell lysates were used as the input and the rest were subjected to immunoprecipitation with anti-V5 antibody (Thermo-Fisher Scientific), followed by the elution with V5 peptide (MedChemExpress, Shanghai, China). The eluted samples were digested with proteinase K before the RNA fragments were extracted by 25:24:2（v/v/v）phenol/chloroform/isoamyl alcohol (pH<5.2). The purified RNA samples were then subjected to the library construction and deep sequencing 1. Use RNA as a template and reverse transcribe it into cDNA using six-base random primers.2. Ligate adapters containing UID (Unique Molecular Identifier).3. Add primers, dNTPs, and DNA polymerase to perform PCR amplification, generating a library that meets the sequencing instrument's requirements.4. Use magnetic beads to recover fragments of the desired size and perform quality control on the constructed library.5. After the library passes quality control, pool different libraries according to effective concentration and the required sequencing data output, then load them onto the sequencing instrument for sequencing