Sample type SRA
Source name U2OS
Organism Homo sapiens
Characteristics cell type: Immortalised osteosarcoma cells
Treatment protocol Cells were cultured in normal growth media. Prior to UV crosslinking, cells were supplemented with 4SU for 14 hours. For UV crosslinking, cells were washed with cold PBS and then irradiated with 365 nm UV at 0.15 J/cm2 in a Stratalinker 2400.
Growth protocol Both HeLa and U2OS cell lines were cultured in Dulbecco’s Modified Eagle Medium (DMEM) with high glucose (4500mg/L), L-glutamine (584mg/L) and sodium pyruvate (110mg/L)(Life Technologies, cat# 11995065), and supplemented with 10% Fetal Calf Serum (Serana, cat# FBS-AU-015) and 100U/ml Penicillin/Streptomycin (Gibco, cat# 15140122). Cells were grown at 37°C in 5% CO2 in a humidified incubator.
Extracted molecule total RNA
Extraction protocol Following UV crosslinking, cells were harvested, lysed in NP-40 lysis buffer and passed through a 32gauge needle. Lysates were cleared by centrifugation and then incubated with RNase T1 (Fermentas). Lysates were pre-cleared using Protein G Dynabeads (Invitrogen), after which RNA-protein complexes containing NONO were immunoprecipitated with NONO antibody (Monoclonal Antibody (MAb) Facility) conjugated to Protein G Dynabeads (Invitrogen). Following immunoprecipitation, beads were washed and then resuspended in dephosphorylation buffer and Calf intestinal alkaline phosphatase (New England Biolabs). Beads were then washed and incubated in polynucleotide kinase buffer and RNA was radiolabelled with Y-32P-ATP (Perkin Elmer). Radiolabelled protein-RNA complexes were removed from Dynabeads then resolved by SDS-PAGE on a NuPAGE Novex 4-12% Bis-Tris Protein Gel. Using 32P radiography RNA-protein complexes corresponding to the sizes of NONO and SFPQ were excised from the gel, and electroeluted in D-Tube Dialyzer midi tubes (Merck Millipore). Electroeluant was digested with Proteinase K (Fermentas) and RNA was then extracted from samples using miRNAeasy Micro Kit (Qiagen).
RNA extracted from PAR-CLIP was converted into a cDNA library using TruSeq small RNA Kit v2 (Illumina) with a size selection range 145-327 bp (RNA of 18-200 nt) and Illumina sequencing with 50bp single reads.