characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: 2022-12-31T00:58:23Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2022-12-31T00:58:23Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA110174290
characteristics.characteristic: External Id
characteristics.text: OXFORD
characteristics.characteristic: INSDC center name
characteristics.text: 2022-12-31T00:58:23Z
characteristics.characteristic: INSDC first public
characteristics.text: 2022-12-31T00:58:23Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS12273104
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-11853:SRSF3 epicardial irCLIP replicate 2
characteristics.characteristic: Submitter Id
characteristics.text: ArrayExpress
characteristics.characteristic: broker name
characteristics.text: immortalised epicardium-derived cell line
characteristics.characteristic: cell line
characteristics.text: epicardial cell
characteristics.characteristic: cell type
characteristics.text: house mouse
characteristics.characteristic: common name
characteristics.text: Protocols: PBS-washed cells were crosslinked with 200mJ/cm2 UV light in a Stratalinker 1800 and harvested by scraping into PBS. An immortalised epicardium-derived cell line (Austin et al., 2008, Dev Dyn) was cultured on 1% gelatine in immorto medium (10% FBS, 1% Penicillin/Streptomycin, ITS supplement, 0.1% IFNγ, DMEM high glucose Glutamax) at 33°C in 5% CO2. Cells were resuspended in lysis buffer (50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 1 % Igepal CA-630, 0.1 % SDS, 0.5 % sodium deoxycholate, protease inhibitor (Sigma Aldrich)) and samples sonicated in a Bioruptor (Diagenode). In the presence of Turbo DNase (Thermo Scientific), RNA in 100ug lysate per sample was partially digested with 1/500 RNase I (Invitrogen). SRSF3 and crosslinked RNA were immunoprecipitated from lysate for 2h at 4°C with rotation using 10ug anti-SRSF3 antibody (ab198291, Abcam). RNA was dephosphorylated with polynucleotide kinase (NEB) at 37°C for 30min. The L3-App-IRD800CWN-biotin linker was ligated to the RNA with T4 RNA ligase (NEB) overnight at 16°C. Eluted SRSF3-RNA complexes were separated on a NuPAGE 4-12% Bis-Tris gel (Invitrogen) and transferred onto a 40μm nitrocellulose membrane. Protein-RNA complexes containing RNA of approximately 70-280nt length were excised and RNA released by proteinase K (Roche) digestion for 60min at 50°C followed by RNA extraction with phenol-chloroform overnight ethanol precipitation. RNA was reverse transcribed with TIGRT-III reverse transcriptase (Ingex) and cDNA/RNA hybrids captured with MyOne Streptavidin C1 Dynabeads (Life Technologies). RNA was degraded by alkaline hydrolysis for 15min followed by cDNA circularization with Circligase II (Epicentre). The cDNA was amplified with Phusion HF polymerase (NEB) and P3/P6 primers and the library was cleaned-up with Select-a-Size DNA Clean & Concentrator kit (ZymoResearch) to remove empty library before a further three PCR cycles using P3/P6Solexa primers. The final library was cleaned up first with Ampure XP beads followed by gel clean-up on a Novex TBE-6% Urea gel.
characteristics.characteristic: description
characteristics.text: mouse embryo stage
characteristics.characteristic: developmental stage
characteristics.text: normal
characteristics.characteristic: disease
characteristics.text: Mus musculus
characteristics.characteristic: organism
characteristics.text: Mus musculus
characteristics.characteristic: organism
characteristics.text: heart
characteristics.characteristic: organism part
characteristics.text: 2
characteristics.characteristic: replicate
characteristics.text: Mus musculus
characteristics.characteristic: scientific_name
characteristics.text: SRSF3 epicardial irCLIP replicate 2
characteristics.characteristic: title