organism: Mus musculus
description: 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.
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2022-12-31
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2022-12-31
attributes.tag: External Id
attributes.value: SAMEA110174290
attributes.tag: INSDC center alias
attributes.value: OXFORD
attributes.tag: INSDC center name
attributes.value: University of Oxford
attributes.tag: INSDC first public
attributes.value: 2022-12-31T00:58:23Z
attributes.tag: INSDC last update
attributes.value: 2022-12-31T00:58:23Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-11853:SRSF3 epicardial irCLIP replicate 2
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell_line
attributes.value: immortalised epicardium-derived cell line
attributes.tag: cell_type
attributes.value: epicardial cell
attributes.tag: common name
attributes.value: house mouse
attributes.tag: developmental stage
attributes.value: mouse embryo stage
attributes.tag: disease
attributes.value: normal
attributes.tag: isolate
attributes.value: not applicable
attributes.tag: organism part
attributes.value: heart
attributes.tag: replicate
attributes.value: 2
attributes.tag: sample name
attributes.value: E-MTAB-11853:SRSF3 epicardial irCLIP replicate 2