Illumina Genome Analyzer II sequencing; RBM20 orchestrates cardiac pre-mRNA
RBM20 orchestrates cardiac pre-mRNA
raw_rbm20_hits_NH_HM_005
Neonatal rat ventricular myocytes (NRVMs) were isolated from SD rats using a modification of the Kasten's technique as described (37). 48 h past isolation, cardiomyocytes were irradiated one time for 400mJ/cm and additional 200 mJ/cm with 254 nm UV light, and lysed in NP40 lysis buffer. After DNase and RNase T1 treatment, the lysate was immunoprecipitated using RBM20 antibody (5) covalently coupled to Protein G Dynabeads (Invitrogen). Radiolabeling of immunoprecipitated RNA, protein-RNA complexes separation, sequencing library preparation and sequencing was done following the PAR-CLIP protocol 4 ug of total RNA was used for library preparation with the TruSeq RNA Sample Preparation Kit (Illumina).