GSM5227083: RPS3A_rep2_Input; Homo sapiens; RIP-Seq
Experiments were performed following a previous published protocol (PMID: 28766298). In brief, >20 million cells were collected for UV crosslink (400 mJoules/cm2, 254 nm), followed by cell lysis, sonication, and RNase I treatment. Anti-HA antibody (BioLegend #901501) was incubated with cell lysates at 4℃ overnight. 2% of samples were saved as paired input before immunoprecipitation (IP) steps. IP samples were washed, followed by RNA dephosphorylation (FastAP, Thermo Fisher; T4 PNK, NEB) and 3' RNA adaptor ligation (T4 RNA ligase, NEB). IP and input samples were run on a PAGE Bis-Tris protein gel and subsequently transferred to a nitrocellulose membrane, from which regions starting from protein size up to 75 kDa above were excised for proteinase K (NEB) treatment and column purification (Zymo). RNA obtained from input samples was also dephosphorylated and ligated to 3' RNA adaptors as performed previously to IP samples. Final RNA samples were reverse transcribed, ligated to a 3' DNA adaptor (T4 RNA ligase, NEB), and PCR amplified to obtain the final library for next generation sequencing. seCLIP-seq protocol