GSM7696469: 22Rv1, Input, replicate 3; Homo sapiens; RIP-Seq
Approximately 5 x107 22Rv1 cells per sample were grown and UV crosslinked at 400 mJoules/cm2 with 254 nm radiation, flash frozen, and stored until use at -80°C. Cells were lysed using 1mL of eCLIP lysis mix and subjected to one round of sonication for 5 minutes with 30 second ON/OFF (total 10 minutes) at 75% amplitude. Rabbit Anti-eIF3B/EF3S9 antibody was then pre-coupled to Anti-Rabbit IgG Dynabeads, added to lysate, and incubated overnight at 4°C. Prior to immunoprecipitation, 2% of the sample was taken as the paired input sample, with the remainder magnetically separated and washed with eCLIP high stringency wash buffers. IP and input samples were cut from the membrane at the relative band size to 75kDa above. RNA adapter ligation, IP-western, reverse transcription, DNA adapter ligation, and PCR amplification were performed as previously described. eCLIP studies were performed according to the single-end seCLIP protocol (PMID: 28766298). The eCLIP cDNA adapter contains a sequence of 10 random nucleotides at the 5¢ end. This random sequence serves as a unique molecular identifier (UMI) after sequencing primers are ligated to the 3¢ end of cDNA molecules. Therefore, eCLIP reads begin with the UMI and, in the first step of analysis, UMIs were pruned from read sequences using umi_tools (v0.5.1. UMI sequences were saved by incorporating them into the read names in the FASTQ files to be utilized in subsequent analysis steps Single-end seCLIP protocol (PMID: 28766298)