GSM8559603: PCBP1_Input_heart_1; Mus musculus; OTHER
eCLIP studies were performed according to the published single-end seCLIP protocol with the following modifications. Approximately 80 mg of frozen embryonic mouse heart tissue was cryogrinded and UV crosslinked at 400 mJoules/cm2 with 254 nm radiation. The equivalence of 75 μg of RNA was lysed using 1 mL of eCLIP lysis mix and subjected to two rounds of sonication for 4 minutes with 30 seconds ON/OFF at 75% amplitude. A pre-validated PCBP1 antibody (Abcam cat# ab168377) was then pre-coupled to Anti-Rabbit IgG Dynabeads (ThermoFisher), added to lysate, and incubated overnight at 4°C. Before 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 of 40kDa to 75kDa above that band size. 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.UMIs were pruned from read sequences using umi_tools (v0.5.1) RNA adapter ligation, IP-western, reverse transcription, DNA adapter ligation, and PCR amplification were performed as previously described.