GSM8795672: DIV14 pyramidal neuron CLIP library; Mus musculus; RIP-Seq
Cells were washed once with DPBS, covered in DPBS and UV crosslinked on leveled ice at 400 mJoules/cm2 with 254 nm radiation. While on ice, cells were collected with a scraper into a tube, washed once with DPBS, pelleted, flash frozen in liquid nitrogen and stored until use at -80 °C. Cell pellet was aliquoted and lysed using 1mL of eCLIP lysis mix. Anti-AGO2 antibody (Eclipse Bioinnovations Inc.) pre-coupled to anti-mouse Dynabeads were washed with eCLIP Lysis Buffer, added to cell lysate or sonicated tissue lysate, and then incubated overnight at 4 °C. After 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 75 kDa above. RNA adapter ligation, IP-western, reverse transcription, DNA adapter ligation, and PCR amplification were performed as previously described in Van Nostrand et al. (2017)