GSM9420289: 70-day-old forebrain organoids, congenital myotonic dystrophy, replicate 2, input; Homo sapiens; OTHER
70-day-old organoids were transferred into ice-cold HBSS (STEMCELL Technologies) in approximately tenfold the original tissue volume and triturated with a micropipette. Dissociated tissues were irradiated three times at 400 mJ/cm² in a Stratalinker 1800 UV crosslinker while keeping the suspension on ice and mixing between each irradiation, and then flash-frozen in liquid nitrogen. eCLIP was performed essentially as described by Van Nostrand et al. (Nature Methods, 2016). Briefly, organoids were lysed and treated with RNase I at a 1:1000 dilution. 2% of each lysate was reserved for preparation of a parallel size-matched input (SMInput) library, while the remaining material was subjected to immunoprecipitation using a mouse anti-MBNL2 antibody (Santa Cruz #sc-136167). Bound RNA fragments in the immunoprecipitates were dephosphorylated and 3'-end-ligated to an RNA adaptor. Protein-RNA complexes from SMInputs and immunoprecipitates were run on an SDS-polyacrylamide gel and transferred to a nitrocellulose membrane. Membrane regions corresponding to a molecular weight between 35 and 50 kDa above the expected size were excised, and RNA was released from the complexes using proteinase K. SMInput samples were dephosphorylated and ligated at the 3' end to an RNA adaptor. All RNA samples (immunoprecipitates and SMInputs) were reverse transcribed using AffinityScript Reverse Transcriptase (Agilent). cDNAs were ligated at the 5' end to a DNA adaptor. cDNA yields were quantified by qPCR, and 100-500 fmol of library per sample were generated using Q5 High-Fidelity PCR Master Mix (New England Biolabs).