GSM4431940: miCLIP replicate 3; Mus musculus; RIP-Seq
Back skin dissected from P0 neonates was treated first with 1x dispase to isolate epidermis, which was then digested with 1:1 0.25% Trypsin-EDTA:versene solution to break down tissue. Single cell suspensions were obtained by filtering through 45 µm cell strainers. Epithelial progenitor cells were isolated by FACS. Total RNA was extracted by TRIzol-LS. Poly(A)+ RNA was extracted with the Dynabeads™ mRNA Purification Kit (Thermo-Fisher, 61006) and treated with the RQ1 RNase-free DNase (Promega, M6101) to remove DNA contamination. 7-8 µg Poly(A)+ RNA was used for each biological replicate (pool of 3 litters of pups) to perform miCLIP. After fragmentation, 1/10 of the sample was saved as input to perform parallel library construction without CLIP. Library construction protocol is as described in Geula et al., 2015, Mapping m6A at Individual-Nucleotide Resolution Using  Crosslinking and Immunoprecipitation (miCLIP)