GSM6523704: epidermis, biol rep 4 - CLIP Sample; Mus musculus; RIP-Seq
Immunoprecipitants or input samples were run on 4-12% Bis-Tris protien gels and transferred to nitrocellulose membrane. RBP:RNP complexes were excised from membrane by cutting at region of membrane between 75kDA to 140kDa in the appropriate lanes. RNA was released from membrane by treatment of the membrane with urea/proteinase K treatment at 37C, and purified using acid phenol/chloroform/isoamyl alcohol Libraries were constructed as described Van Nostrand et al Nature Methods 2016. RNA adapters ligated to the RNA were A01 and B01, following reverse transcription DNA adapter ligated to the cDNA was rand3Tr3. Libraries were amplified by PCR using NEB Q5 High Fidelity Mastermix with primers containning Illumina P5 and P7 indices. Libraries (175-350bp) were purified from 3% low melt agarose gel using Qiagen minelute gel extraction kit eCLIP-Seq