GSM5668156: TCFL5-eCLIP-rep2; Mus musculus; RIP-Seq
Lysates were clarified from UV light-treated testicular  seminiferous tubules. protein-RNA complexes were isolated with antibody. RNA adapter X1A and X1B were ligated to RNA 3' ends dephosphorylated by FastAP Alkaline Phosphatase (EF0652, Thermo Fisher Scientific). Immunoprecipitation beads were eluted for 10 min at 70°C using washing buffer. Sample were analyzed by NuPAGE (4%–12% gradient precast gels run with MOPS buffer). Cross-linked RNA-protein complexes were transferred onto nitrocellulose membrane (1060008, Amersham Protran Premium GE). Then the membrane was cut from the protein band to 75 kDa above it and RNA was purified using acid phenol/chloroform/isoamyl alcohol (25:24:1) (P1011, Solarbio). RNA adapter RiL19 was ligated to input RNA 3' ends dephosphorylated by FastAP Alkaline Phosphatase. AR17 primer was used to reverse transcript all RNA and Rand103Tr3 adapter was ligated to cDNA 5' ends. The ligated cDNA were purified with MyONE Silane beads (37002D, Thermo Fisher Scientific), quantified by qRCR, and amplified with Q5 PCR master mix (M0492L, NEB). The amplified cDNA were run in 3% low melting temp agarose gel (50180, Lonza), cut from 175bp to 350bp, and extracted with QIAGEN gel extraction kit. The cDNA libraries were sequenced on an Illumina NovaSeq.