GSM6330105: NIH3T3, Input; Mus musculus; RIP-Seq
cells were crosslinked with ultraviolet (UV) at 254 nm (400 mJ/cm2), followed by lysis in lysis buffer (50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate, freshly added protease inhibitor) and digestion with RNAase I (Ambion). Immunoprecipitation was performed using anti-HP1 alpha antibody (Abcam, ab109028) and M-280 sheep anti-rabbit IgG beads (Invitrogen). After 3' RNA adapter ligation, samples were run on protein gels and transferred to membranes (PVDF membrane for western blotting, nitrocellulose membrane for library preparation). A region 75 kDa above the HP1 alpha band on nitrocellulose membrane was cut and treated with proteinase K to isolate RNA. RNA was converted to cDNA followed by 3' DNA adapter ligation. Then the sample was PCR amplified and fragment size selected by agarose gel electrophoresis and gel extraction. eCLIP-seq libraries were prepared through 3' RNA adapter ligation, western blotting, HP1 alpha band isolation, RNA convertion to cDNA, 3' DNA adapter ligation, and PCR amplification.