GSM2758638: PAR-CLIP_PHC1_r2; Homo sapiens; RIP-Seq
300e6 HEK293T cells with overnight 4SU incubation were washed once in cold PBS and crosslinked at 365nm and a total energy of 0.15 J/cm2. Cells were scraped and resuspended in cold PBS. After centrifugation at 500 rcf for 5 minutes, pellet was resuspended in lysis buffer (50 mM HEPES pH 7.5, 150 mM KCl, 5 mM CaCl2, 1 % NP-40) and incubated 15 minutes on ice. Lysate was treated with 20 U/mL Fermentas RNase T1 and 15 U/mL Fermentase RNase free DNase I for 15 minutes at RT and then sonicated for 15 cycles (30 sec on / off) on a Diagenode Bioruptor on high. Lysate was cleared at 21’000 rcf and 4 °C for 10 minutes. IP was performed for 3 hours with 100 uL streptavidin magnetic myONE T1 beads (Thermo Fisher). Beads were washed 3 times in lysis buffer and treated with RNase T in 100 uL at 20 U/uL for 15 minutes at RT. After three more lysis buffer and two dephosphorylation buffer washes (50 mM Tris-HCl pH 7.9, 100 mM NaCl, 10 mM MgCl2) RNA was dephosphorylated at 37 °C in 100 uL NEB buffer 3 with 0.5 U / uL CIP for 10 minutes. 500 uL lysis buffer were added for beads to rebind during 10 minutes on ice. Three more lysis buffer washes and two PNK-buffer (70 mM Tris-HCl, pH 7.6, 10 mM MgCl2) washes were followed by phosphorylation with radioactive 32P at 37 °C in 100 uL of PNK buffer with 5 mM DTT, 17 nM (10 uCi) 32P-ATP and 0.5 U/uL PNK. After 30 minutes 5 uM normal ATP were added for additional 5 minutes. 1 mL of lysis buffer was added and beads are allowed to rebind during 10 minutes on ice. Beads were washed 5 times with lysis buffer, boiled in SDS-DTT solution and run on a NuPage Novex 4-12 % bis-tris gel for 1.5 hours at 150 V in MOPS buffer. Gel was imaged for 30 minutes with a phosphorimaging cassette and bands of interest excised. Elution of Protein-RNA complex was performed three times in 400 uL MOPS buffer using d-tube dialyzer midi MWCO 3.5 kDa each for 1 hours at 100 V and 5 minutes at 100 V reversed polarity. All subsequent steps were carried out exactly as described Hafner M. et al 2010. Library was prepared similary to Illumina small RNA protocol. We adhered to Hafner et al 2010.
GEO Accession
GSM2758638