GSM9631343: PAR-CLIP-seq-HEK293T-SMD2IP-1; Homo sapiens; OTHER
PAR-CLIP 4SU treatment and UV crosslinking:Add 4SU (MCE, HY-W011793) to final conc. of 200 uM to culture medium, 37℃, 14 hr. For each 15 cm plate, remove medium and wash twice with 5 ml ice-cold PBS. Then place the plate on ice, 365 nm, 1500*100 uJ/cm2, crosslink twice. Collect cells in 2 ml ice-cold PBS. Centrifuge to collect cell pellet. Finally freezing with liquid nitrogen. Lysate preparation:Thaw cell pellet on ice, add 2 times vol lysis buffer containing 50 mM HEPES (pH 7.5), 150 mM KCl, 2 mM EDTA, 0.5% (v/v) NP40, 0.5 mM DTT, complete EDTA-free protease inhibitor cocktail, mix well, incubate on ice for 10min with periodic perturbation. Then Cells were lysed by sonication with 6 cycles of 5 seconds on / 5 seconds off at 30% amplitude, while keeping samples on ice to prevent overheating. Centrifuge 15000g for 15min 4℃ to collect supernatant. IP:Add Monoclonal anti-FLAG ®M2（Sigma-Aldrich，F3165）to cleared lysate. Rotate for 4h at 4℃. Prepare Protein A/G Magnetic Beads by washing twice with IP-wash buffer containing 50 mM HEPES-KOH (pH 7.5), 300 mM KCl, 0.05% (v/v) NP40, complete EDTA-free protease inhibitor cocktail. Then add beads to lysate, rotate for 3h at 4℃. 2nd RNase T1 digestion:Resuspend beads in 1 vol of IP wash buffer containing 50 mM HEPES-KOH( pH 7.5), 300 mM KCl, 0.05% (v/v) NP40, 0.5 mM DTT, complete EDTA-free protease inhibitor cocktail, add RNase T1 (Thermo Fisher，EN0541) to a final conc. of 10U/μl and incubate for 8min at 22℃, then chill reaction on ice for 5min. Wash beads 3 times in High Salt Buffer containing 50 mM HEPES-KOH(pH 7.5), 500 mM KCl, 0.05% (v/v) NP40, 0.5 mM DTT, complete EDTA-free protease inhibitor cocktail. Then change to new tube during the last wash. Dephosphorylation:Wash beads once with Dephosphorylation buffer containing 50 mM Tris-HCl (pH 7.9), 100 mM NaCl, 10 mM MgCl2, 1 mM DTT, then resuspend in 1volumn of commercial dephosphorylation buffer (beads volume). Add Quick CIP (NEB, M0525V) to final concentration of 0.5U/μl, incubate for 15min at 37℃ (remove 5'P). Library construction:Total RNA was extracted using TRIzol™ reagent according to the manufacturer's instructions. RNA-seq library preparation was conducted using the Illumina Stranded Total RNA Prep with Ribo-Zero Plus kit, following the manufacturer's protocol.