title: GSM8680409: SBP2, Input, rep3; Homo sapiens; RIP-Seq
library_construction_protocol: Cells were lysed in 0.5 ml of NP40 lysis buffer (50 mM HEPES, pH 7.5, 150 mM KCl, 2 mM EDTA, 1 mM NaF, 0.5% (v/v) NP40, 1 mM DTT, protease inhibitor cocktail, 100 U/ml RNaseOUT recombinant ribonuclease inhibitor) per 10cm dish, and rotated for 30 min at 4°C. After centrifuging at 20,000 g at 4°C for 10 min, the supernatants were collected. Protein concentrations were determined using the BCA method. 4 mg protein with 120 μl anti-flag magnetic beads (Biomake) were used for immunoprecipitation at 4°C for 5h with rotation. Before starting the incubation, 1/5 of the sample was taken as Input. Beads were washed three times with IP wash buffer (50 mM HEPES-KOH, pH 7.5, 300 mM KCl, 0.05% (v/v) NP40, 1 mM DTT, protease inhibitor cocktail), and further washed three times with high-salt wash buffer (50 mM HEPES-KOH, pH 7.5, 500 mM KCl, 0.05% (v/v) NP40, 1 mM DTT, protease inhibitor cocktail). RNA from both Input and IP samples was extracted according to the instructions provided with the TRIzol reagent (Thermo Fisher Scientific), followed by DNA removal with RNase-free DNase (Promega). RNA-seq libraries were prepared with the VAHTS Universal V6 RNA-seq Library Prep Kit for Illumina (Vazyme Biotech), following the manufacturer's instructions using 1 µg of RNA.