GSM5775609: MDAMB231_RIP-seq_YTHDC1_IP_rep2; Homo sapiens; RIP-Seq
Cells were washed with cold PBS and UV-crosslinked 3× at 120,000 μJ/cm2 using a 254nm lamp. Nuclear isolation buffer (320mM sucrose 10mM Tris-HCl pH 7.5, 5mM MgCl2, 1% Triton X-100 vol/vol) was added onto the dish and the cells were detached with a cell scraper. The cells were incubated on ice for 20 minutes on ice followed by pelleting of nuclei by centrifugation at 1500 × g for 15 minutes at 4°C. The pellet was resuspended in RIP buffer (150mM NaCl, 25mM Tris-HCl pH 7.4, 5mM EDTA, 0.5mM DTT, and 0.5% Tergitol NP40) containing HaltTM Protease (Thermo Fisher Scientific) and RiboLock (Thermo Fisher Scientific) RNase inhibitors and sonicated using a Qsonica Q800R3 sonicator to isolate nuclear contents. The lysate was centrifuged at 15,000 × g for 10 minutes at 4°C and the supernatant was pre-cleared by incubation with protein A/G beads for 1 hour at 4°C. The amount of protein in each sample was quantified using the Bio-Rad Protein Assay Kit (Bio-Rad). For each immunoprecipitation reaction, 5 μg of anti-flag antibody (F3165, Sigma-Aldrich) or normal mouse IgG (12-371, Millipore) was conjugated to 50 μl Protein A/G magnetic beads (Thermo Fisher Scientific) for 4 hours at 4°C. The beads were washed twice with RIP buffer and incubated with supernatant containing 500 μg protein from nuclear lysate overnight at 4°C. Meanwhile, supernatant containing 50 μg of protein (10% input) was saved for each RNA or protein input sample. The next day, the immunoprecipitation reactions were washed 3x with RIP buffer and resuspended in 100 μl PBS. For isolation of immunoprecipitated protein, 20 μl of beads were transferred to another 1.5 ml tube and 6.7 μl of 4x Laemli buffer was added and the sample was incubated at 95°C for 5 minutes. The remaining beads were then treated with DNase 1 (Thermo Fisher Scientific) followed by Proteinase K (Thermo Fisher Scientific) to remove unwanted DNA and uncrosslinked protein. The tube was then placed on a magnetic stand and the supernatant containing the immunoprecipitated protein fraction was transferred to a new tube and frozen at -80°C. Trizol was added to the remaining 80 μl of beads and RNA was isolated. Samples were rRNA depleted followed by library construction using KAPA Stranded mRNA-Seq Kit (Illumina Platforms) (KR0960m Kapa Biosystems) with 10 cycles of PCR amplification and sequenced on an Illumina Hiseq2500.