GSM8111684: xz50, liver, Input; Mus musculus; RIP-Seq
The liver tissue was lysed and homogenized at 4℃ for 1 h in buffer solution consisting of 1% SUPERaseln, 1% protease inhibitor cocktail, 150 mM KCl, 0.5 mM DTT, 2 mM EDTA, 10 mM Tris-HCl, and 0.5% NP-40. After centrifugation at 12,000 rpm for 10 min, the supernatant was collected in a 1.5 mL centrifuge tube. To preserve the input, 50 μL of lysate was stored in 1 mL Trizol at -80℃.  For immunoprecipitation, 5 μg of the YTHDF3 antibody was combined with 40 μL of protein A beads (Sigma), dissolved in 140 μL of PBST buffer (PBS and 0.05% Tween), and incubated for 1 h at 4°C. As a control, 5 μg of normal rabbit IgG was added to another 40 μL of protein A beads in parallel. Half of liver lysate was added to protein A-anti YTHDF3 mixture, while the other half was added to protein A-IgG mixture. After thorough mixing, the mixture was continuously agitated for 3 h at 4°C. The mixture underwent eight washes with 500 μL ice-cold NT2 buffer (200 mM NaCl, 1% SUPERaseln, 1% protease inhibitor cocktail, 50 mM Tris-HCl, 2 mM EDTA, 0.05% NP-40, 0.5 mM DTT).  To elute the samples, 100 μL of protease K buffer (150 mM NaCl, 12.5 mM EDTA, 2%(w/v) SDS, 100mM Tris-HCl) and 20 μL thermo protease K were added to each sample, followed by incubation at 55℃ for 40 min. Using a magnetic separator, the supernatant was collected, and Trizol was added to preserve it as IP sample. RNA was extracted from both input and IP samples for sequencing purpose. The library was generated using the SMARTer® Stranded Total RNA-Seq Kit v2 (Takara) with three biological replicates for each sample.