GSM7720912: mESC, WT, caRNA, m5C-IP, rep3; Mus musculus; OTHER
Total RNAs from whole cell or the chromatin-associated fractions were randomly fragmented by incubation at 94 °C for 4 minutes using 1× fragmentation buffer (NEB E6186A). Fragmentation was stopped by adding 1× Stop Solution. Spike-in RNAs were added to each sample. Four microgram anti-m5C antibody (Diagenode, MAb-081-100) was conjugated with 30 μL of protein G beads (Invitrogen, 1003D) in 300 μL IP buffer (10 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.05% Triton X-100 (v/v)) for two hours at 4 °C on a rotating wheel. The same procedure was performed for a control reaction using mouse IgGs (Abcam ab37355). Bead-antibody complexes were washed three times with IP buffer and finally brought to 250 μL with IP buffer. After heat denaturation and quick chill on ice, 10-μg samples of RNA were added to the bead-antibody complexes and incubated with 1 μL SUPERase•In™ RNase Inhibitor (Invitrogen, AM2694) overnight at 4 °C on a rotating wheel. After several washes with IP buffer, RNA was incubated in 100 μL elution buffer (5 mM Tris-HCl pH 7.5, 1 mM EDTA, 0.05% SDS, and 200 μg Proteinase K (Invitrogen 25530049)) for 1 hour at 50 °C. Beads were removed by centrifugation in a microcentrifuge, and the supernatant was purified with RCC-5 without size selection. Libraries were constructed with SMARTer® Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian (TaKaRa Bio, 634411) according to the manufacturer's instructions.