title: GSM8377592: K562, WT, caRNA, m5C-IP, MeRIP, r1; Homo sapiens; RIP-Seq
library_construction_protocol: Fractionation of mESCs and K-562 cells was performed following the published protocol with the optimized concentration of NP-40 (MilliporeSigma, 492018) for each cell line. In brief, 5 × 106 to 1 × 107 724 cells were harvested and washed with 1 mL cold PBS/1 mM EDTA buffer, then centrifuged at 4 °C with 500 g to collect the cell pellet. 200 μL ice-cold lysis buffer (10 mM Tris726 HCl, pH = 7.4, 0.05% NP-40, 150 mM NaCl) were added to the cell pellet and incubated on ice for 5 minutes, then gently pipetted up the cell lysate over 2.5 volumes of chilled sucrose cushion (24% RNase-free sucrose in lysis buffer) and centrifuged at 4 °C with 15,000 g for 10 minutes. All the supernatant was collected as cytoplasmic fraction and the nuclei pellet was washed once by gently adding 200 μL ice-cold PBS/1 mM EDTA to the nuclei pellet without dislodging the pellet. The nuclei pellet was resuspended in 200 μL prechilled glycerol buffer (20 mM Tris-HCl, pH = 7.4, 75 mM NaCl, 0.5 mM EDTA, 0.85 mM DTT, 0.125 mM PMSF, 50% glycerol) with gentle flicking of the tube. Then an equal volume of cold nuclei lysis buffer (10 mM HEPES, pH = 7.6, 1 mM DTT, 7.5 mM MgCl2, 0.2 mM EDTA, 0.3 M NaCl, 1 M urea, 1% NP-40) was added, followed by vigorous vertexing for 5 seconds twice. The nuclei pellet mixtures were incubated for 2 minutes on ice, then centrifuged at 4 °C with 15,000 g for 2 minutes. The supernatant was collected as the soluble nuclear fraction (nucleoplasm). The pellet was gently rinsed with cold PBS/1 mM  EDTA without dislodging and then collected as the chromosome-associated fraction. caRNA was extracted with standard TRIZol protocol. Fractionation of LK cells were performed similar to ES cells with minor modification. Briefly, LK cells were cultured in vitro for 2 hours followed sorting from autoMACS® Pro Separator, and then ice-cold lysis buffer (10 mM Tris-HCl, pH = 7.4, 0.15% IGEPAL 741 ® CA-630, 75 mM NaCl) was used to separate cytoplasmic fraction. caRNAs were fragmented by soniation on a Bioruptor pico (Diagenode) device with 30 cycles, 30s on/30s off. Then in vitro transcribed spike-in RNA from firefly luciferase mRNA or renilla luciferase mRNA with different m5C levels were added to the immunoprecipitation reaction. Immunoprecipitated RNA was extracted by using Dynabeads™ MyOne™ Silane (Invitrogen, 37002D). Library construction was performed according to the standard protocol of SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian (TaKaRa bio, 634417). MeRIP-seq