GSM8403157: GLORI_3KR_rep1; Homo sapiens; OTHER
The cells were collected, washed with 1 ml cold PBS buffer containing 1 mM EDTA, and centrifugated at 500 g and room temperature for 5 min. Added 200uL ice-cold cytoplasmic lysis buffer (10 mM Tris-HCl pH=7.5, 0.1% IGEPAL®CA-630, 150 mM NaCl) to the cell pellet and incubated on ice for 5 min, then added 2.5 volumes of chilled sucrose cushion (24% RNAse-free sucrose in cytoplasmic lysis buffer). Subsequently, centrifuge at 15,000g and 4 ℃ for 10 min. The supernatant was collected as the cytoplasmic fraction. Added 100uL of prechilled glycerol buffer (20 mM Tris-HCl pH=7.9, 75 mM NaCl, 0.5 mM EDTA, 0.85 mM DTT, 0.125 mM PMSF, 50% glycerol) to resuspend the nuclei pellet, then added 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% IGEPAL®CA-630) and vigorously vortexed. The mixtures of nuclei pellets were incubated on ice for 2 min, then centrifuged at 4°C with 15,000 g for 2 min. The supernatant is soluble nucleoplasm, and the pellet is chromatin fraction. The chromatin fraction was dissolved  in 1ml of TRI Reagent™ (AM9738, Invitrogen, USA), and was heated at 50 °C with shaking at 1,000 rpm for 20 minutes until the pellet completely dissolved. Then, caRNAs were extracted according to the manufacturer's instructions for TRI Reagent™.  For GLORI reaction,genomic DNA is removed from caRNAs by using DNase Ⅰ (EN401, Vazyme). Subsequently, non-ribosomal caRNAs were further enriched by using the Ribo-MagOff rRNA Depletion Kit (N420-01, Vazyme Biotech, ChinaVazyme). Then, GLORI was carried out according to previously published. Briefly, caRNAs were fragmented at 94 °C for 30 s. Then, guanosine of fragmented caRNAs was protected in a protection buffer (1.32 M glyoxal, 50% DMSO) at 50 °C for 30 min. Subsequently, add 10 μl saturated H3BO3 solution and incubate at 50 °C for 30min. Next, add deamination buffer (750 mM NaNO2, 40 mM MES buffer, and 2.08 M glyoxal) and incubate at 16 °C for 8 h to deaminize adenine. Finally, add deprotection buffer (0.25 M TEAA and 25% deionized formamide) and incubate at 95 °C for 10 minutes. For the library construction of GLORI products, SMARTer Stranded Total RNA-Seq Kit v2 (Takara) was used according to the manufacturer's protocols. Sequencing was performed at GeneMind Biosciences Company (Shenzhen, China) through the SURFSeq 5000 platform on PE100 mode.