GSM9105192: GLORI-seq, T24_DMSO1; Homo sapiens; RNA-Seq
Total RNA from T24 cells was isolated with TRIzol. Small RNA fractions were depleted using the MEGAclear Transcription Clean-Up Kit (Thermo Fisher Scientific). Enrichment of mRNA was performed twice using the Dynabeads mRNA Purification Kit (Thermo Fisher Scientific). mRNA was then fragmented by incubation for 3 min at 94 °C using the NEBNext Magnesium RNA Fragmentation Module (New England Biolabs). Fragmented mRNA was then DNase I-treated and purified by the RNA Clean & Concentrator-5 kit (Zymo Research). Fragmented RNA samples were then subjected to the chemical deamination protocol. For RNA protection, 100 – 200 ng of fragmented mRNA supplemented with a synthetic Spike-In RNA oligonucleotide were added into protection buffer (1.32 M Glyoxal solution, 50 % DMSO, prepared in water) and incubated for 30 min at 50 °C in a thermal cycler. The RNA was further stabilized by adding 10 µL of freshly prepared saturated boric acid at RT, followed by incubation for 30 min at 50 °C in a thermal cycler. Protected RNA was then added into 50 µL of freshly prepared deamination buffer (1.5 M NaNO2, 80 mM MES (pH 6.0), 1.76 M Glyoxal solution, prepared in water), and incubated for 8 hours at 16 °C in a thermal cycler. After deamination, RNA was purified by ethanol precipitation ON at – 80 °C. Pelleted RNA was washed twice using 75 % ethanol and air-dried for 5 min at RT. Then, the RNA pellet was resuspended in 50 µL of deprotection buffer (500 mM triethylammonium acetate (pH 8.6), 47.5 % deionized formamide, prepared in water) and incubated for 10 min at 95 °C in a thermal cycler. Deprotected RNA was purified by ethanol precipitation for at least 30 min at – 80 °C. Pelleted RNA was washed twice using 75 % ethanol and air-dried for 5 min at RT. The RNA pellet was then resuspended in 50 µL of water and further purified using the RNA Clean & Concentrator-5 kit (Zymo Research). For sequencing library preparation, a two-step RNA end-repair protocol was pursued. RNA 3'-end dephosphorylation was performed by Antarctic phosphatase (New England Biolabs) treatment in a total reaction volume of 20 µL. The reaction was incubated for 30 min at 37 °C and inactivated for 2 min at 80 °C. Subsequently, RNA 5'-end phosphorylation was performed by T4 Polynucleotide Kinase (New England Biolabs) treatment in a total reaction volume of 50 µL. The reaction was incubated for 30 min at 37 °C, inactivated for 20 min at 65 °C, and purified using the RNA Clean & Concentrator-5 kit (Zymo Research). The libraries were then prepared using the NEBNext Small RNA Library Prep Set for Illumina in combination with the NEBNext Multiplex Oligos for Illumina (Index Primer Sets 1 and 3) (New England Biolabs). Size selection of the libraries was performed via TBE-PAGE using 8 % TBE polyacrylamide gels (Thermo Fisher Scientific) selecting sequencing competent molecules in a size range of 160 – 250 bp.