title: GSM8627507: Tumoral1; Homo sapiens; RNA-Seq
library_construction_protocol: Patient tissue samples were homogenized by disruption using the TissueRuptor II (Qiagen). Total RNA from homogenized tissue samples and HEK293T 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.