GSM9643832: PRAISE-seq-HEK293T-nontreated-Ctrl1; Homo sapiens; OTHER
PRAISE-seq Pre-mRNA purification:Ribosomal RNA was depleted using the Ribo-off rRNA Depletion Kit (Human/Mouse/Rat) (Vazyme, N406) according to the manufacturer's protocol. Sulfite/bisulfite treatment:The 85% sulfite/15% bisulfite solution was prepared freshly by adding 2.58 g of potassium sulfite (Aladdin, P112413) and 0.3 g sodium bisulfite (Oxoid, 10019018) to 8 ml of RNase-free water and the solution was vortexed to clear (pH value is ~7.5). The 100 mM hydroquinone was prepared freshly by adding 11.01 mg of hydroquinone (Oxioid, 10011317) to 1 ml of RNase-free water. For sulfite/bisulfite treatment, 5 µl of RNA fragments was dissolved in 50 µl sulfite/bisulfite solution, which is a 100:1 mixture of 85% sulfite/15% bisulfite solution and 100 mM hydroquinone, and subjected to heat incubation at 70 °C for 5 h. The reaction mixture was purified by passing it through Spin Column (Sangon, B518688). Desulfonation:Purified RNA was transferred to a new 1.5 ml nuclease-free tube and adjusted to 100 μl with RNase-free water, then incubated with an equal volume of 1 M Tris–HCl (pH 9.0) at 75 °C for 30 min. The reaction was then immediately stopped by chilling on ice and ethanol precipitation. Add a coprecipitant (Takara, 9094) to aid in precipitation during the settling process. After ethanol precipitation, the RNA was resuspended in 10 µl of RNase-free water, and the concentration of RNA was quantified by Qubit RNA HS Assay Kit (Invitrogen, Q33231). Take 50 ng RNA (~1 µl) as 'treated' sample for subsequent library construction. Library construction:'Treated' RNA sample and 'untreated' RNA sample were subjected to library construction. For first-strand cDNA synthesis, Maxima H minus Reverse Transcriptase (Thermo Fisher, K1651) was used. Second, we used RNase H to remove mRNA (Thermo Fisher, EN0201) and finally, for second-strand cDNA synthesis, VATHS Universal V10 RNA-seq Library Prep Kit for Illumina (Vazyme, NR606) was used. Twelve cycles were used for untreated and treated RNA in the second round of PCR. High-throughput sequencing was performed on the Illumina xplus system.