title: GSM8585574: TTseq_DMSO_rep2; Homo sapiens; OTHER
library_construction_protocol: The TRIzol-cell mixture was collected, and total RNA was extracted via TRIzol/chloroform extraction followed by isopropanol precipitation. RNA concentration was measured using the Qubit RNA BR Assay Kit (Thermo Fisher, Q10210). For each sample, 1 µg of S. cerevisiae 4TU-labeled RNA was mixed with 100 µg of purified 4SU-labeled RNA. The total volume was adjusted to 100 µL with RNase-free water, and 20 µL of 1 M NaOH was added per sample. Samples were incubated on ice for 20 minutes to fragment the RNA, and RNA fragmentation was stopped by adding 80 µL of 1 M Tris-HCl, pH 6.0. The samples were purified using Micro Bio-Spin P-30 gel columns (Bio-Rad, 7326250) to neutralize NaOH and prevent further fragmentation. The column-purified RNA was biotinylated in a 250 µL reaction containing 10 mM Tris-HCl, pH 7.4, 1 mM EDTA, and 5 mg of MTSEA biotin-XX linker (Biotium, BT90066) for 30 minutes at room temperature in the dark. Biotinylated RNA was purified by phenol-chloroform extraction and isopropanol precipitation. The RNA was then denatured at 65°C for 10 minutes and purified using the μMACS Streptavidin Kit (Miltenyi, 130-074-101) according to the manufacturer's instructions. The purified nascent RNA was further cleaned using the RNeasy MinElute kit (QIAGEN, 74204) and used to prepare sequencing libraries. TTchem-seq libraries were generated using the KAPA RNA HyperPrep Kit (Roche, 08098093702) with the fragmentation step omitted. A total of 75 ng of nascent RNA was denatured at 65°C for 5 minutes. The rest of the library preparation followed the manufacturer's instructions, with two SPRI bead purifications (Beckman Coulter, A63881) performed using bead-to-sample ratios of 0.95x and 1x.
identifiers.namespace: GEO
identifiers.id: GSM8585574_r1