title: GSM8239462: HEK293T cells, DHX29-KO, 0 h, rep3; Homo sapiens; RNA-Seq
library_name: GSM8239462
library_construction_protocol: Control and DHX29-deficient HEK293T cells were plated and maintained in the culture media supplemented with 25 μM 4-thiouridine (s4U, Sigma Aldrich) for 24 h. Then cells were washed with DPBS and the culture media were replaced with fresh ones that contained 2.5 mM uridine (Sigma Aldrich). After 0, 3, and 6 h of incubation, cells were lysed in TRIzol, and total RNA was isolated according to the manufacturer's protocol under reducing conditions (supplemented with 0.1 mM DTT). Samples were protected from light whenever possible. Then 10 µg of total RNA was incubated in the reaction mix containing 10 mM iodoacetamide (Sigma Aldrich), 50% DMSO, and 50 mM NaPO4 (pH 8). Samples were incubated at 50 °C for 15 min, followed by the addition of DTT (20 mM). The alkylated RNA samples were purified using RNA Clean & Concentrator-5 (Zymo Research). RNA sequencing libraries were generated using QuantSeq 3' mRNA-Seq V2 Library Prep Kit (FWD) with Unique Dual Indices (12nt, Lexogen) according to the manufacturer's protocol. Sequencing was performed using NextSeq 500 Sequencer and NextSeq 500/550 High-Output v2 Kit (Illumina).