title: GSM7213775: WT, 24h dTAG, rep2; Homo sapiens; RNA-Seq
library_construction_protocol: Cells were harvested by TRIzol (Invitrogen, 15596018) and stored at -80°C until processing. Chloroform was added to TRIzol lysate and separated into aqueous and organic phases by centrifugation per manufacturer instructions, and then the aqueous fraction was extracted using the RNA Clean & Concentrator-5 (Zymo, R1013) with on-column DNase I digestion. RNA was checked for purity by Nanodrop and was quantified by Qubit RNA broad range assay (Invitrogen, Q10210). For acute depletions, SLAM-seq96 was performed as described with minor modifications, with 4-thiouridine labeling of nascent transcription for the last 2 h prior to harvest. Briefly, RNA was extracted using the Direct-zol RNA miniprep kit (Zymo, R2052), modified to include 0.1 mM DTT in wash buffers and 1 mM DTT to the water for elution, with protection from light. Four ug of RNA was then alkylated with 10 mM iodoacetamide (G Biosciences, 786-078) dissolved in ethanol at 100 mM, in 50% v/v DMSO, 50 mM NaPO4 pH 8 for 15 min at 50°C. Alkylation was quenched by addition of 1 ul of 1 M DTT, and alkylated RNA was extracted by RNA Clean & Concentrator-5 kit. RNA-seq libraries were prepared using the QuantSeq 3' mRNA-Seq Library Prep FWD kit (Lexogen, 113.96) using 500 ng of input RNA and ~15 cycles of amplification, with unique dual indices.