GSM6235277: SLAM-seq_S9CC47_R6_0h; Homo sapiens; OTHER
Total RNA was extracted using Trizol reagent, phenol-chloroform extraction was performed, and the aqueous phased was used as input to the Quick-RNA Miniprep kit. During RNA extraction with Quick-RNA Miniprep kit, 0.1 mM DTT was added to the the RNA wash and RNA pre-wash buffers, but the on-column DNase I step was skipped. RNA was eluted in H20 with 1 mM DTT, quantified with Qubit RNA Broad Range assay (ThermoFisher, Q10211), and > 2 ug total RNA was used as input to the alkylation reaction. Alkylation was performed in dark tubes after which light exposure was allowed, and after quenching RNA was purified and subjected to on-column DNase I digestion using the RNA Clean & Concentrator-5 kit (Zymo, R1013). 500 ng alkylated RNA was used as input to QuantSeq 3' mRNA-Seq Library Prep Kit FWD with unique dual index (UDI) add-on (Lexogen, 113.96), with 15 cycles of PCR amplification. Library size distributions were confirmed by separation on a PAGE gel and staining with SYBRGold and pooled based on quantifications from Qubit dsDNA High Sensitivity Kit (ThermoFisher , Q32854).