GSM8829541: A2.2_5nM_SLAM-seq_rep1; Homo sapiens; OTHER
RNA extraction was performed using the RNeasy Plus Mini Kit (Qiagen, 74134) according to the manufacturer's instructions. Total RNA was subjected to alkylation with 10 mM iodoacetamide (IAA, Sigma-Aldrich, I6125) for 15 minutes and RNA was re-purified by ethanol precipitation. The purified RNA was quantified using a Qubit RNA broad range assay (Invitrogen, Q10210). 400 ng of alkylated RNA were used as input for generating 3'-end mRNA sequencing libraries using the QuantSeq 3'mRNA-Seq Library Prep Kit FWD for Illumina (Lexogen, 113.96) and PCR Add-on Kit for Illumina (Lexogen, 020). The distribution of RNA fragments was analyzed using High Sensitivity D1000 assay (Agilent, ScreenTape 5067-5548 and Reagents 5067-5585) in Agilent 4200 Tapestation. Libraries were pooled at 4 nM, and shallow sequencing was performed using MiSeq (Illumina) to estimate the required read counts per sample, aiming for conditions where more than 3% of reads contained ≥2 T>C conversions. Deep sequencing was performed on an Illumina NovaSeq SP flow cell, generating 150 bp single-end reads.