title: GSM8633399: eTAM-seq_RNA_WT_replicate1; Escherichia coli; RNA-Seq
library_name: GSM8633399
library_construction_protocol: Total RNA was extracted using TriZol reagent. 5µg total RNA of each sample was saved as the input control fraction. All RNA fractions were rRNA depleted using the siTools rRNA depletion kit, and the E. coli ribopool. TadA8.20 was overexpressed and purified following a previously published protocol (PMID: 36593412). 50 ng of rRNA-depleted E. coli RNA was subjected to the eTAM-seq workflow. Briefly, RNA was deaminated with 200 pmol of TadA8.20 in 20 μL 1 × deamination buffer supplemented with 10% (v:v) SUPERase•In RNase Inhibitor at 53 °C for 1h. The treated RNA was purified using RNA Clean & Concentrator kits (Zymo), and the deamination reaction was repeated twice at 44 °C. TadA8.20-treated RNA was built into libraries using the NEBNext Ultra Directional RNA Library Prep Kit for Illumina (New England Biolabs, catalog no. E7760L), following the manufacturer's protocol (section 4 for purified mRNA, skipping fragmentation). The input library was constructed using the same kit with 50 ng RNA as input. Libraries were quantified using the KAPA Library Quantification Kit (KAPA Biosystems, catalog no. KK4824) and sequenced on a NextSeq 2000 instrument (R1 25 cycles, R2 97 cycles, Illumina, catalog no. 20024906).