title: GSM8335805: KpnDRT2_WT_uninfected_dRNA-seq_treated; Escherichia coli str. K-12 substr. MG1655; RNA-Seq
library_construction_protocol: Total RNA was extracted using TRIzol (Thermo Fisher Scientific). For dRNA-seq library preparation, 1 µg of total RNA was fragmented in 1× NEBuffer 2 by heating at 92 °C for 1.5 min. DNase treatment was performed with 1 μL TURBO DNase (Thermo Fisher Scientific) in the presence of 1 μL SUPERase•In RNase Inhibitor (Thermo Fisher Scientific) for 10 min at 37 °C. Samples were treated with 1 μL T4 PNK in 1× T4 DNA ligase buffer (NEB) at 37 °C for 30 min and purified using the Zymo RNA Clean & Concentrator-5 kit. Samples were split in two and either treated to enrich primary transcripts or used as untreated controls. To enrich primary transcripts with tri-phosphorylated 5′ ends, samples were treated with 1 μL of Terminator Exonuclease (Biosearch Technologies) in 1× Terminator Reaction Buffer A (Biosearch Technologies) supplemented with 0.5 μL SUPERase•In RNase Inhibitor (Thermo Fisher Scientific). Reactions were incubated at 30 °C for 1 hour and stopped by adding EDTA to a final concentration of 5 mM. Samples were purified using the Zymo RNA Clean & Concentrator-5 kit, and then treated with 2 μL RppH (NEB) in 1× NEBuffer 2 supplemented with 1 μL SUPERase•In RNase Inhibitor (Thermo Fisher Scientific). Reactions were incubated at 37 °C for 30 min and purified using the Zymo RNA Clean & Concentrator-5 kit. Illumina sequencing libraries were prepared using the NEBNext Small RNA Library Prep kit.  For Term-seq library preparation, 1 µg of total RNA was treated with 1 µL TURBO DNase in 1× TURBO DNase Buffer (Thermo Fisher Scientific) supplemented with 1 µL SUPERase•In RNase Inhibitor (Thermo Fisher Scientific) for 10 min at 37 °C, followed by cleanup using the Zymo RNA Clean & Concentrator-5 kit. Ligation of an i7 Illumina adapter to RNA 3′ ends was performed using the NEBNext Small RNA Library Prep kit, followed by cleanup using the Zymo RNA Clean & Concentrator-5 kit. Samples were fragmented in 1× NEBuffer 2 by heating at 92 °C for 1.5 min, then treated with 2 μL RppH (NEB) in the presence of 1 μL SUPERase•In RNase Inhibitor (Thermo Fisher Scientific) for 30 min at 37 °C. This was followed by treatment with 1 μL T4 PNK in 1× T4 DNA ligase buffer (NEB) at 37 °C for 30 min and cleanup using the Zymo RNA Clean & Concentrator-5 kit. Illumina library preparation continued with the remainder of the NEBNext Small RNA Library Prep protocol after the initial i7 adapter ligation step.  Libraries were sequenced on an Illumina NextSeq 500 in paired-end mode with 75 cycles per end.