GSM9320897: Escherichia coli MG1655, wild-type with pTOPO_CMY145, biol rep2; Escherichia coli str. K-12 substr. MG1655; RNA-Seq
RNA extraction was performed using the zymo kit for quick RNA extraction. DNase treatment was carried out using the Turbo DNase kit from Invitrogen. After quality checking with a TapeStation (Agilent), RNA samples were sent to Genewiz. rRNA depletion was performed using NEBNext rRNA Depletion Kit (Bacteria). RNA sequencing library preparation was performed using NEBNext Ultra II Directional RNA Library Prep Kit for Illumina by following the manufacturer's recommendations (NEB, Ipswich, MA, USA). Briefly, enriched RNAs were fragmented for 15 minutes at 94 °C. First strand and second strand cDNAs were subsequently synthesized. The second strand of cDNA was marked by incorporating dUTP during the synthesis. cDNA fragments were end repaired and adenylated at 3'ends, and universal adapters were ligated to cDNA fragments, followed by index addition and library enrichment by limited-cycle PCR. The incorporated dUTP in second strand cDNA quenched the amplification of second strand, which helped to preserve the strand specificity. Sequencing libraries were validated using NGS Kit on the Agilent 5300 Fragment Analyzer (Agilent Technologies, Palo Alto, CA, USA), and quantified by using Qubit 4.0 Fluorometer (Invitrogen, Carlsbad, CA).