GSM4278254: MqsR_10_minute_expression; Escherichia coli; RNA-Seq
RNA was extracted using Trizol (Invitrogen) and Direct-zol RNA MiniPrep kit (Zymo). Briefly, samples with rRNA subtraction with treated with bacterial Ribo-Zero kit (Illumina) or comparable homebrew kit. Next, RNA was fragmented with RNA fragmentation reagents (Ambion). First strand cDNA synthesis was conducted using random primers and Superscript III (Invitrogen). To enable strand specificity, second strand synthesis was conducted using dUTP instead of dTTP with RNase H, E. coli DNA ligase, and E. coli DNA polymerase. Ends were repaired with T4 DNA polymerase, Klenow DNA polymerase, and T4 PNK. 3' ends were adenylated with the Klenow fragment (3'>5' exo-). Y-shaped adapters were ligated and 9-12 cycles of PCR were conducted with standard Illumina primers with multiplexing indexes. Libraries were extracted from an acrylamide gel and submitted for sequencing at the MIT BioMicro Center using standard Illumina protocols.
GEO
GSM4278254
GEO Accession
GSM4278254