GSM4237306: Ecoli_chloramphenicol_diy_depletion; Escherichia coli; RNA-Seq
RNA was extracted using Trizol (Invitrogen) and Direct-zol RNA MiniPrep kit (Zymo). See publication for complete protocol. Briefly, samples with rRNA subtraction with treated with either a custom ribosome depletion technique or the Ribo-Zero kit (Illumina). 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. Amplified libraries were cleaned up with AMPure XP beads and submitted for sequencing at the MIT BioMicro Center using standard Illumina protocols.