GSM5550326: E. coli Δ10 - targeting - rep 2; Escherichia coli; RNA-Seq
Total RNA was isolated from cells pelleted at 1 hour post RFP induction. Cell lysis was done using Max Bacterial Enhancement Reagent (Invitrogen) for 4 min and then with TRIzol reagent (Invitrogen) for 5 min. RNA was extracted by chloroform and precipitated with isopropanol. RNA pellets were washed with 70% ethanol and then dissolved in nuclease free water and then treated with Turbo DNA-free kit (Invitrogen). Total RNA samples were treated with MICROBExpress Bacterial mRNA Enrichment Kit (Invitrogen) for rRNA depletion prior to library preparation. To obtain both primary (5' PPP) and processed (5' P/5' OH) transcripts, RNA samples were treated with RNA 5' Pyrophosphohydrolase (RppH) (NEB) for 30 min at 37C which removes pyrophosphate from 5' end from triphosphorylated RNA to leave a 5' monophoshphate RNA. Fragmentation was carried out by sonication using the Covaris protocol to obtain fragments of 200 nt size. T4 PNK (NEB) treatment was done to obtain 5'P ends for adapter ligation during library preparation. Samples were purified using the Zymo Research Oligo Clean and Concentrator kit. Library preparation for RNA sequencing was done using the NEBNext Multiplex Small RNA Library Prep Set for Illumina according to manufacturer's protocol. BluePippin size selection was done using 2% agarose gel cassette (Sage Science) to select for 100 - 600 bp products. QC at each step was carried out by both Qubit and fragment analyzer. RNA sequencing was performed using Illumina NextSeq High-Output kit 2 × 35 bp paired end read length.