title: HS100R_POP1_RNA - Transcriptome of del hns-stpA mutant population of E. coli K12 MG1655 evolved under laboratory conditions in LB at 37C for 100 hour
design: We generated the double mutant of hns and stpA  (Δhns-stpA) in Escherichia coli  K12-MG1655 using  λ-red recombinase method (Datsenko and Wanner). The double mutant was evolved under laboratory condition at 37°C in LB .  Evolved population collected at  different time points such as ~ 100 hours and ~250 hours were collected and frozen.  For RNA extraction the overnight cultures were inoculated in 100 mL of fresh LB to bring the initial OD of the fresh culture to 0.03 and the flasks were incubated at 37°C with shaking at 200 rpm. Two biological replicates were performed for each sample. Samples were collected at the mid-exponential phase of growth. Slight modifications of previously published protocols, based on those recommended by the manufacturer for the TRIzol (Invitrogen) bacterial RNA isolation kit, were used. Briefly, cells were pelleted down at 8000 rpm for 10 minutes at 4°C. Pellets were washed with autoclaved DEPC-treated RNAse- free water and cells were snap-frozen in liquid nitrogen and stored at -80 ̊ C until required. Cells were homogenized with sterile pestle followed by RNA extraction using TRIzol reagent according to manufacturer’s instruction until the 70% ethanol wash step. Total RNA was treated with DNase I (Invitrogen Cat No. 18068-015) according to the manufacturer’s instruction. Further precipitation of RNA and ribosomal RNA cleanup was achieved by MICROBExpress bacterial mRNA purification Kit. Total RNA was suspended in 10μL of RNAse free water (Ambions, Cat No. AM9932). Concentration and the quality of the RNA were determined on a Nanodrop and by visualization under agarose gel respectively. Sequencing libraries were prepared using TruSeq RNA sample preparation kit v2 (Illumina, Catalog No. RS-122-2001) according to the manufacturer’s guidelines. Prepared libraries were checked for quality on an Agilent Bioanalyzer, and sequenced for 50 cycles – from one end – on an Illumina HiSeq1000 platform.