Antisense transcription and its roles in response to environmental changes in E. coli K12

Antisense RNAs (cis-asRNAs) in prokaryotes are more pervasive than originally thought. However, little is known about their expression patterns and functions. Here we determined transcriptomes and proteomes of E. coli at multiple time points in five culture conditions using directional RNA-seq and tandem mass spectrometry. Overall design: A frozen stock of Escherichia coli K12 strain MG1655 was thawed, inoculated in LB medium in a test tube by 1:100 dilution and cultured overnight at 37°C and 250 rpm. The cells were then transferred to fresh LB medium in a flask by 1:100 dilutions, and cultured at 37°C and 250 rpm. When the cells grew to an optical density at 600 nm (OD600) of 1.0, they were spun down at 3,200g for 25 min. For the minimal medium MOPS culture (MOPS), the cell pellets were resuspended in the same volume of MOPS medium (100ml of 10X MOPS mixture, 880ml of sterile H2O, 10ml (0.132M) KH2PO4 and 10ml of 20% glucose, Teknova, Hollister, CA). For heat shock treatment (HS), the cell pellets were resuspended in the same volume of MOPS medium, and incubated at 48°C and 250 rpm. For phosphorus-starvation treatment (M-P), the cell pellets were resuspended in the MOPS medium without KH2PO4. For carbon-starvation treatment (M-C), the cell pellets were resuspended in the MOPS medium without glucose. Each culture was done in three replicates in parallel, and an equal volume of cells was collected from each replicate to minimize experimental variations during sampling. Three milliliters of such pooled cell suspension were collected in a test tube containing 1.5ml RNA Later (Invitrogen) immediately after the cell pellets were resuspended in the indicated medium (0 min) and at the indicated time points thereafter (HS: 15min, 30min, 1h, 2hrs and 4hrs; MOPS, M-C and M-P: 1hr, 2hrs, 4hrs, 6hrs; M-C: 1hr, 2hrs, 4hrs, 6hrs). For the LB culture, samples were collected when the cell suspension OD600 reached to 0.5, 1.0 and 3. Cells were spun down at 6,000g for 8 mins (-4°C), and the pellets were resuspended in 1.5 ml of RNAlater. The samples were stored at -80°C until use.