RNA-Seq of ARES: 5'-enriched pool
Cell material from 192 E. coli GeneEE clones were combined. With this pool, a 10 mL overnight pre-culture in LB was inoculated. From this culture, the main culture was inoculated with a starting OD600 nm of 0.2 which was then grown until mid-log phase. For RNA isolation, samples of this culture were harvested by quick centrifugation, removal of the supernatant, flash-freezing in liquid nitrogen, and were stored at 80 C. RNA was isolated and a transcription start site (TSS) library was prepared as described by Pfeifer-Sancar et al (2013) with one modification: Instead of the unspecific loop adapter, the oligo AGACGTGTGCTCTTCCGATCTGACCGTTCACAGAACCTTCC was used for reverse transcription. Sequencing was done in a 75 nt run on Illumina MiSeq sequencing machine.