GSM7721010: Wt_r1_DNA_gel; Escherichia coli K-12; OTHER
Gel-extracted DNA corresponding to the polyP dependent, Hfq-associated high molecular weight fraction on native gels was recovered by crushing the gel slice with a pipette tip, adding 200 microliters of PAGE elution buffer (0.5 M ammonium chloride, 0.03 M sodium acetate, 5 mM EDTA, 0.1% Tween20) and incubating for 6 hours at 37 C with shaking. At the end of the incubation, the sample was centrifuged 1 minute at 16,100xg at 4 C, and then an additional 100 microliters of PAGE elution buffer was added and vortexed, and the supernatant combined with the additional 200 microliter eluate. 600 microliters of ice cold ethanol were added to each sample, followed by overnight incubation at -80 C to precipitate the DNA.   After precipitation, the DNA was recovered by spinning 10 minutes at 16,100xg at 4 C, washing the pellet with 1 mL 95% ethanol, re-spinning, and then removing all ethanol from the pellet and allowing it to dry. The pellet was then resuspended in 200 microliters TE (pH 7.5). Half of the recovered material was treated with RNase A (final concentration 0.25 mg/mL) and purified using a Zymo DNA C&C kit according to the manufacturer's instructions. The recovered DNA was prepared for sequencing using a NEBNext Ultra 2 DNA sequencing preparation kit with UMI adapters