GSM4746332: Stationary rep1; Escherichia coli; RNA-Seq
Samples were treated with Tween (0.2%) and chloramphenicol (5 µg/ml) then immediately filtered out of solution and resuspended in lysis buffer containing 25 mM Tris pH 8.0, 25 mM NH4Cl, 10 mM MgOAc, 0.8 % Triton X-100, 0.1 U/µL RNase-free DNase I, 1 U/μL. Then immediately frozen in liquid nitrogen and stored at -80 C.  Frozen pellets were thawed on ice and centrifuged for 20 minutes at 16,000 x g at 4 °C with 0.3% Sodium Deoxychorate to pellet particulate matter. The supernatant was then removed and RNA was column purified (Qiagen miRNeasy cat. No. 217004) before removing rRNA (Invitrogen RiboMinus cat no. K155004) Superase-In, ~60 U/µl ReadyLyse Lysozyme, 1.55 mM Chloramphenicol, and 17 μΜ 5′-guanylyl imidodiphosphate (GMPPNP). cDNA libraries were prepared and multiplexed according to manufacturer's protocols: NEBNext Ultra II cat no. E7103S. Libraries were then pooled and sequenced by Illumina HiSeq paired-end sequencing.