title: GSM1477268: LB_WT; Listeria monocytogenes EGD-e; RNA-Seq
library_construction_protocol: 10µg of total RNA was ribodepleted and non-directional libraries were constructed using a random hexamer priming approach fully described in the manuscript For all RNA isolations, 25 ml cultures of L. monocytogenes were grown to an OD600 of 0.3 – 0.5 and subsequently pelleted by centrifugation at 4000 rpm for 20 mins in 50 ml Falcon tubes with swing bucket rotor. Pellets were resuspended in 1ml Tri Reagent (Sigma, 93289), transferred to 2 ml Lysing Matrix Tubes (MP Biomedicals, 6911) and mechanically lysed by bead beating in a FastPrep apparatus (45 secs, speed 6.5 followed by an additional 30 secs, speed 6.5). Subsequently tubes were centrifuged for 5 min at 8000 X G, 4° C in a tabletop centrifuge to separate beads from lysates. Lysates were drawn off and transferred to 2 ml Eppendorf tubes. RNA isolation proceeded according to the manufacturers instructions. Briefly, 200 μl Chloroform (Carlo Erba Reagents, 438601) was added to the lysate, shaken and incubated for 10 minutes at room temperature followed by centrifugation for 15 min at 13,000 X G, 4° C. The upper aqueous phase was removed and transferred to a new 1.5 ml Eppendorf tube and RNA was precipitated by the addition of 500 μl Isopropanol and incubation at room temperature for 5-10 mins. RNA was pelleted by centrifuging for 10 mins at 13,000 X G, 4° C. The supernatant was discarded and the pellet was washed twice with 75% ethanol. RNA pellets were resuspended in 50 μl water.