Bacterial cell culture, recovery, and lysis

We used the following clones for generating RNA-seq and Ribo-seq datasets: Ara-1 – 11330, Ara+1 – 11392, Ara-2 – 11333, Ara+2 – 11342, Ara-3 – 11364, Ara+3 – 11345, Ara-4 – 11336, Ara+4 – 11348, Ara-5 – 11339, Ara+5 – 11367, Ara-6 – 11389, Ara+6 – 11370. Bacteria were cultured in medium as per the recipe on the LTEE website (http://myxo.css.msu.edu/ecoli/dm25liquid.html) supplemented with 4 g/L glucose instead of the typical 25 mg/L. Each culture was grown in 50 mL in a shaking incubator at 37°C at 125 rpm until an OD600 of 0.4–0.5 was reached. This took between 1.5 and 4 hr, depending on the line. Cells were recovered via vacuum filtration and immediately frozen in liquid nitrogen (LN2). Frozen pellets were stored at –80°C until lysis. A mortar and pestle were chilled to cryogenic temperatures with LN2 for lysis. The pellet was ground to a powder while submerged in LN2. Once pulverized, 650 µL of lysis buffer was added to each sample and ground further. Lysis buffer contained the following: 20 mM Tris pH 8, 10 mM MgCl₂, 100 mM NH₄Cl, 5 mM CaCl₂, 1 mM chloramphenicol, 0.1% v/v sodium deoxycholate, 0.4% v/v Triton X-100, 100 U/mL DNase I, 1 µL/mL SUPERase-In (Thermo Fisher Scientific AM2694). The frozen lysate was allowed to thaw until liquid, then incubated for 10 min on ice to allow complete lysis. Afterward, the lysate was centrifuged at 20,000× g for 10 min at 4°C, and the supernatant recovered and transferred to a new tube. Each sample was split into two for RNA-seq and Ribo-seq libraries.