0.22 m centrifugal filters

Bands containing inverse toeprints corresponding to stalled ribosomes from the initiation codon to the last NNN codon were excised from the gel with a clean scalpel. Gel pieces were crushed through a 5 ml syringe into 15 ml Falcon tubes and 10 ml of gel elution buffer (10 mM Tris-HCl, pH 8.0, 500 mM Na-Acetate, and 0.5 mM Na-EDTA) were added. The tubes were incubated on a tube rotator at room temperature overnight. Gel debris were separated from the extraction solution by filtering through 0.22 µm centrifugal filters (Millipore). Each sample was then concentrated to ~1 ml using a SpeedVac. DNA was precipitated in 5 ml Eppendorf tubes using 1 ml of isopropanol with 3.7 µl GlycoBlue (Thermo Fisher Scientific Scientific) and incubating at -80°C overnight. After precipitation, DNA was recovered by centrifugation in a ThermoScientific Heraeus Multifuge X3R centrifuge at 20,000xg for 30 min at 4°C using a Fiberlite F15-8x50cy rotor (ThermoScientific). The supernatant was removed, and DNA pellets were resuspended in 20 µl H 2 O (molecular biology grade, Milipore) for subsequent addition of the next-generation sequencing (NGS) adaptors.