NTHi375 was grown for 12 h on PVX agar. Two to five colonies were inoculated into 10 mL CDM-2, in the absence or presence of Trp and/or Ser, grown for 12 h at 100 r.p.m., diluted into 20 mL fresh CDM-2 to OD600=0.07, and grown to OD600=0.6 at 200 r.p.m. Next, 7 mL bacterial cultures were recovered, pelleted (4,000 r.p.m, 4 min), flash frozen, and stored at -80C. Bacterial RNA was isolated using NucleoSpin RNA kit (Macherey-Nagel) as specified by the manufacturers instructions. Briefly, 5x109 CFU (3 mL cultures) were pelleted by centrifugation at 14,000 r.p.m. for 5 min, resuspended in 100 L TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8) containing 1 mg/mL lysozyme by vigorous vortexing, and incubated at 37C for 10 min. Cells were lysed with 350 L Buffer RA1 and 3.5 L -mercaptoethanol. Lysates were filtered through NucleoSpin Filter units to reduce viscosity and mixed with 350 L 70% ethanol. RNA was applied to NucleoSpin RNA columns, salt was removed using membrane desalting buffer (MDB), one on-column rDNase treatment step was included, samples were cleaned with RAW2 and RA3 buffers, and RNA was elutedRNA quality was evaluated using RNA 6000 Nano LabChips (Agilent 2100 Bioanalyzer, Santa Clara, CA) and sequenced on an Illumina HiSeq platform with 2  150 bp reads by Admera Health. Sixty million 2  150 bp reads were generated for the three replicates of each sample type