title: GSM8948093: Wild type Haemophilus influenzae RdkW20 in anaerobiosis replica 1; Haemophilus influenzae; RNA-Seq
library_construction_protocol: l  RNA for sequencing was isolated from bacteria grown in sBHI to OD600=0.3. H. influenzae was grown for 12 h on PVX agar. Two to five colonies were inoculated into 10 mL sBHI, grown for 12 h at 90 r.p.m., diluted into 20 mL fresh sBHI to OD600=0.05, and grown to OD600=0.3 at 180 r.p.m. Next, 7 mL bacterial cultures were recovered, pelleted (4,000 r.p.m, 4 min), flash frozen, and stored at -80ºC. Bacterial RNA was isolated using NucleoSpin RNA kit (Macherey-Nagel). Alternatively, bacterial suspensions collected from PVX agar anaerobic growth were normalized to OD600=0.4 in sBHI, 5 mL aliquots were transferred to 50 mL-Falcon tubes with 20 mL sBHI and grown to OD600=0.3 also in static anaerobiosis. Next, 25 mL bacterial cultures were recovered, pelleted (4,000 r.p.m, 4 min), flash frozen, and stored at -80ºC. Total RNA was isolated using TRIzol reagent (Invitrogen). In both cases, to prevent any DNA interference, a second DNase digestion was performed using RNase-free DNase and cleaning on a RNeasy Mini column (Qiagen). Purified RNA was quantified on a Nanodrop OneC (Thermofisher Scientific), libraries were checked for quality control with RNA 6000 Nano LabChips (Agilent 2,100 Bioanalyzer), and sequenced by Admera Health on an Illumina HiSeq platform with 2×150 bp reads, estimated 20M PE reads per sample, 10M in each direction. Three replicates per sample type were sequenced.