GSM4567123: S. aureus and HUVEC 1hpi, Replicate 2; Staphylococcus aureus; Homo sapiens; RNA-Seq
Pellets were resuspended in 700 μl acetate buffer (20 mM NaOAc, 1 mM EDTA, 0.5% SDS, pH 4.5) and transferred to FastPrep tubes containing lysing matrix B. Cells were lysed in a Thermo Savant FastPrep FP120 Cell Homogenizer at 6.0 m/sec for 3 x 40 sec with 1 min pause on ice in between runs, and then centrifuged for 5 min at 20,000 g. Lysate was transferred to tubes containing 500 μl phenol solution (2:1 phenol in water adjusted to pH 4.5 with NaOAc) and 100 μl chloroform followed by incubation at 65 °C at 1100 rpm in a thermomixer for 15 min with vortexing every 2 min. After centrifugation, the aqueous phase was transferred into tubes containing 500 μl chloroform followed by vortexing, centrifugation, and transferring to new tubes. The RNA was precipitated using 96% EtOH and 10vol% 3M NaOAc on ice for ≥1 hour. Tubes were centrifuged for 60 min at 4°C, and pellets were washed in ice cold 70% EtOH followed by centrifugation for 10 min. Supernatant was removed and pellets were then air-dried and dissolved in sterile ddH2O. RNA quantity was measured using Nanodrop spectrophotometer, and RNA quality was then verified by fragment analysis using a Fragment Analyzer (Agilent), followed by RiboZero Gold (MRZE724, Epidemiology/Illumina) treatment on the 3 samples from each group to remove both human and bacterial rRNA. After DNase treatment, library preparation was performed using the NebNext® UltraTM Library prep kit (NEB) followed by quality assessment by the fragment analyzer
GEO Accession
GSM4567123