GSM8070194: USA300 LAC* cells, WT/m⏀11 biological replicate A; Staphylococcus aureus; RNA-Seq
For RNA extraction, cells were isolated with centrifugation and resuspended in 1 mL Trizol (Invitrogen, #15596026), then transferred to lysing matrix B (MP Biomedicals, #116911050) tubes for mechanical disruption at 6 m/s for 30 seconds three times. After disruption, samples were centrifuged for 10 minutes at 12,000 rpm at 4°C and the upper phase was transferred into a new RNA-free tube containing 500 µL ice cold Trizol, gently mixed, and incubated for 5 minutes at room temperature, then 200 µL chloroform was added and samples were centrifuged at 12000 rpm for 15 minutes at 4°C. The aqueous phase was mixed with 500 µL isopropanol and transferred to RNeasy column (Qiagen #74004) for washing and RNA elution per manufacturer protocol. Extracted total RNA was visualized on the Agilent 2100 Bioanalyzer system using a Bioanalyzer Nanochip run with the Prokaryote setting. RIN scores for the total RNA measured from 9 to 10. Total RNA (500 ng per sample) was input into the Illumina stranded Total RNA Prep, Ligation with Ribo-Zero Plus kit (96rxn, cat # 20040529). Libraries were prepared according to manufacturer's instructions. PCR Amplification was run with 11 total cycles. Following completion of the preparation, library samples were visualized on the Agilent 4200 Tapestation System using High Sensitivity DNA Screentape. The concentration of each library was assessed by Qubit using the High Sensitivity DNA Kit. The samples were pooled equimolar and the pool was sequenced on the Illumina Novaseq 6000 system on one lane of the SP 100 cycle flow cell kit. The library pool was sequenced as paired end 50 bases.