GSM9285290: dliaFSR, untreated, replicate 3; Enterococcus faecalis; RNA-Seq
For RNA extraction, cultures were harvested and treated with RNAprotect (Qiagen, USA) to stabilize RNA. Then, cells were lysed with 20 mg/mL lysozyme (Sigma-Aldrich, USA) in lysis buffer (10 mM Tris-HCl at pH 8, 50 mM NaCl, 1 mM EDTA, 0.75 M Sucrose) at 37°C for 1 h. Following centrifugation, the pellet was resuspended in 1 mL of TRIzol and 200 µL of chloroform, and incubated at 4°C for 2 min. After centrifuging at 12,000g at 4°C for 15 min, the top aqueous phase was mixed thoroughly with an equal volume of 80% ethanol. Total RNA was then isolated using the RNeasy Mini Kit (Qiagen, USA) according to the manufacturer's recommendations. DNA was depleted via on-column DNase I treatment (Qiagen, USA). RNA quality (RINe ≥8) was assessed using the RNA ScreenTape system (Agilent Technologies, USA), while DNA contamination (≤10%) was quantified using the Qubit RNA BR and dsDNA HS Assay kits (Invitrogen, USA). cDNA library preparation and ribosomal RNA depletion was performed by the in-house sequencing facility using the Illumina Total RNA Prep with Ribo-Zero Plus kit (Illumina, USA).