GSM1908518: 20B; Lactococcus garvieae; RNA-Seq
Frozen cell pellets obtained from cultures at 9 h were suspended in 500 µL of cold Tris-EDTA buffer. Then, 25 µL of 20% SDS, 500 µL of phenol at pH 4, 3.5 µL of β-mercaptoethanol, and 600 mg of Zirconium beads were added. The solution was milled in a Retsch MM200 mixer mill (VERDER Group, Haan, Germany) at 30 hertz for two 2-minute runs with 2-minute cooling on ice in between. Next, 200 µL of chloroform were added. The solution was gently hand-mixed and centrifuged at 13,000 x g for 20 min at 4°C. Extraction of RNAs was then performed on the aqueous and translucent phase using NucleoSpin RNA-L or RNA-II kit (Macherey-Nagel, GmbH & Co. KG, Düren, Germany) following the suppliers instructions. Then we treated RNA extracts with rDNAse I using an Ambion DNA-free™ Kit (Ambion, Inc., Austin, Texas, USA) following the suppliers instructions. Absence of residual DNA was confirmed by qPCR with S. aureus gyrB gene primers. If needed, rDNAse I treatment was repeated until Cycle threshold (Ct) were > 30. The RNA extracts were quantified using a Nanodrop™ 2000C (Thermo Fisher Scientific Inc., Waltham, Massachusetts, USA). Ribosomal RNAs were depleted from the total RNA (2 x 5 µg of RNA by sample) using a Magnetic Kit RiboZero for Gram Positive Bacteria (Illumina Inc., San Diego, California, USA) according to the manufacturer's instructions. The quality and concentration of RNA in each sample were assessed using a RNA 6000 pico kit (Agilent Technologies, Santa Clara, California, USA). RNA libraries were prepared by the MGX Plateform (Montpellier GenomiX, CNRS, Montpellier, France) using Illumina kit (TruSeq Stranded mRNA Sample Preparation kit), devices and standard protocols (Illumina Inc.)