GSM1611962: CSP_15_II; Streptococcus mutans UA159; RNA-Seq
Pelleted bacteria were washed with 500 µl nuclease free water and centrifuged for 2 min at 13.000 rpm. The supernatant was removed and the pellet resuspended in LM-lysis buffer containing 2.5 mg lysozyme and 100 U mutanolysin (pH=7.4). The resuspension was shaken in a heating block at 25°C and 1400 rpm for 45 min. 700 µl of Qiazol Lysis solution (Qiagen, Germany) was added and the mixture transferred to a falcon tube containing sterile 0.1 µm glass beads (Carl Roth, Germany). The falcon tubes were vortexed for 3 min and centrifuged at 6000 rpm at RT to pellet the glass beads. The supernatant was transferred to a new tube and 200 µl of chloroform was added. Samples were vortexed for 15 sec, incubated for 2 min at RT and centrifuged for 20 min at 13.000 rpm and 4°C. The upper aqueous phase (approximately 550 µl) was transferred to a new tube and 870 µl of ethanol was added and mixed thoroughly. RNA extraction was carried out using the MiRNeasy Kit (Qiagen, Germany) according to the manufacturer’s instructions. To remove genomic DNA the optional on-column DNaseI digestion using the DNase I Kit (Qiagen, Germany) was performed for 45 min. After the washing steps the RNA was eluted in 50 µl of nuclease free water supplied with the kit. To test the integrity of the isolated total RNA and the enriched mRNA, samples were analyzed using the Agilent 2100 Bioanalyzer and the RNA 6000 Pico Kit (Agilent, Germany). The Illumina ScriptSeq™ v2 RNA-Seq Library Preparation Kit was used according to the manufacturers protocol
GEO Accession
GSM1611962