Illumina HiSeq 2500 paired end sequencing; Sphingosine survival response of Staphylococcus aureus and Staphylococcus epidermidis
Sphingosine survival response of Staphylococcus aureus and Staphylococcus epidermidis
bacterial cultures grown to OD600 0.5, treated with 5um D-sphingosine or equivalent ethanol (lipid carrier) for 20 min, cells harvested by centrifugation and stored in RNAlater to stabilise RNA until RNA extraction bacteria grown in 50 ml THB in 250 ml flasks at 37 degrees C with shaking at 250 RPM  5um D-sphingosine or equivalent ethanol (lipid carrier) for 20 min immediately prior to cell harvesting pelleted cells lysed with lysostaphin, mutanolysin and proteinase K, RNA extracted using RNeasy kit (Qiagen) DNase treated then cleaned with RNeasy MinElute clean up kit (Qiagen) depletion of rRNA with Ribo-zero for Gram positive bacteria, RNA concentration normalised then strand specific ScriptSeq kit (Epicentre) used to generate libraries
Experimental Factor: compound
D-sphingosine
Experimental Factor: dose
5
Experimental Factor: strain
Newman