B. vulgatus in vivo mono-culture repilcate 5
Upon sacrafice, mouse ceca were suspended in 5 mL of RNAlater (Ambion) and vortexed vigorously. The suspension was centrifuged briefly (15s) at 500 x g to remove debris. The remaining suspended bacteria and small particulate material was pelleted (15 minutes, 10,000 x g) and stored at -20C with fresh RNAlater. Pellets were resuspended and lysed in 2 mL of denaturing buffer (4 M guanidium thiocyanate, 25 mM sodium citrate, 0.5% N-laurylsarcosine, 1% N-acetyl cysteine, 0.1 M 2-mercaptoethanol), divided into 500 ?L aliquots and 4 ?L of 1 M sodium acetate (pH 5.2) added to each aliquot. An equal volume of buffer saturated phenol (pH 4.3) was added to each sample, and incubated with frequent mixting at 64C for 10 minutes. The phenol was separated from the solution by the addition of 1 mL of chloroform, followed by a 15 minutes incubation on ice, and a 18,000 x g centrifugation at 4C for 30 minutes. RNA was precipitated from the aqueous layer with 1/10 volume 3 M sodium acetate, 1.2 ?L of 500 mM DEPC-treated EDTA and 2 volumes of cold ethanol with incubation at -80C overnight. The RNA was pelleted at 4C by centrifugation, washed 4 times with 80% ethanol and resuspended in 100 ?L of RNAse-free dH2O.