Impact of Spore Forming (SF) Community of bacteria on the small intestine

To investigate the impact transcription that the Spore Forming (SF) Community of bacteria have on the small intestine, B6 mice on a autoclaved diet that were germfree (GF) or colonized with the SF community were investigated. Using laser capture microdissection the intestinal epithelial cells on the villi and the crypts were dissected out and bulk RNA seq was performed. The duodenum of the small intestines from male B6 mice with different mictobiotas (GF, and SF) were dissected out, cleaned, and flushed with OCT and placed in cryo mold, and embed in OCT. Frozen sections 9 um thick were cut from the blocks and mounted on PEN slides. Working one slide at a time, slides were taken out of the freezer, fixed with 70% EtOH, stained with Methyl Green and Eosin Y, dehydrated in xylene, and then within 1 hour of being taken out of the freezer, villi intestinal epithelial cells were dissected out using a Laser Capture Microdissection scope. After tissue was collected 350uL of Qiagen buffer RLT+ beta mercaptone ethonal was added and incubated at room temperature for 30 min for lysis before being stored in the -80C. RNA was extracted with the RNeasy micro kit from Qiagen with on column DNase I digest and eluted in 14uL RNAse free H2O. Samples have been stored at -80C.