Illumina HiSeq 2500 sequencing; RNA-seq of human intestinal organoids colonized with E. coli and other immature intestinal tissues
RNA-seq of human intestinal organoids colonized with E. coli and other immature intestinal tissues
Human ES cell line  H9 (NIH registry #0062) was obtained from the WiCell Research Institute. Stem cells were maintained on Matrigel (BD Biosciences, San Jose, CA) in mTeSR1 medium (STEMCELL Technologies, Vancouver, Canada). hESCs were passaged and differentiated into human intestinal organoid tissue as previously described (Spence et al 2011, McCracken et al 2011). HIOs were maintained in ENR media without antibiotics prior to microinjection experiments. Microinjections were performed using a protocol modified from Leslie and Huang et al 2015. Briefly, HIOs were injected using thin wall glass capillaries (TW100F-4, World Precision Instruments, Sarasota FL) shaped using a P-30 micropipette puller (Sutter Instruments, Novato CA). Pulled microcapilaries were mounted on a Xenoworks micropipette holder with analog tubing (BR-MH2 & BR-AT, Sutter Instruments) attached to a 10 ml glass syringe filled with sterile mineral oil (Fisher Scientific, Hampton NH). Fine control of the micropippette was achieved using a micromanipulator (Narishge International Inc., East Meadow NY) and microinjection was completed under 1-2X magnification on an SX61 stereo dissecting scope (Olympus, Tokyo Japan). HIOs suspended in Matrigel (Corning Inc., Corning NY) were injected with approximately 1 ul solution. In bacterial microinjection experiments, the HIO culture media was removed immediately following microinjection and the cultures were rinsed with PBS and treated with ENR media containing penicillin and streptomycin to remove any bacteria introduced to the culture media during the microinjection process. After 1 h at 37C, the media was replaced with fresh anti-biotic free ENR. RNA was isolated using the mirVana RNA isolation kit and following the \"Total RNA\" isolation protocol (Thermo-Fisher Scientific, Waltham MA). RNA library preparation and RNA-sequencing (single-end, 50 bp read length) were performed by the University of Michigan DNA Sequencing Core using the Illumina Hi-Seq 2500 platform.
Experimental Factor: developmental stage
late embryonic stage
Experimental Factor: growth condition
enteroid derived from primary epithelium
Experimental Factor: environmental stress
none
Experimental Factor: infect
none
Experimental Factor: compound
none
Experimental Factor: time
not applicable
Experimental Factor: xenograft
not applicable