high throughput sequence alignment protocol
High throughput sequence alignment protocol was based on the TopHat/Cufflinks pipeline. First, sequences were aligned to the reference genome (Hg19) with TopHat.
normalization data transformation protocol
Each sample was independently processed with Cufflinks in order to generate an initial transcriptome. We used the Cuffmerge tool to merge the private transcriptomes into a single reference, and at the same time annotated known genes and extended partial transcripts. This common transcriptome was used in a second pass with Cufflinks, which quantified each transcript and gene (known or novel) in each sample. The reference annotation used was based on the UCSC knownGenes table.
nucleic acid sequencing protocol
Completed libraries were sequenced on an Illumina HiSeq2000, generating around 20 million high quality 50 base long reads per sample.
Prior RNA extraction, total tissue collected in RLT Plus buffer were lysed using a FastPrep machine.
nucleic acid extraction protocol
Total RNA was extracted using an RNeasy Plus Micro Kit (Qiagen) according to manufacturer guidelines.
sample collection protocol
Total tissue was collected in RLT Plus buffer (Quiagen) supplemented with 0.1% 2-Mercaptoethanol in FastPrep lysing Matrix D tubes.
nucleic acid library construction protocol
The libraries were constructed according to manufacturer guidelines. (Illumina). Briefly, 350 to 900 ng of total RNA determined by Qbit high sensitivity spectrofluorometric measurement (Invitrogen) was poly-A selected and reverse transcribed using Illuminas TruSeq RNA library preparation kit V2. Each sample was fitted with a unique adapter containing a 6 base molecular barcode for high level multiplexing. Prior sequencing, a 12 cycles of PCR amplification was performed on the samples.
Human Intestinal Organoids (HIOs) were generated and maintained as previously described in Spence, J.R., Mayhew, C.N., Rankin, S.A., Kuhar, M.F., Vallance, J.E., Tolle, K., Hoskins, E.E., Kalinichenko, V.V., Wells, S.I., Zorn, A.M., et al. (2011). Directed differentiation of human pluripotent stem cells into intestinal tissue in vitro. Nature 470, 105-109. Briefly, line H1 embryonic stem cells (WiCell Research Institute, Inc.) were grown in feeder-free conditions in Matrigel (BD Biosciences) coated six-well Nunclon surface plates (Nunc) and maintained in mTESR1 media (Stem Cell Technologies). For induction of definitive endoderm (DE), cells were passaged with Accutase (Stem Cell Technologies) and plated at a density of 65,000 cells per well in 24-well Nunc plates. Cells were allowed to grow in mTESR1 media for two days before treatment with 100 ng/ml of Activin A for three days as previously described. DE was then treated with hindgut induction medium (RPMI 1640, 100x NEAA, 2% dFCS,) for four days with 100 ng/ml FGF4 (R&D) and 3 M Chiron 99021 (Tocris) to induce formation of mid-hindgut spheroids. Spheroids were then plated in Growth Factor Reduced (GFR) Matrigel and maintained in intestinal growth medium (Advanced DMEM/F-12, N2 supplement, B27 supplement, 15 mM HEPES, 2 mM L-glutamine, penicillin-streptomycin) supplemented with 100 ng/ml EGF (R&D) to generate human intestinal organoids (HIOs). Media was changed twice weekly thereafter. HIOs were replated in fresh Matrigel every 14 days. HIOs were prepared for transplantation as previously described in Watson, C.L., Mahe, M.M., Munera, J., Howell, J.C., Sundaram, N., Poling, H.M., Schweitzer, J.I., Vallance, J.E., Mayhew, C.N., Sun, Y., et al. (2014). An in vivo model of human small intestine using pluripotent stem cells. Nat Med 20, 1310-1314. Briefly, single matrigel embedded HIOs were transplanted into the mesentery of the mice. Mice were anesthetized with 2% inhaled isoflurane (Butler Schein), and the abdomen of the mouse was then shaved, and prepped in sterile fashion using isopropyl alcohol and povidine-iodine. A midline incision of approximately 2 cm was made in order for the intestine to be accessed. A small pocket was created in the mesentery and the HIO placed within. The skin was closed in a double layer and the mice were given a subcutaneous injection of Buprenex (0.05 mg/kg; Midwest Veterinary Supply) for pain management. Ten to twelve weeks following engraftment, the mice then underwent a secondary surgery with similar preparations. During this procedure, the engrafted tHIO was cut and briefly flushed before insertion of the gelatin capsule within the lumen of the tHIO. Mice were sacrificed and tissue harvested 14d postoperatively.