Illumina HiSeq 2500 sequencing; RNA-seq of human epithelium-only colonic and small intestinal organoids derived from intestinal resections, biopsy specimens and cryopreserved crypts
RNA-seq of human epithelium-only colonic and small intestinal organoids derived from intestinal resections, biopsy specimens and cryopreserved crypts
Normal gastrointestinal tissues were collected from deceased donors, or from biopsy specimens, and was approved by the University of Michigan Medical School Institutional Review Board (IRB). Small and large intestinal epithelium organoid cultures were established and maintained as according to Dame et al. (Dame, MK et al. Development. 2018: 1-16.), and Tsai et al. (2Tsai, YH, Czerwinski, M, et al. CMGH. 2018: 218-222). To establish organoids, the mucosa was separated from resected surgical tissue and incubated with 10 mM dithiothreitol for 15 minutes (Sigma-Aldrich) prior to cold incubation with 8 mM ethylenediaminetetraacetic acid (Sigma-Aldrich) for 75 minutes. Pure crypts were detached from the lamina propria by snap-shaking, washed three times in cold DPBS and cryopreserved in growth medium containing 10% FBS and 10% DMSO. Cryopreserved crypts were later seeded into 8 mg/mL Matrigel (Corning) and expanded. The growth medium consisted of 50% L-WRN conditioned medium (as described in Miyoshi and Stappenbeck (Nature Protocols. 2013: 2471-2482)) and 50% basal medium composed of advanced Dulbecco's modified Eagle Medium/F12, 2 mM GlutaMAX, 10 mM HEPES, 1x N2 supplement, 1x B27 supplement without retinyl acetate, 1 mM N-Acetyl-L-Cysteine, 10 µM Y27632, 100 ng/mL EGF, 500 nM A83-01, and 10 µM SB202190, 50 units/mL penicillin/streptomycin, and 100 µg/mL Primocin. Cultures were supplemented with 2.5 µM CHIR99021 for the first ten days of establishment. 10 mM Nicotinamide was used at the initial establishment of the specimens in enteroid culture, and then removed for continued expansion. Medium changes occurred daily. Cultures were split every 5-7 days using mechanical dissociation from previous Matrigel by a 1 mL pipet, followed by re-plating in fresh Matrigel in 10 µL droplets. CHIR99021 was included for 24 hours following each passage. All plasticware, excluding culture plates, were coated in 0.1% bovine serum albumen in DPBS (Sigma; A8806) to prevent tissue/organoids/cells from adhering to surfaces. All lines were confirmed to be negative for mycoplasma and were tested for the presence of viruses (CLEAR with Infectious Disease PCR Panel, Charles River Laboratories). Lines were authenticated to match source tissues by short tandem repeat analysis at the University of Michigan DNA Sequencing Core. Small and large intestinal epithelium organoids were grown in normal growth media prior to sequencing. Organoids were isolated from Matrigel droplets by mechanical disruption of the Matrigel with a 200 μL pipet tip in cold DPBS, rotated for 15 minutes at 4°C in 2 mM EDTA, and washed three times in cold DPBS with 3-minute centrifugations at 250xg, 4°C. RNA isolation was conducted using the RNeasy Micro Kit (Qiagen 74004) with on-column DNase digestion as described by the manufacturer. Duplicate organoid samples (exception Prep_duo-89; n=1) were prepared for RNA isolations for each cell line, designated -1 and -2. Libraries were created with Takara SMARTer Stranded Total RNA Sample Prep Kits (634876, Takara Bio) as described by the manufacturer.