GSM7802742: Mesentery, uninfected control of WT H18N11 experiment, bat 2; Artibeus jamaicensis; RNA-Seq
The small and large intestines were cut longitudinally to expose the lumen, intestines were then cut into 0.5 to 1.0 cm pieces and washed four times with 10 mL wash solution consisting of 1×PBS, 5 mM EDTA under vigorous shaking. Subsequently, 20mL of predigestion buffer (Hanks' balanced salt solution (HBSS) containing 20mM HEPES, 5mM EDTA, 5% fetal calf serum (FCS) and 1mM Dithiothreitol (DTT)) were added and incubated at 37°C for 20minutes with constant rotation. After vortexing for 10 sec, the pieces were drained through a 70-µm cell strainer and the supernatant was saved. Cells in the supernatant were washed three times by adding wash solution and subsequent centrifugation at 350g for 5min. The predigestion step was then repeated as described above. After the second round of predigestion, 20mL of HBSS containing 20mM HEPES was added to the intestinal pieces and incubated for 20 min under continuous rotation followed by vigorous vortexing for 10sec. The pieces were sieved through a 70-µm cell strainer, and the supernatant was washed with wash solution as described above. All cell fractions contained epithelial cells and were pooled. Intestinal pieces were resuspended in digestion buffer (HBSS with 20mM HEPES, 1.25mM CaCl2, 1mM MgCl2, 5mM EDTA, 5% FCS, Lamina Propria Dissociation Kit enzymes D, R, and A, were added according to the manufacture's protocol) and incubated at 37°C for 30 min with constant rotation. The intestinal tissue was transferred into a gentleMACS C Tube and processed with a gentleMACS using the manufacturer's program. Perfusion solution was added and the pieces were strained through a 70-µm cell strainer. The supernatant was processed and washed as described above. This second fraction contained the cells of the lamina propria. Intraepithelial and lamina propria fractions of each bat were counted using trypan blue and cells were mixed in a 1:1 ratio for each bat. To prepare a single-cell suspension of the mesentery, the mesentery was tamped through a cell strainer in a Petri dish containing 5 mL of perfusion solution. A total of 12,000 cells per sample were processed with a Chromium Next GEM Single Cell 5' Kit v2 (10x Genomics) to achieve a recovery of at least 8000 cells.