GSM8862419: Duodenal epithelium; Control; H. polygyrus (7 d.p.i.); Mus musculus; RNA-Seq
To generate muscularis single-cell suspensions, the distal 12 centimeters of small intestines were dissected on ice, cut longitudinally along the mesenteric border, and laid flat (luminal side down) on C-fold towels dampened with ice-cold holding buffer (HBSS without Ca2+ and Mg2+, 5% FCS, 10mM HEPES) on a metal tray in an ice bucket. Muscularis propria were isolated from lamina propria by stereomicroscope-aided microdissection performed on an ice-cold metal block lined with buffer-dampened C-fold towels, rinsed 2-3x in holding buffer to limit epithelial cell contaminants, minced with scissors, and digested in digestion buffer (HBSS with Ca2+ and Mg2+, 5% FCS, 10mM HEPES, 100 μg/mL DNase I, 200 μg/mL Liberase TH) for 15-30 minutes at 37°C in flasks with stir-bars spinning at 550 rpm. After digestion, samples were gently triturated with 3-5 passes through an 18G needle attached to a syringe and single-cell suspensions were strained through 40μm nylon meshes, washed, and further processed for fluorescence-activated cell sorting (FACS). To enrich for muscularis glial and stromal cells, our input consisted of 50% single, live, CD45neg, Itgβ1+ cells and 50% all single, live cells, purified by FACS. Biological replicates (n = 2; 1 female, 1 male) from each respective group were pooled into the same 10x Genomics channel for single-cell encapsulation. To generate duodenal crypt epithelium single-cell suspensions, the proximal 10 centimeters of small intestines were dissected on ice, cut longitudinally along the mesenteric border, and luminal contents were gently removed. Intestines were cut into ~1 cm segments and placed in epithelial dissociation buffer (PBS with 5mM EDTA) for 30 min on ice with mild shaking. The first fraction of buffer was discarded, and fresh epithelial dissociation buffer was added to the samples and tubes were kept on ice for 30 min with occasional vigorous shaking. The second fraction of buffer was saved and the previous step was repeated with a third fraction of epithelial dissociation buffer. The second and third fractions containing epithelial cells enriched for crypts were combined and strained through a sterile 70 μm mesh filter and pelleted by centrifugation. Epithelial crypts were further dissociated with the addition of 10 mL of pre- warmed (37°C) TrypLE solution (Gibco) for 3 min. Ice-cold DMEM/F-12 media supplemented with 5% FBS was added to quench the reaction and cells were filtered through a sterile 40 μm mesh strainer. Cells were counted and resuspended in PBS supplemented with 0.4% BSA for 10x Genomics scRNA-seq encapsulation. Sorted muscularis cells and duodenal epithelial cells were each pelleted and resuspended in PBS with 0.4% BSA. A maximum of 10,000 cells from each group was loaded on a Chromium controller (10X Genomics) following the protocol for 3' v3.1 dual index chemistry single cell RNA sequencing (CG000315_RevE and attached products, 10x Genomics). For the muscularis, cells sorted from the control Il13ra1 flox group (~10,000 sort-enriched cells pooled from 1 male and 1 female mouse) and the Calb2ΔIl13ra1 cKO group (~10,000 sort-enriched cells pooled from 1 male and 1 female mouse) were loaded into two separate channels of the Chromium controller (10X Genomics). For epithelial samples, cells from the control Il13ra1flox group (~5,000 manually counted cells from 1 female mouse; male sample compromised) and the Calb2ΔIl13ra1 cKO group (~10,000 manually counted cells pooled from 1 male and 1 female mouse) were loaded into two separate channels, respectively, of the Chromium controller (10X Genomics). Libraries were prepared according to manufacturer's instructions, pooled in equimolar ratios and sequenced on a NovaSeq sequencer (Illumina) in the following read configuration: R1:28, I1:10, I2:10, R2:90bp.