biostudies-arrayexpressKatherine WaltonMus musculushttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-15222Whole intestines were harvested from fetuses from timed pregnant matings for wildtype littermates and endothelial specific Notch inhibited mutants (Jax strain #032613). Fetal stages were confirmed according to the Theiler staging chart (https://www.emouseatlas.org/emap/ema/staging_criteria/staging_criteria.html). Whole intestines (from the common bile duct through the cecum) were collected at E16-E16.5. Male and female intestines from each stage were pooled and dissociated to single cells for single cell RNA sequencing as previously described (Miller et al. 2020 Dev Cell). 705-KW-1 and 705-KW-2 were whole intestine. 2482-KW1 and 2482-KW2 samples were enriched with CD31+ MACS beads. 3593-KW1 and 3593-KW2 samples were enriched with CD31+ MACS beads and depleted for CD45.biostudies-arrayexpressSample Collection - Cell dissociations were carried out similar to previously published methods (Miller et al., 2020). To dissociate mouse fetal tissue to single cells, tissue was mechanically minced into small fragments, and in a petri dish filled with ice-cold 1X HBSS (with Mg2+, Ca2+). This tissue was then transferred to a 15 mL conical tube. Dissociation enzymes and reagents from the Neural Tissue Dissociation Kit (Miltenyi, cat. no. 130-092-628) were used, and all incubation steps were carried out in a refrigerated centrifuge pre-chilled to 10ºC unless otherwise stated. All tubes and pipette tips used to handle cell suspensions were pre-washed with 1% BSA in HBSS to prevent adhesion of cells to the plastic. Tissue was treated for 15 minutes at 10ºC with Mix 1. Mix 2 was added to the digestion, and tissue was incubated for 10 minute increments at 10ºC until digestion was complete. After each 10 minute incubation, tissue was agitated using a P1000, and tissue digestion was visually assessed under a stereo microscope. This process continued until the tissue was fully digested. Cells were filtered through a 70 µm filter coated with 1% BSA in 1X HBSS, spun down at 500g for 5 minutes at 10ºC and resuspended in 500µl 1X HBSS (with Mg2+, Ca2+). 1 mL Red Blood Cell Lysis buffer (Roche cat. No 11814389001) was then added to the tube and the cell mixture was placed on a rocker for 15 minutes at 4ºC. Cells were spun down (500g for 5 minutes at 10ºC), and washed twice by suspension in 2 mLs of HBSS + 1% BSA followed by centrifugation. Cells were counted using a hemocytometer, then spun down and resuspended (if necessary) to reach a concentration of 1000 cells/µL and kept on ice. Samples were then enriched with CD31+ MACS beads. Cells were counted using a hemocytometer, then spun down and resuspended (if necessary) to reach a concentration of 1000 cells/µL and kept on ice. Single cell droplets were immediately prepared on the 10x Chromium according to manufacturer instructions at the University of Michigan The Advanced Genomics Core, with a target of capturing 5,000 cells. Single cell libraries were prepared using the Chromium Next GEM Single Cell 3’ Library Construction Kit (v3) according to manufacturer instructions.Sequencing - All single-cell RNA-sequencing was performed with an Illumina NovaSeq 6000 by the University of Michigan DNA Sequencing core.Sample Collection - Cell dissociations were carried out similar to previously published methods (Miller et al., 2020). To dissociate mouse fetal tissue to single cells, tissue was mechanically minced into small fragments, and in a petri dish filled with ice-cold 1X HBSS (with Mg2+, Ca2+). This tissue was then transferred to a 15 mL conical tube. Dissociation enzymes and reagents from the Neural Tissue Dissociation Kit (Miltenyi, cat. no. 130-092-628) were used, and all incubation steps were carried out in a refrigerated centrifuge pre-chilled to 10ºC unless otherwise stated. All tubes and pipette tips used to handle cell suspensions were pre-washed with 1% BSA in HBSS to prevent adhesion of cells to the plastic. Tissue was treated for 15 minutes at 10ºC with Mix 1. Mix 2 was added to the digestion, and tissue was incubated for 10 minute increments at 10ºC until digestion was complete. After each 10 minute incubation, tissue was agitated using a P1000, and tissue digestion was visually assessed under a stereo microscope. This process continued until the tissue was fully digested. Cells were filtered through a 70 µm filter coated with 1% BSA in 1X HBSS, spun down at 500g for 5 minutes at 10ºC and resuspended in 500µl 1X HBSS (with Mg2+, Ca2+). 1 mL Red Blood Cell Lysis buffer (Roche cat. No 11814389001) was then added to the tube and the cell mixture was placed on a rocker for 15 minutes at 4ºC. Cells were spun down (500g for 5 minutes at 10ºC), and washed twice by suspension in 2 mLs of HBSS + 1% BSA followed by centrifugation. Cells were counted using a hemocytometer, then spun down and resuspended (if necessary) to reach a concentration of 1000 cells/µL and kept on ice. Single cell droplets were immediately prepared on the 10x Chromium according to manufacturer instructions at the University of Michigan The Advanced Genomics Core, with a target of capturing 5,000 cells. Single cell libraries were prepared using the Chromium Next GEM Single Cell 3’ Library Construction Kit (v3) according to manufacturer instructions.Nucleic Acid Extraction - Immediately after tissue dissociation, single cell libraries were prepared on the 10x Chromium with a target of 5,000 cells. Chromium Single Cell 3’ Library & Gel Bead Kit v3, 16 rxns PN-120237 was used.Sample Collection - Cell dissociations were carried out similar to previously published methods (Miller et al., 2020). To dissociate mouse fetal tissue to single cells, tissue was mechanically minced into small fragments, and in a petri dish filled with ice-cold 1X HBSS (with Mg2+, Ca2+). This tissue was then transferred to a 15 mL conical tube. Dissociation enzymes and reagents from the Neural Tissue Dissociation Kit (Miltenyi, cat. no. 130-092-628) were used, and all incubation steps were carried out in a refrigerated centrifuge pre-chilled to 10ºC unless otherwise stated. All tubes and pipette tips used to handle cell suspensions were pre-washed with 1% BSA in HBSS to prevent adhesion of cells to the plastic. Tissue was treated for 15 minutes at 10ºC with Mix 1. Mix 2 was added to the digestion, and tissue was incubated for 10 minute increments at 10ºC until digestion was complete. After each 10 minute incubation, tissue was agitated using a P1000, and tissue digestion was visually assessed under a stereo microscope. This process continued until the tissue was fully digested. Cells were filtered through a 70 µm filter coated with 1% BSA in 1X HBSS, spun down at 500g for 5 minutes at 10ºC and resuspended in 500µl 1X HBSS (with Mg2+, Ca2+). 1 mL Red Blood Cell Lysis buffer (Roche cat. No 11814389001) was then added to the tube and the cell mixture was placed on a rocker for 15 minutes at 4ºC. Cells were spun down (500g for 5 minutes at 10ºC), and washed twice by suspension in 2 mLs of HBSS + 1% BSA followed by centrifugation. Cells were counted using a hemocytometer, then spun down and resuspended (if necessary) to reach a concentration of 1000 cells/µL and kept on ice. Samples were then enriched with CD31+ MACS beads and depleted for CD45. Cells were counted using a hemocytometer, then spun down and resuspended (if necessary) to reach a concentration of 1000 cells/µL and kept on ice. Single cell droplets were immediately prepared on the 10x Chromium according to manufacturer instructions at the University of Michigan The Advanced Genomics Core, with a target of capturing 5,000 cells. Single cell libraries were prepared using the Chromium Next GEM Single Cell 3’ Library Construction Kit (v3) according to manufacturer instructions.Library Construction - Immediately after tissue dissociation, single cell libraries were prepared on the 10x Chromium with a target of 5,000 cells. Chromium Single Cell 3’ Library & Gel Bead Kit v3, 16 rxns PN-120237 was used.OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesData Transformation - Data matrices for further analysis were generated using the CellRanger pipeline (v1.5.1) under the standard parameters by the University of Michigan Advanced Genomics Sequencing Core.UnknownTranscriptomicsGenomicsProteomicsIllumina NovaSeq 6000RNA-seq of coding RNA from single cellsMus musculusVascular Connections to Mesenchymal Clusters Coordinate Intestinal Villus Morphogenesis and PatterningKatherine WaltonXiangning DongDong X*, Reed TT*, Zhao Z, Islam H, Gumucio DL, Walton KDfalseRNA-seq of mouse fetal intestines comparing wildtype littermates with Notch inhibited mutantsWhole intestines were harvested from fetuses from timed pregnant matings for wildtype littermates and endothelial specific Notch inhibited mutants (Jax strain #032613). Fetal stages were confirmed according to the Theiler staging chart (https://www.emouseatlas.org/emap/ema/staging_criteria/staging_criteria.html). Whole intestines (from the common bile duct through the cecum) were collected at E16-E16.5. Male and female intestines from each stage were pooled and dissociated to single cells for single cell RNA sequencing as previously described (Miller et al. 2020 Dev Cell). 705-KW-1 and 705-KW-2 were whole intestine. 2482-KW1 and 2482-KW2 samples were enriched with CD31+ MACS beads. 3593-KW1 and 3593-KW2 samples were enriched with CD31+ MACS beads and depleted for CD45.2025-08-01T00:00:00Z2025-06-30T12:36:24.915Z2025-06-30T12:36:24.915ZE-MTAB-15222ERP174325EFO_0002944EFO_0004170EFO_0005684EFO_0005518EFO_0003816EFO_0004184