title: Single cell RNA sequencing of small intestine in germ-free and specific pathogen free mice
design: Single cell RNA sequencing of small intestine in germ-free and specific pathogen free mice
library_construction_protocol: Small intestine epithelium. Whole small intestine was dissected from the animal, and tissue was cut longitudinally to remove contents and vortexed in clean 4 °C Ca/Mg-free PBS to wash it. This was repeated until solution was clear. 20 ml of 4 °C Dissociation Buffer (Ca/Mg-free HBSS, 2.5 mM EDTA pH 8) was added and incubated shaking at 4 °C for 40 min to dissociate epithelial layer. Tissue was removed and epithelium was collected by centrifugation at 500g for 3 min. To obtain single cell solution from crypts we resuspended the pellet in 10 ml of prewarmed 0.25% Trypsin-EDTA solution and incubated for 20 min at 37 °C while shaking. Then we added 20 ml of cold Ca/Mg-free PBS, and the samples were then filtered using 65-μm mesh. Finally, cells were collected at 500g 4 °C for 5 min and resuspended in cold Ca/Mg-free PBS with 0.04% BSA. Small intestine lamina propria. Whole small intestine was dissected from the animal, Payers patched were dissected and removed and tissue was cut longitudinally to remove contents. Then it vortexed in clean 4 °C Ca/Mg-free PBS to wash it. This was repeated until solution was clear. 12 ml of 37 °C Dissociation Buffer (Ca/Mg-free HBSS, 5 mM EDTA pH 8) was added and incubated shaking at 37 °C for 5 min to dissociate epithelial layer. Then tissue was vortexed epithelial layer stripping and moved to fresh 10 ml of Dissociation Buffer and incubated shaking at 37 °C for 15 min. Then, the tissue was placed in cold Ca/Mg-free HBSS, washed three times using vortex and dried on clean paper. To disintegrate colon tissue was first chopped with scissors and subsequently incubated shaking in Digestion Buffer (RPMI, 10% FCS, 0.625 mg/ml collagenase D, 1 mg/ml dispase and 0.04 mg/ml liberase TM) at 37 °C for 35 min. 10ml of cold Ca/Mg-free HBSS was then added to stop digestion and cells were passed through 85μm metal mesh. Then cells were collected by centrifugation at 500g for 8 min at 4 °C. To lyse erythrocytes pellets were resuspended in 1 ml ACK buffer and incubated for 1 min at room temperature. Then 10ml of Ca/Mg-free PBS was added, cells were centrifuged at 500g for 8 min. To remove dead cells and to ensure depletion of epithelium we resuspended the pellet in 100 μl of Ca/Mg-free PBS with 1% FCS and incubated for 5 min with 1 μgl/ml anti-CD16/32 (Biolegend) to block Fc receptors before adding the 1:100 EPCAM APC-Cy7 antibody (G8.8, Biolegend). The cells were incubated for 30 min on ice, then 1:10.000 dilution of DAPI was added and stained for additional 3 min. Then 10 ml of Ca/Mg-free PBS was added, and the cells were centrifuged for 8 min at 500g. The supernatant was removed,and the cells were resuspended in Ca/Mg-free PBS, filtered through a 40μm mesh and DAPI negative and EPCAM negative cells were sorted on BD FACSAria III. Sorted cells were then centrifuged 8 min at 500g, resuspended in Ca/Mg-free PBS with 0.04% BSA and counted. according to 10x genomics Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 User Guide according to 10x genomics Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 User Guide