title: Single cell RNA sequencing of colon in germ-free and specific pathogen free mice
design: Single cell RNA sequencing of colon in germ-free and specific pathogen free mice
library_construction_protocol: Colon epithelium. Whole colon 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. 25 ml of 37 °C Dissociation Buffer (Ca/Mg-free HBSS, 2 mM EDTA pH 8, 1 mM DTT) was added and incubated shaking at 37 °C for 30 min to dissociate epithelial layer. Tissue was removed and epithelium was collected by centrifugation at 500g for 3 min. The cells were then incubated for one hour on ice in 500 μl Miltenyi Dead Cell Removal kit beads, after which 5.5 ml of kit Binding Buffer was added to each sample. The samples were filtered and then loaded onto LS-columns and the flow-through was collected. The flow-through was then centrifuged 5 min 500g and was resuspended in Ca/Mg-free PBS with 0.04% BSA and samples proceeded directly to scRNAseq. Colon lamina propria. Whole colon was dissected from the animal and cut longitudinally to remove contents and tissue was vortexed in clean 4 °C Ca/Mg-free PBS to wash it. This was repeated until solution was clear. 10 ml of 37 °C Dissociation Buffer (Ca/Mg-free HBSS, 3 mM EDTA pH 8, 1 mM DTT) was added and incubated shaking at 37 °C for 15 min to dissociate epithelial layer. Then tissue was vortexed epithelial layer stripping and moved to fresh 10ml of Dissociation Buffer and incubated shaking at 37 °C for 20 min. Then, the tissue was placed in cold Ca/Mg-free HBSS, washed twice 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, 0.8 mg/ml collagenase D, 1.6 mg/ml dispase) at 37 °C for 35 min. 10 ml of cold Ca/Mg-free HBSS was then added to stop digestion and cells were passed through 85-μm 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 10 ml 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 1μg/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 PBS, filtered through a 40-μm 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