GSM8527699: Salmonella 24h post-infection, Batch 1, Replicate 1; Mus musculus; RNA-Seq
Small intestine was extracted and rinsed in cold Phosphate buffered saline (PBS). The tissue was opened longitudinally and sliced into small fragments roughly 2 mm long followed by incubation with 20mM EDTA-PBS on ice for 75 min. Then, the tissue was shaken vigorously, and the supernatant was collected as fraction 1 in a new conical tube. The tissue was incubated in fresh PBS, and a new fraction was collected every 5 min. Fractions were collected until the supernatant consisted almost entirely of crypts. The fractions 3-4 (enriched for crypts) were filtered through a 70 µm filter, centrifuged at 400g for 5 min, and dissociated with TrypLE Express (Gibco) for 1 min and 30 seconds at 37°C. The single-cell suspension was then passed through a 40μm filter. Cells for each condition or mouse were stained with the Dapi-NucBlue (ThermoFisher, cat no. R37605), CD45 (Biolegend, 30-F11), Biotin anti-mouse CD326 (EpCAM) antibody (cat #118204) followed by staining using TotalSeq™ PE Streptavidin (B0951 - B0955). Isolated IEC were sorted into a cooled 15 mL tube with 0.04% BSA in PBS using a Sony SH800 cell sorter. 20,000 cells EpCAM+ CD45- (IEC), EpCAM+ CD45- GFP+ (Invaded IEC) were sorted for each mouse or condition and pooled following manufacturer's instructions. 30,000 single-cell suspension was loaded onto Next GEM Chip G targeting 15,000 cells and then ran on a Chromium Controller instrument to generate GEM emulsion. Single-cell 3' RNA-seq libraries and cell surface protein libraries were generated according to the manufacturer's protocol (Chromium Single Cell 3' Reagent Kits User Guide (v3.1 Chemistry Dual Index). Final libraries were quantified using NEBNext Library Quant Kit for Illumina (NEB) and high sensitivity D5000/D1000 TapeStation (Agilent). Libraries were pooled according to targeted cell number, aiming for ~20,000 reads per cell for gene expression libraries and ~5,000 reads per cell for cell surface protein libraries. Pooled libraries were sequenced on a NovaSeq 6000 instrument using an S1 100 cycles reagent kit (Illumina). A total of 8000 cells were loaded into each well of a Chromium Next GEM Chip G and run on the Chromium Controller (10X Genomics) for Gel Bead-in-emulsion (GEM) generation. The samples were processed according to the CG000317 Chromium Next GEM Single Cell 3' v3.1 (Dual Index) Rev. D protocol (10X Genomics) for RNA library construction.