GSM6644992: murine splenic immune cells, WT STm infected, isotype control antibody [C2_CtrlAb]; Mus musculus; RNA-Seq
All samples subjected to scRNA-seq were prepared and FACS-enriched using the following procedures. STm infected spleens were harvested and digested in buffer containing HBSS + Ca2+ + Mg2+ + 50 μg/mL DNase (Roche) + 25 μg/mL Liberase TL (Sigma) at 37 °C for 25 min, mixing at 200 rpm. EDTA was added to 5 mM final concentration to stop digestion reaction and cells were washed with RPMI containing 10% FCS. Following RBC lysis, splenocytes were washed twice RPMI containing 10% FCS.  Splenocytes were stained with an antibody mixture for surface markers (CD11b Alexa fluor 647, CD11c PECy7, Ly6C PerCP Cy5.5, Ly6G FITC, CD3 APC efluor 780, CD19 APC efluor 780, and NK1.1 APC efluor 780) for 25 minutes on ice, washed twice with RPMI containing 10% FCS, then resuspended in the same buffer with 1:2000 DAPI. Splenocytes were then FACS-enriched on a BD FACSAria cell sorter. A permissive gating strategy was utilized to simultaneously enrich CD11b+CD11c+Ly6C+ macrophages and capture other splenocytes for sequencing. Sorting gates were set tightly for size/scatter, singlet, and living cells but more loosely for CD3/CD19/NK1.1-, CD11b+, Ly6G-, Ly6C+, and CD11c+ cells. The viability of sorted cells were checked using Trypan blue staining and hemocytometer inspection under a light microscope. Samples had viability greater than 90%. Cells were resuspended to a concentration to 500-1200 cells/uL, partitioned, and captured for sequencing on a 10x Chromium Controller. Libraries were prepared by the Stanford Functional Genomics Facility (SFGF) using 10X Genomics 3' GEX v3.1 kit and sequenced on the Illumina HiSeq4000 platform to a depth of ~40,000 - 50,000 reads/cell. 10X Genomics 3' GEX v3.1 kit.