scRNA-sequencing of embryonic and neonatal immune cells isolated from various organs
scRNA-sequencing of embryonic and neonatal immune cells isolated from various organs
Blood was collected by bleeding into heparin-Phosphate Buffered Saline (PBS; D8537, Sigma-Aldrich; 50 µl of 100 IU/ml heparin (585661, LEO Pharma) in 500 µl of PBS) from euthanized E17.5 and newborn mice. Testes, kidneys, and liver were gently minced with scissors and digested with 50 µg/ml DNase 1 (10104159001, Roche) and 1 mg/ml Collagenase D (11088866001, Roche) in Hank's Buffered Saline (HBSS; RNBF2378, Sigma-Aldrich) solution at 37 °C for 15 to 45 min. Bone marrow cells were collected by carefully crushing and rinsing the femora (and from embryos also tibiae) with HBSS, followed by sequential filtering through silk cloth (pore size 77 µm, 77-48Y115, Marabu Scandinavia) until no visible aggregates were detected. Finally, all isolated cells were washed with 500 µl of PBS and suspended in PBS and filtered through a silk cloth.  Cells were stained for flow cytometry without fixation and sorted with a Sony SH800 Cell Sorter (100 µm nozzle, purity mode). Single cells were gated with FSC-H vs. FSC-W and SSC-H vs. SSC-W to avoid doublets, and dead cells were excluded. Live single CD45+ cells (kidney, testis) or CD45+ CD3- CD45R- cells (liver, BM, blood) were sorted into the RPMI-1640 medium containing 2% (v/v) FCS. Freshly sorted CD45+ cells were processed immediately according to 10X Genomics guidelines (CG000126_Guidelines for Optimal Sample Prep Flow Chart RevA).  If no treatment is specified, samples were collected from embryos or newborn mice in steady state conditions. For embryonic dexamethasone treatment or LPS exposure experiments, pregnant dams at E17.5 were injected i.p. with either 5 mg/kg of dexamethasone (D2915, Sigma-Aldrich) in PBS or 100 µg/kg LPS (from E. coli O55:B5; L4524, Sigma-Aldrich) in 0.9% (w/v) NaCl solution or the equivalent volume of the respective solvent. Animals were euthanized 2 or 4 hours after dexamethasone or LPS treatment, respectively, as described above. Single-cell RNA-sequencing libraries were prepared according to the manufacturer's instructions (CG00052 RevB) using 10X Genomics Chromium Single Cell 30 Library and Gel Bead Kit v3 and Chromium Single Cell A Chip Kit.  Single-cell RNA-sequencing libraries were prepared according to the manufacturer's instructions (CG00052 RevB) using 10X Genomics Chromium Single Cell 30 Library and Gel Bead Kit v3 and Chromium Single Cell A Chip Kit. 