GSM6046026: WT_Female_Infected; Mus musculus; RNA-Seq
GSM6046026
Single-cell preparation of brain-derived cells were obtained from freshly dissected brain cells from infected and uninfected mice. Brain cortices were dissected in cold Hibernate A medium (BrainBits LLC) and sliced to approximately 0.5 mm before transferred to a 15 mL falcon tube with Hibernate A and B27 medium (HABG) (Thermo Fischer Scientific). Collected tissue samples in 15 mL conical tubes were warmed up to 30°C in a shaking water bath for 8 minutes, before the HABG supernatant replaced with activated papain (34 U/mL, Worthington Biochemical Corporation) and the tubes placed back into the shaking water bath for tissue digestion (30°C, 150 rpm) for 30 minutes. Cells were released from the digested tissues by trituration using a fire-polished Pasteur pipette. Released cells were collected in the supernatant and filtered through a 70 mm MACS Smart Strainer (Miltenyi Biotec) into a new 15 mL conical tube. The single cell suspension was layered onto a Optiprep density gradient to separate cells from debris, after centrifugation (800 x g, 15 min, 22°C). The debris fraction was collected, and the gradient material diluted with HAGB before tubes were centrifuged (200 x g, 5 min, 22°C). The supernatant was aspirated, and ACK lysis buffer (Thermo Fisher Scientific) was added to the cell suspension remove any remaining red blood cells (5 min, RT). Hank's balanced salt solution (Thermo Fisher) was added to the cell suspension containing the lysis buffer and tubes were centrifuged (200 x g, 5 min, 22°C). To remove dead cells from the single cell suspension, a Dead cell removal kit (Miltenyi Biotec) was used as directed by the vendor. Cells were resuspended in PBS with 0.04% non-acetylated BSA prior to the single-cell sequencing step using Chromium Single-cell 3ʹ GEM, Library & Gel Bead Kit v3 (10× Genomics) on a 10x Genomics Chromium Controller following manufacturers protocol. Single-cell library preparation was performed using Chromium Single Cell 3′ GEM, Library & Gel Bead Kit v3 (10x Genomics, Pleasanton, CA, USA; Catalog no. 1000075) following the manufacturer's protocol.