GSM8477275: Aged_PICS; Mus musculus; RNA-Seq
Mice were euthanized using CO2 fixation prior to collecting organs, blood, and lymphoid organs. Spleens were processed into single cell suspensions and filtered through 40 µm cell strainer followed by hemolysis. Cells were blocked with Fc-block CD16/32 (93) and stained with viability dye zombie aqua, CD45 (30-F11), TCRB (17A2), B220 (6D5) (BioLegend), using methods described above for surface staining. For collection of singlet cells, live CD45+ TCRB+ CD19- T cells, live CD45+ TCRB- CD19+ B cells, and live CD45+ TCRB+ CD19+ PICs were sorted using a MoFlo Astrios Sorting Machine, at the Princess Margaret Hospital Flow Cytometry Facility, University Health Network (Toronto, Canada), and collected into Eppendorf tubes containing a 500 uL cushion of RPMI-1640 (Wisent) with 10% heat-inactivated FBS (Wisent). Samples were processed according to protocols for 5' v2 chemistry 10X Genomics at the Princess Margaret Genomics Centre. Library construction was performed according to the manufacturer's instructions (single cell 5' v2 protocol, 10X genomics).