GSM8247648: Y_iso; Mus musculus; RNA-Seq
Mice were anesthetized with 87.5 mg per kg ketamine and 12.5 mg per kg xylazine and transcardially perfused with ice-cold phosphate-buffered saline. For collection of splenocytes, spleens were removed, mechanically dissociated with a syringe plunger over a 70 μm cell strainer and washed with 10 ml of MACS. Cells were centrifuged and RBC lysis was performed (155 mM NH4Cl, 1 mM KHCO3 and 0.1 mM EDTA) with 2 ml for 5 minutes room temperature (RT). Subsequently, cells were washed, spun 500 RCF for 5 minutes, and resuspended in MACS. ScRNAseq analysis was performed on splenocytes isolated from four mice per group. Splenocytes were isolated as described above, blocked (2.4G2), then labeled with CD45.1 (A20, 1:200) and C45.2 (104, 1:200) antibodies in MACS for 30 minutes on ice. Samples were washed, then stained with a live/dead marker (Hoechst 33342, 1:10,000) before filtering through a 35 μm FACS tube filter sorting on a BD FACSAria Fusion with a 70 μm nozzle and with a flow rate of 1–2.5. Cells were first gated by forward and side scatter, then gated for doublets with height and width. 40,000 cells per sample that were Hoechst- and CD45.1+ or CD45.2+ were collected for sequencing analysis and pooled into 6 groups (CD45.1+ young isochronic, CD45.1+ young heterochronic, CD45.2+ young heterochronic, CD45.1 + aged heterochronic, CD45.2+ aged heterochronic, and CD45.2+ aged isochronic). Samples were delivered to the UCSF-IHG Genomics Core for analysis with the 10x Genomics Chromium Single Cell Expression Solution 3′ kit with Feature Barcode Technology (v.3.1). The Genomics Core prepared cells for 10x Genomics Chromium single-cell capture. 25,000 cells were loaded per sample. cDNA libraries were prepared according to the standard 10x Genomics protocols. The final library pool was sequenced to a depth of 30,000 cDNA reads per cell and 3,000 ADT reads per cell on the NovaSeq X 10B system at the UCSF CAT Core.