GSM9369826: KI Day 2; Mus musculus; RNA-Seq
Under sterile conditions, the freshly collected tissues were washed twice with pre-cooled RPMI 1640 medium containing 0.04% BSA. The tissues were then mechanically dissociated into approximately 0.5 mm³ fragments using surgical scissors and transferred into freshly prepared enzymatic digestion solution. The digestion mixture contained RPMI 1640 (Conring, cat.no. 10-040-CVR), 0.04% BSA (MACS, cat.no. 1000076), and 0.2% collagenase II (Gibco, cat.no. 17101015). The samples were incubated at 37°C for 30-60 minutes with gentle inversion every 5-10 minutes. The digested cell suspension was filtered through a BD 40 μm cell strainer (Falcon, cat.no. 352340) 1-2 times. The filtrate was centrifuged at 300 × g for 5 minutes at 4 ° C. The cell pellet was resuspended in appropriate medium, mixed with an equal volume of red blood cell lysis buffer (Miltenyi, cat.no. 130-094-183), and incubated at 4°C for 10 minutes. After centrifugation at 300 × g for 5 minutes, the supernatant was discarded. The pellet was washed once with medium followed by another centrifugation at 300 × g for 5 minutes, and the final supernatant was removed. CD45+ immune cells were enriched using mouse CD45 MicroBeads (Miltenyi, cat.no.130-052-301） accordding to the manufacturer's protocol. Finally，the cells were re-suspended with 1ml RPMI 1640 medium (Conring, cat.no.10-040-CVR) with 0.04% BSA added. Single-cell suspension concentration and cell viability were then evaluated by Luna-FL cell counter (Logos Biosystems，Korea) or Trypan Blue staining method. The freshly prepared single-cell suspension was adjusted to a concentration of 700-1200 cells/μl. Library preparation and loading were performed following the manufacturer's protocol for the 10× Genomics Chromium Next GEM Single Cell 3 ʹ Reagent Kits v3.1 (cat.no.PN-1000268). The constructed libraries were sequenced on the Illumina Nova 6000 PE150 platform for high-throughput sequencing.