GSM8816206: CITEseq_hippocampal cells, batch4_HTO; Mus musculus; OTHER
Mice were anesthetized using CO₂ and transcardially perfused with ice-cold perfusion buffer (PB+), which contained Hibernate A-minus Calcium (HA-Ca), Actinomycin D, and Triptolide. Following perfusion, brains were dissected, and hippocampi were isolated, minced, and transferred into PB+. The minced tissue was enzymatically digested using an activated papain solution (papain, DNase I, and DXB+) at 37°C for 30 minutes with gentle shaking. After digestion, the tissue was gently triturated using pipettes until no visible clumps remained. DNase I was added to the suspension, which was then centrifuged at 300g for 5 minutes at room temperature. To inactivate papain, the cell pellet was resuspended in ovomucoid solution and centrifuged at 300g for 10 minutes. The cell suspension was subsequently passed through a pre-wetted 70 µm filter and rinsed with HA-Ca buffer, followed by centrifugation at 300g for 10 minutes at 4°C. For further purification, the cell suspension was layered onto a Percoll gradient (70%-30%) and centrifuged at 300g for 30 minutes at 22°C. Single cells were collected from the 70%-30% interface, resuspended in Hibernate A-Low Fluorescence (HA-LF) buffer containing inhibitors, and centrifuged again. Cells were counted using a hemocytometer. For blocking, 1 million cells were resuspended in 100 µL Staining Buffer (2% BSA/0.02% Tween in PBS), followed by the addition of 10 µL Fc Blocking reagent and incubation at 4°C for 10 minutes. Hashing antibodies (1 µg) were added, and the mixture was incubated at 4°C for 30 minutes. The cells were then washed three times with Staining Buffer, centrifuged at 400g for 5 minutes at 4°C, and resuspended in PBS for downstream applications. Method of single cell capture, library preparation and sequencing Cell suspension, (concentration and percent viability determined by staining Trypan Blue Stain (0.4%) (ThermoFisher Scientific PN-T10282) and Countess II Automated Cell Countess), was loaded into Chromium Next GEM Chip G (10X Genomics PN-1000127) with a targeted recovery of 16,000 to 20,000 cells per well and processed with Chromium Controller (10X Genomics). Single-cell RNAseq libraries were prepared with Chromium Single Cell 3' Reagent Kit (v3.1 CG000204 Rev C; 10X Genomics PN-1000128) and Chromium i7 Multiplex Kit (Single Index; PN-120262) following the standard throughput manual workflow. Single-cell HTO libraries were prepared following CITE-seq & Cell Hashing Protocol (New York Genome Center Technology Innovation Lab, version 2019-02-13).  The single-cell RNA-seq libraries were sequenced on an Illumina NovaSeq 6000 with paired-end reads (28 cycles Read 1, 8 cycles i7, 91 cycles Read 2).