Chromium nuclei isolation kit

Nuclei were isolated from brain-derived live CD45⁺ and CD45^– cells using the Chromium Nuclei Isolation Kit (10X Genomics) using the manufacture’s instructions. Following isolation, nuclei were stored on ice and used immediately for subsequent library preparation. A barcoded 3′ sc gene expression library and ATAC library were prepared from single-nuclei suspensions using the Chromium Single Cell Multiome kit (10X Genomics) following the manufacturer’s instruction. The libraries were quantified by fluorometry on a Qubit instrument (LifeTechnologies) and by qPCR with a Kapa Library Quant kit (Kapa Biosystems-Roche) prior to sequencing. The libraries were sequenced on a NovaSeq 6000 sequencer (Illumina) with paired-end 150 bp reads with approximately 35,000 reads pairs per nuclei for gene expression and 25,000 read pairs per nuclei for ATAC libraries.

Since scRNAseq needs to be performed on fresh tissue, snRNAseq was chosen instead given its ability to be performed on previously frozen samples. snRNAseq samples were stored in liquid nitrogen for less than 5 months before processing. Nuclei were isolated using the Chromium Nuclei Isolation Kit (10X Genomics). snRNAseq was performed using the 10× Genomics Chromium X platform with 10× Genomics 3′ gene expression kit. Cell count and viability of the nuclei were verified on the ThermoFisher Countess 3 cell counter and EVO imager microscope. Single nuclei were parsed and proceeded into library preparation following manufacturer protocol. Quality control of the libraries was performed on the Agilent BioAnalyzer and Kapa library quantification. Samples were sequenced on the Illumina Novaseq6000 SP and data was packaged using the 10× Genomics CellRanger pipeline. The snRNAseq and spatial transcriptomic samples were patient-matched to increase the reliability of results during single-cell mapping and analysis.

Nuclei isolation for Single-Cell ATAC-Seq was performed according to the manufacturer’s manual (Chromium Nuclei Isolation Kit, 10x Genomics). Briefly, frozen hippocampal tissue was homogenized in 500 μl of pre-chilled lysis buffer using Dounce homogenizers. The homogenate was then transferred into pre-chilled Nuclei Isolation Columns, and the columns were centrifuged at 16000*g for 30 seconds at 4 °C. The flowthrough was briefly vortexed for 10 seconds, followed by centrifugation at 500*g for 3 min at 4 °C. The nuclear pellet was resuspended in 500 μl of Debris Removal Buffer and centrifuged at 700*g for 10 min at 4 °C. The nuclear pellet was then resuspended in 1 ml of Wash Buffer, centrifuged at 500*g for 5 min at 4 °C, and finally resuspended in 50 μl of Resuspension Buffer. The Single Cell ATAC-Seq protocol, comprising of the Transposition, GEM Generation, Barcoding, and Library Construction steps were carried out according to the manufacturer’s protocol (Chromium Next GEM Single Cell ATAC Kit, 10x Genomics). Libraries were sequenced in paired end in Illumina Novaseq.