***All sample drop-offs must be scheduled ahead of time due to the sensitivity of the 10X Protocol.***
Cryopreserved PBMCs or tissues are to be kept at -80C or on dry ice, to preserve sample integrity, from the time of collection to the time of delivery.
The input material for the single-cell multiome ATAC + Gene expression workflow is a clean nuclei suspension. This nuclei suspension may come from tissue or a cell suspension. Whenever possible, dissociate tissues into a cell suspension before isolating nuclei. However, for frozen and hard to dissociate tissues it is necessary to go straight from tissue to nuclei. If you start with a low viability cell suspension, sorting for live cells and/or removing dead cells, with the dead cell removal kit, before nuclei isolation may improve sample quality. After nuclei isolation, stain cells with the viability dye to determine quality. Less than 5% of cells should be alive after nuclei isolation. Sorting after nuclei isolation may be necessary to remove the debris.
Minimum cell/nuclei sample requirements:
The optimal input nuclei concentration for most 10x Genomics Single Cell assays is 700–1,200 nuclei/μl.
*If possible, bring the input nuclei suspension to a concentration that is optimal for the dynamic range of counting technique used (manual or automated), allows for 2–3 reproducible counts (where the standard
deviation is <25%), and requires pipetting 2–5 μl (Single Cell Multiome ATAC + Gene Expression and Single Cell ATAC Assays) into the Single Cell Master Mix.
*Nuclei recovery from healthy tissues typically ranges from 5,000–15,000 nuclei/mg. Both disease state and cell type composition can impact nuclei recovery.
*Resuspend nuclei in a low initial volume (50–200 μl) during final resuspension step if nuclei yield is expected to be low or is unknown.
*DO NOT resuspend nuclei in a volume <50 μl regardless of input tissue
mass