Sample Guidelines scRNA

Handbook: cell preparation for single cell protocols

Cell viability: optimal > 90 % (minimum acceptable 80%)

No (or minimal) cell debris.
If necessary, use one or more of the following: Dead cell removal kit, FACS or cell strainers

Cell concertation: between 700-1200 uL/cells (target 10K cells) or 1300-1600 uL/cells (target 10k-20k cells)

Cell size: < 30 µm, cells larger than 30 might cause clogging of 10X chip

Resuspension buffer: 1X PBS + 0.04% BSA (BSA concentration may be up to 1 % depending on the cell type).

Cell cryopreservation is supported by both 10X and BD Rhapsody technologies. Protocols may vary depending on the cell type. Here is a general guideline for cell preparation for single cell protocols. We recommend that you use a test sample to verify that this protocol is suitable for your samples and to check viability after thawing, as cryopreservation can have a major impact on cell viability.

Thawing: One or more protocols may be applicable for a specific sample type and the most appropriate protocol should be used for thawing single cell suspensions for use with 10x Genomics Single Cell assays. This handbook describes different protocols depending on the sample type.

This protocol outlines how to isolate, wash, and count single nuclei from both single cell suspensions and tissue for use with 10x Genomics Single Cell RNA protocol (without kit). The optimal starting input is 2 x10⁶ cells.

The Chromium Nuclei Isolation Kit is an all-in-one solution for the standardized isolation of nuclei from frozen tissue for use in 10x Genomics Single Cell assays.

Quality control of isolated nuclei: Here are some tips on how to check the quality of your nuclei preparations. From the figure below sample A and B are acceptable.

Fixation of cells or nuclei can be performed following the Chromium Next GEM Single Cell Fixed RNA Sample Preparation Kit protocol. The optimal input is 300K cells or 500K nuclei. After fixation, the optimal input to start hybridization is 200K cells or 500K nuclei. When multiplexing, the cell/nucleus input can be lowered.

If the starting material is tissue, follow the ‘Tissue Fixation & Dissociation for Chromium Fixed RNA Profiling‘ protocol to obtain the final cell/nuclei suspension. Check the list of tissues tested and the corresponding cell yields. The optimum input may vary from tissue to tissue, but generally 25 mg is a good starting point.

Removal of dead cells in fixed samples samples is possible when using a LIVE/DEAD™ fixable dye.

Below are the genes excluded from the human and mouse probe set:
– TCR joining and variable regions
– IG joining and variable regions
– Ribosomal proteins
– Mitochondrial ribosomal proteins
– Read-through genes
– KIR and HLA genes (due to their inherent allelic diversity)
– Non-coding RNA

Nuclei from primary cells or cell lines can be isolated using the ATAC or Multiome protocols respectively. The recommended input is 100k cells.

Nuclei from tissue can be isolated using the Chromium Nuclei Isolation Kit for both ATAC and Multiome. Alternatively, nuclei can be isolated without the kit following ATAC or Multiome protocol. Check if your tissue is suitable for these protocols

When performing ATAC or Multiome with BD Rhapsody, nuclei can be isolated from cells (PBMC and cell lines) or frozen tissue using the linked protocol.

The use of RNAse inhibitor is mandatory when performing a Multiome assay. Use the recommended brands for both 10X and BD protocols.

Use these tools to estimate the number of cells you will need to sequence to capture the desired cell population/type.

a. scPower: http://scpower.helmholtz-muenchen.de/
b. How Many Cells: https://satijalab.org/howmanycells
c. SCOPIT: https://alexdavisscs.shinyapps.io/scs_power_multinomial/