10X Chromium Nuclei Isolation Processing Workflows with Voyager

The nuclei isolation solution from 10X genomics is a protocol for isolating nuclei from frozen samples for single nuclei sequencing. The sample prep workflow has the advantage of avoiding long enzymatic incubations and being accessible without specialized equipment. The workflow includes cell lysis, nuclei isolation in a spin column, and debris removal. Importantly, isolated nuclei are compatible with Chromium Single Cell ATAC, Single Cell Multiome ATAC + Gene Expression, and Single Cell Gene Expression assays without modifications to the single cell workflows. Nuclei suspension concentration, viability, and quality can be assessed with a nucleic acid fluorescent dye and follow-up with an fluorescent automated counter or microscope.

Pros and cons

Pros:

• Applicable to frozen samples
• Capture more nascent transcripts
• Sensible to tissues with multiple nuclei in one cell

Cons:

• Lose spatial information
• More expensive and less flexible than open source technologies

Getting Started

The vignettes below provide examples of processing raw data using a workflow that includes seqspec, gget, and kallisto/bustools to generate a count matrix. We process the output of various transcriptomics technologies into a SpatialFeatureExperiment(SFE) object for use with Voyager.

Vignette	Colab Notebook	Description
Preprocess raw data	Colab Notebook	Fetch reference data with gget, process raw data with seqspec, generate a count matrix with kallisto-bustools
Create a SFE object	Colab Notebook	Download data, create SFE object

Analysis Workflows

The analysis tasks include basic quality control, spatial exploratory data analysis, identification of spatially variable genes, and computation of global and local spatial statistics. Accompanying Colab notebooks are linked.

Vignette	Colab Notebook	Description
Nuclei Basic QC	Colab Notebook	Basic qc and preprocessing