Best practices for using Illumina Single Cell Nuclei Isolation Kits for Illumina Single Cell kits

The Illumina Single Cell Nuclei Isolation kit provides a pre-formulated set of reagents for isolation of nuclei from frozen tissue and is designed to be compatible with various tissue types. The isolated nuclei are compatible with Illumina Single Cell 3’ RNA Kits.

Assessing Nuclei Quality

The quality of isolated nuclei significantly impacts downstream single-cell workflows. Nuclei quality should always be assessed before input into Illumina Single Cell and the nuclei isolation workflow should be repeated if sufficient quality is not obtained.

Visual inspection of nuclei is recommended to determine the quality and the concentration of a nuclei suspension before input into Illumina Single Cell 3’ RNA kits. Users should quantify generated nuclei suspensions with a fluorescent nucleic acid stain, like Acridine Orange or Propidium Iodide (AO/PI), with a fluorescence-capable automated counter. Always conduct replicate counts to ensure accuracy. Use of trypan blue is not recommended.

A high-quality nuclei suspension will have minimal debris and aggregates (Figure 1A). For tissues with high amounts of debris, a smaller tissue input is recommended to reduce debris carryover. High-quality nuclei with intact membranes will appear round and smooth (Fig 1 B) while nuclei with compromised membranes will appear disjointed with an indication of blebbing (Fig 1 A).

General Recommendations for Nuclei

The quicker cells are processed to encapsulated nuclei, the better. Minimal centrifugation and keeping the preps ice cold the entire time is recommended. (Keep all buffers, reagents, equipment, consumables, and plasticware on ice throughout the isolation.) When isolating nuclei for many samples, it is best to prep samples in small batches through Capture and Lysis (10-15 minutes of hands-on time), then use the stopping point to prep another batch of samples. The samples can then be processed together through the remaining Illumina Single Cell workflow steps to eliminate batch effects. Try to minimize the amount of time between completing nuclei isolation and starting Capture and Lysis. After isolation, nuclei will start to degrade and clump.

Do not freeze and store isolated nuclei (freeze/thaw cycles disrupt the nuclear membrane) unless fixing nuclei first according to the demonstrated DSP-Methanol Fixation Protocol for Nuclei for Illumina Single Cell. Refer to the Fixation section of the Training Packet for further information.

Always conduct replicate counts to verify accuracy. It is not recommended to use trypan blue, as debris can easily be counted as nuclei, which will overestimate the count (leading to a lower-than-expected capture rate), especially when size gating is not implemented.

Size Gating Recommendations (Nuclei)

Size gating recommendations using an automated fluorescent cell counter with nuclei isolated from various mouse tissue types:

Minimum size gate: 4 µm

Maximum size gate: 11 µm (16 µm for brain)

For more information refer to the Training Packet.

For any feedback or questions regarding this article (Illumina Knowledge Article #9450), contact Illumina Technical Support [e-mail].