Best Practices for Nuclei Isolation with alternative nuclei isolation protocols for Illumina Single Cell
It is not required to use the Illumina Single Cell Nuclei Isolation kit for sample preps of nuclei to be compatible with the Illumina Single Cell workflow. Alternative workflows from other vendors or lab developed workflows can be used as long as the recommendations in the Training Packet for Alternative Nuclei Isolation protocols are followed. Recommendations are reviewed below.
Recommendations
When using alternative nuclei isolation protocols, it is necessary to include final concentrations of 0.8 U/μl RNase inhibitor and 1X protease inhibitor into the nuclei extraction buffer/nuclei lysis buffer during tissue lysis. Failure to do so can result in degraded cDNA.
After nuclei extraction is complete, centrifuge the nuclei suspension (preferably, 500 × g for 5 minutes at 4°C) to pellet and aspirate the supernatant. Use a P200 to carefully remove as much supernatant as possible without disturbing the pellet.
Important note:using a fixed angle centrifuge for pelleting nuclei for wash steps can result in excessive sample loss. Tubes compatible with swinging bucket centrifuges should be used to result in a flat pellet.
Add 1 ml of 1X Nuclei Suspension Buffer (1X NSB), mix well and spin at 500 × g for 5 minutes at 4°C.
Next, aspirate supernatant without disturbing the pellet and resuspend nuclei using 1X NSB.
Note: centrifuge speeds/times listed may need to be adjusted for non-mammalian sample types.
It is possible to use other kits or alternative protocols for nuclei isolation, but the extra time-consuming steps to minimize large debris to prevent clogging of microfluidic devices (eg, sucrose or other density gradients, magnetic bead purifications) can be harsh on the nuclei. This can increase nuclei leakage and prevent transcripts from paranuclear membranes from being captured.
Using filter steps is a gentle method for debris removal and is optimal for PIPseq technology, which has no instrument clogging issues from cellular or nuclear debris. After Dounce homogenizer steps, it is recommended to use two filter steps to remove debris. It is recommended to start with a 40 μm gravity filter. If the first filter step takes more than ~3 minutes, optionally use an Uberstrainer with negative pressure for the first filter step (but this will let through more debris), then immediately follow with a 40 μm gravity filter step to remove any remaining debris, which should then go much quicker.
For the second filter step, it is recommended to use a smaller sized filter, such as a 10 μm Uberstrainer while applying negative pressure. For nuclei from mouse brain or other larger nuclei sample types, a 20 μm Uberstrainer may be used. The faster this step can be completed, the better quality the isolated nuclei will be. Using negative pressure is preferable to gravity filtration.
When using an alternative nuclei isolation kits or protocols, the RNase inhibitor is user supplied when adding it to the nuclei extraction buffer or when formulating the 1X Nuclei Suspension Buffer (NSB). The Illumina Single Cell 3’ RNA kits provide enough RNase inhibitor to add with the samples into the PIP tube during Capture and Lysis.
Recommended alternative RNase Inhibitors include: Protector RNase Inhibitor (40 U/μl) and RiboGrip RNase Inhibitor (220 U/μl) are optimal for nuclei, with RiboGrip being the best choice for challenging sample types, such as those with lower RNA content and/or higher endogenous RNase (eg plants).
Enough Bovine Serum Albumin (BSA) is provided in the kits for the final nuclei suspension, but if users would like to include BSA in the 1X NSB used for wash steps (this is optional, and not used internally), additional BSA (molecular-biology grade) should be purchased.
DSP-Methanol fixation is an alternative option that can be used to help protect the mRNA in isolated nuclei before the Illumina Single Cell workflow and is especially recommended if it is not possible to proceed immediately to Capture and Lysis following nuclei isolation.
Refer to the Sample Preparation section of the Training packet for more information.
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