Prepare Nuclei Suspension
Illumina recommends that you prepare nuclei suspensions using the Illumina Single Cell Nuclei Isolation Kit to ensure consistent, high-quality nuclei across frozen mammalian tissues. Refer to the Illumina Single Cell Nuclei Isolation Kit Product Documentation for instructions.
If using an alternative nuclei isolation protocol, use the provided NSB to prepare NSB working solution (refer to Prepare NSB Working Solution) for the final nuclei suspension. Substitute NSB working solution for the wash steps in the alternative nuclei isolation protocol (at least 1 ml of NSB working solution in up to two wash steps). This kit only includes enough BSA for the final nuclei suspension, so make sure to add user-supplied BSA (1% final concentration) and RNase inhibitor (0.8 U/µl final concentration) if needed for the wash steps.
Alternative Nuclei Isolation Protocols
| • | When using alternative nuclei isolation protocols, it is necessary to include 0.8 U/µl RNase inhibitor final concentration and 1X protease inhibitor into the nuclei extraction buffer/nuclei lysis buffer during tissue lysis. |
| • | After nuclei extraction is done, centrifuge nuclei suspension (500 × g for 5 minutes at 4℃) to pellet and aspirate supernatant. Use a 200 µl pipette to carefully remove as much supernatant as possible without disturbing the pellet. |
Using a fixed angle centrifuge for pelleting nuclei for wash steps can result in excessive sample loss. Use tubes compatible with swinging bucket rotor centrifuges to ensure a flat pellet.
| • | Add 1 ml of NSB working solution, mix well and spin at 500 × g for 5 minutes at 4°C. |
| • | Aspirate supernatant without disturbing the pellet and resuspend nuclei using NSB working solution. |
| • | If unable to leave less than 100 µl supernatant remaining, increase the wash volume to 2 ml. |
The wash step removes reagents inhibitory to the assay from the nuclei suspension (eg, FBS, BSA > 1%, high salt or calcium, carryover organic solvents). Failure to remove inhibitory reagents can cause cDNA QC failure.
Refer to Prepare NSB Working Solution for instruction on preparing Nuclei Suspension Buffer for various applications. Refer to the Illumina Single Cell Nuclei Isolation Kit product documentation for recommendations on the resuspension volume per mg tissue for various tissue types. It is not recommended to process more than 50 mg of tissue per sample at once to achieve optimal lysis.
After preparing the nuclei suspension, place samples on ice and proceed immediately to Capture and Lysis. Do not freeze isolated nuclei. If not proceeding directly to capture and lysis, it is recommended to fix nuclei to prevent mRNA degradation. Refer to Prepare Nuclei Suspension for details.
To prepare 1 ml of NSB working solution, combine the volumes from the following table. Prepare sufficient volume for the final resuspension with NSB working solution per sample. Exact volumes required might be sample size and type specific to achieve optimal nuclei concentration.
Refer to DSP-Methanol Fixation for Nuclei Protocol Instructions for the volume to use for the final suspension before fixation.
|
Component Name |
NSB Working Solution Volumes for Fresh Nuclei (µl) |
NSB Working Solution Volumes for Fixed Nuclei (µl) |
|---|---|---|
|
Nuclei Suspension Buffer |
167 |
167 |
|
Nuclease-Free Water |
773 |
833 |
|
25% BSA |
40 |
Not applicable |
|
RNase Inhibitor (40 U/µl) |
20* |
Not applicable |
|
Total |
1000 |
1000 |
* Alternatively, you can use 40 µl RNase inhibitor if provided at 20 U/µl and 753 µl nuclease-free water. Refer to RNase inhibitor recommendations in User-Supplied Consumables & Equipment.
