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Recorded webinar: sNuc-Seq – High throughput RNA profiling of single nuclei with Nadia

Description

sNuc-seq allows researchers to profile gene expression in difficult to isolate cells as well as archived tissues. Dolomite Bio has developed and validated a new protocol for high throughput sNuc-Seq on the Nadia Instrument. This webinar will discuss:

  • Dolomite Bio’s approach to profiling single nuclei in a high throughput method using the Nadia Instrument
  • The benefits of using the sNuc-Seq application, how to overcome the challenges and how to implement it yourself
  • Top tips for Sample preparation, droplet optimization and library preparation
  • Presentation of sNuc-Seq data from both Dolomite Bio and Imperial College London (ICL)
  • A research case study form ICL: Using brain nuclei from post-mortem tissue to investigate Motor Neuron Disease

Questions and Answers

1. What is the maximum scale of cells and samples?

The largest cell size which we currently recommend can be encapsulated on the Nadia instrument is approximately 40µm. Larger cells will increase the risk of blockages occurring in the microfluidic junction or cartridge filter.The range of sample volumes which can be run on the Nadia instrument is between 100 and 250µl.

2. Do I need to buy a different cartridge to conduct sNuc-Seq on Nadia?

No, you will not need to buy separate Nadia cartridges to access sNuc-Seq as an application. As of our last update, all of our previous scRNA-Seq Nadia cartridges are now capable of performing either scRNA-Seq or sNuc-Seq as standard. Simply insert the cartridge into Nadia and you will be prompted to choose which protocol you wish to use. For further information on this feature, consult our Nadia User Manual.

3. Do we need to change parameters for encapsulating nuclei or are they the same as for single cells?

The protocol parameters for sNuc-Seq on Nadia are largely the same as the parameters for scRNA-Seq aside from the incubation time following dropletisation. This incubation time is increased for nuclei to allow more thorough lysis.Additionally, the protocol for isolating single nuclei prior to encapsulation is different and requires more time than the protocol for singulating whole cells. Both of these differences are covered in more detail in our newest sNuc-Seq for Nadia protocol.

4. Is there a way to limit mRNA background in the sNuc-Seq application?

Background mRNA can be limited at a few different steps of the protocol if this is a persistent problem. mRNA background can most effectively be limited during the nuclei isolation step, where tissue homogenisation should be conducted in a clean environment, exposing the lysate to the air for a minimum amount of time. Ideally, this can be conducted in a fume hood and on ice to avoid contamination. Make sure to aspirate off sufficient supernatant when spinning down and resuspending the nuclei in EZ buffer. Both of these steps are detailed in the sNuc-Seq on Nadia protocol. During sample loading on the Nadia, the integrated magnetic stirrers in the Nadia cartridge will agitate your nuclei samples. Depending on precise conditions, you may wish to decrease the stirrer speed to disturb your nuclei less. This decreases the chance of the stirrers causing damage to the nuclei and releasing mRNA into the sample solution where it can be encapsulated and contribute to junk mRNA. After sequencing has occurred, we also recommend that users construct a knee-plot with their unaligned reads by plotting their cellular barcodes against the cumulative reads assigned to those barcodes. From here, setting a threshold at the inflection point of this knee-plot can allow users to omit beads which occupied a droplet without a cell but that were still bound to background mRNA. More details on this pipeline and how to construct knee-plots are available in our NGS guide.For further information on this feature, consult our Nadia User Manual.