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Thoughts on ERCC Spike Ins?

Posted By: Winegard, on Jan 9, 2017 at 8:23 AM

Hello 10Xers,

 

As we get more and more customers wanting to run 10X sc-RNA-seq we are getting more questions regarding the possible use of ERCC Spike Ins as controls.  Anyone have thoughts on this?  Is anyone currently using them (We have not for any of our 10X experiments but have for other single cell experiments).  My concern is that the spike ins will end up in every droplet and therefore cause issues with cell counts from Cell Ranger....

 

Thanks in advance,

 

Neil

 

4 Replies

Re: Thoughts on ERCC Spike Ins?

Posted By: jens-10x, on Feb 21, 2017 at 8:00 AM

Hi Neil,

 

We are currently working on a Demonstrated Protocol that outlines the use of ERCCs with our Single Cell assay.
If you want to proceed prior to our protocol release, I have provided some details below that you should consider before moving forward:

 

ERCC spike-ins can be added to the master mix, but keep in mind that they will distribute evenly across all ~80K GEMs generated in a channel, not just the ones containing cells. Please note that Cell Ranger supports alignment to ERCC references, but not yet further analysis of the results. You may therefore have to disregard the summary outputs and directly analyze the unfiltered gene-barcode matrix.

 

The optimal load will depend on GEM volume, number and expected losses due to dead volume. For your reference, we estimate that diluting the ERCC mix 1:10 and using 3 uL of that in 100 uL of master mix will result in a mean of 169,000 RNA molecules per GEM.

 

Lastly, if your customers are interested in learning more about ERCCs with our system I recommend reading the attached paper where we employ ERCCs to directly measure cDNA conversion rates.

 

 

We will have many more details in an official Demonstrated Protocol that we intend to release in the near future.

 

Best,

Jens

Re: Thoughts on ERCC Spike Ins?

Posted By: james, on Feb 22, 2017 at 6:31 AM

Hi Jens,

Has 10X taken a look at the more complex RNA controls from Lexogen (SIRVs) or the Garvan (SEQUINS)?

We're thinking about which control to standardise to and the ERCCs are going to get a 2.0 revamp anytime soon so using the current controls would be useful to get experience but might not be so great long-term.

James.

Re: Thoughts on ERCC Spike Ins?

Posted By: ebahl, on Oct 19, 2017 at 9:57 AM

Hi Jens,

 

Thank you for your detailed response to this question. Are there any updates regarding the mentioned official Demonstrated Protocol?

 

Thanks,

Ethan

Re: Thoughts on ERCC Spike Ins?

Posted By: cassie, on May 27, 2019 at 1:36 AM

To whom it may concern,

 

I try to calculate the amount of original ERCC molecule, but I could not get the figure as Fig 2d in the paper from attachment.

Calculation of the amount of ERCC molecure is as follows, could you please help me check out which part goes wrong?

602214.15 * 3 * concentration.mix1 / 1000

 

Thanks for any information you may provide.

 

Best regards,

Cassie