Runing a mix of samples using Hashtags in different 10x chip lanes
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15 months ago
sccastillo • 0

Hi there,

I have the possibility of using Hashtag antibodies to multiplex four samples. These four samples correspond to two conditions where each condition have two biological replicates. I'm planing to load 40,000 cells in total between all samples to yield around 20,000 cells. I know that some people do superloading on the 10x lanes but I would like to just split the sample mix and use two lanes instead. However, I'm not sure about the approach to demultiplex the samples. Would it be possible? Since everything will be split in two lanes.

Alternatively, I could just put together two samples and run them separately, but here I'm afraid of batch effects between 10x lanes (although 10x says that the batch effect is very low).

I wonder if anyone have thoughts about these two approaches. I would really appreciate any recommendation.

Thanks a lot!

Steph

RNA-Seq 10x hashtag • 722 views
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If your samples are going to use standard 10x indexes (SI-GA-A1, SI-GA-A2 etc. for 4 samples) then you should be able to make a pool and run it on two lanes. Unless I am misinterpreting your question.

I know that some people do superloading on the 10x lanes

What does that mean? I am not familiar with that term.

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I think the poster means lanes on the 10X instrument, not lanes on the Illumina instrument.

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I see. Well then this is not a bioinformatics question. Following 10x tech support's recommendation would be the way to go.

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My question is if it would be possible to demultiplex 4 samples that are in different 10x lanes.

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Yes, I mean lanes in the 10x chip. The superloading is to load more cells than the recommended by 10x, >20,000 cells. The risk is that the multiplet rate increases, and you lose more cells.

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