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When one is preparing cells for the 10x genomics single cell RNA Seq platform, the manual says to dilute the cells (in media or 1x PBS) into water for a final volume of 46.6ul to achieve your targeted cell recovery. Do users mix their cell suspension in with water, then pipette into the mastermix at this step?
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In the 3' v2 protocol, 10x recommended making your mastermix, then adding water to the mastermix and pipette mixing, and then finally adding your cells to the mastermix with water.
The newest 3' v3 protocol, they do not specify an order at that step, but I have been doing it in the same order as the v2 without issue. -
Thanks Joe. It seems very confusing that they would have written so clearly in the v2 manual but this critical description is removed from the v3 manual! I wonder who made such a terrible decision to remove it. Those who pick up the v3 manual and not know of v2 would not be able to see through this. If that is the order required, it should be given in both manuals.Comment
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Love this - good data definitely starts from good input, and poor input can only give relatively poor data. I particularly like the mention of Nanodrop/absorbance based methods for quantification. It's such a toss up if you'll get an accurate reading or what amounts to a randomly generated number, and a lot of library/sequencing related issues can be traced back to poor quant.Yesterday, 11:43 AM
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I’m not a sequencing expert. I’m a purification scientist who uses NGS to evaluate workflows my group develops. With this perspective, we think about the sample first and the NGS workflow second. The sequencer is an exceptionally honest reporter, but it can only report on what you give it, so whether you get clean, interpretable data from an NGS workflow is largely determined before you begin.
Here are nine questions we think about, in roughly the order they matter, before...
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