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Old 12-03-2016, 10:32 AM   #1
seqgirl123
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Default Smart-Seq v4 ultra low RNA kit

Hello,

I would be using this kit for single cell RNA in a 96 well plate. I will be preparing the reactions in a plate. I would like to know if anyone has spiked in ERCC or Array Control RNA Spikes into the lysis mastermix or any internal RNA controls into the reaction, and how did you do this? Did you spike it into the lysis mastermix, and if yes, at what volume of this versus the other lysis reagents? Preferably i will like to use ERCC spike ins, how do I spike them into the reaction setup?

My other question is I would like to have the RT step barcoded per single cell. Where can I get 96 barcodes and how to add them into the RT step of this workflow?

Last edited by seqgirl123; 12-03-2016 at 10:38 AM.
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Old 12-04-2016, 01:47 PM   #2
cmbetts
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I haven't done it for that kit specifically, but I like putting the ERCCs into the lysis mix. That way the performance of the ERCCs can be a better reflection of RNases coming in with the cells and other things that are proxies for reaction efficiency. Ideally, you want them to take up as little volume as possible and not to be adding too many that they swamp out your cell's signal. Using ~200k mRNA/cell, you can calculate how many ERCC molecules would make up an appropriate percentage of reads and add them to the lysis buffer before aliquoting to the wells, which will allow you to have such a low volume added per well that you can effectively run the protocol without modification.
If you're only looking for cell barcodes, they have a 3' DE version of the kit available, which might fit your needs off the shelf. If you want cell barcodes + UMI, you'll have to order yourself. There was a paper in PLoS One a while ago, don't have the citation handy, that has several lists of >= 2 hamming distance barcodes that can be used for cell labels that can be added to the RT step, or you could pull them directly out of the methods section of the SCRB-Seq paper.
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Old 12-05-2016, 05:49 AM   #3
seqgirl123
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cmbetts, thank you for your reply. I will be adding ERCC to the lysis mix in single cells. I prefer ERCC too. Do you know where I can find how to dilute ERCC into single cell lysis reactions?

Please post the link to the PLoS paper or any of those papers that have the barcodes listed. I need to order the cell barcodes + UMI asap. I will greatly appreciate any help!
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Old 12-07-2016, 11:57 AM   #4
seqgirl123
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Hello,

I have found a paper here http://www.nature.com/nmeth/journal/...meth.2772.html that the supplementary table 2 "list of custom oligonucleotides" they published the full list of their Tn5 sequences.

Has anyone used these sequences for the smart-seq v4 kit?

Are there better sequences?

Last edited by seqgirl123; 12-07-2016 at 12:07 PM.
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Old 03-16-2017, 01:39 PM   #5
hideandSEQ
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Quote:
Originally Posted by seqgirl123 View Post
Hello,

I have found a paper here http://www.nature.com/nmeth/journal/...meth.2772.html that the supplementary table 2 "list of custom oligonucleotides" they published the full list of their Tn5 sequences.

Has anyone used these sequences for the smart-seq v4 kit?

Are there better sequences?
I've never tried that method, although I wish we did because amplification bias often plagues our C1 data.

I believe that was the first UMI method designed for use on a C1 but since then there have been papers that claim to be better. I think the TSO from the Islam paper left something to be desired in RT, and the UMI barcodes could be more complex

This one is the most recently published one I've found for modifying the C1, they claim their method is even better.
https://academic.oup.com/nar/article...ve-single-cell

Semi-related, this paper is an excellent guide for using the C1. I think they go into why they believe normalizing gene expression by spike ins is a poor choice.

http://www.nature.com/articles/srep39921
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