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Old 08-06-2017, 05:51 AM   #7
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Location: HongKong

Join Date: Aug 2017
Posts: 1

Originally Posted by Conradona View Post
Dear all,

I am preparing single-cell libraries according to the method described in Macaulay I, et al. G&T-seq: Parallel sequencing of single-cell genomes and transcriptomes. Nature Methods, 2015.

The amplified cDNA seems to look fine on the tapestation when I add no ERCC spike-in controls.

But I get strange double peaks when I add the controls and the tapestation screentape gets overloaded. The paper says to use 1 μl of a 1:250,000 dilution and I went up to 1:500,000 and 1:1,000,000 but get the same results.

The project leader tells me to just dilute more and more but I am suspicous that this won’t solve the problem.

Thanks in advance for any hints!
Hi Conradona,

Recently, I am working with single cell pick with Smart-seq2 method and encounter the same problem as you. Without adding ERCC, the fragment analyzer shows good quality of amplified cDNA. However, after adding ERCC into lysis buffer, a strange double peaks appears as you said, even the same as negative control.

Could you tell how you solve this problem in the end?

Thank you very much.

Best regards.
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