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

Join Date: Aug 2017
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Quote:
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!
Conny
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.
Alex
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