G&T-seq

[firsal]

Hi,
I want to try G&T-seq protocol for sequencing RNA and DNA from one single cell. I have already experience in using Smart-seq 2 and MDA protocols.

But I'm interested to know if someone has tried this protocol and if it's easy to set up, especially the first part of the method to capture mRNA using magnetic beads.
In general what are the limitations and difficulties in this protocol?

Tnx
F

[pmiguel]

"G&T", to me means "Gin and Tonic". But I presume you are abbreviating "Genomics and Transcriptomic"?

--
Phillip

[Conradona]

Hi F,
you might have noticed that there is a detailed protocol for G&T-seq online now since last week, published in nature protocols: “Separation and parallel sequencing of the genomes and transcriptomes of single cells using G&T-seq”. setting the method up with this detailed info shouldn’t be too difficult. I managed before but it took me a while and I will include some modifications as soon as possible. One of the main problems in the beginning was the MgCl2 concentration in the reverse-transcription mastermix. It’s too high in the original paper from last year and I didn’t get any amplified cDNA at all. I tried the MgCl2 concentration like in the smart-seq2 paper and it worked. There are some other minor things corrected in the new paper.
Good luck!
conny

[firsal]

Hi Conny,

Thanks for your comments. I also noticed the Mgcl2 is high as I used higher Mgcl2 in my smartseq2 protocol once and it didn't worked.

F