Tweet
Hi
The 10x genomics join scATAC-seq and scRNA-seq (Single Cell Multiome ATAC + Gene Expression, as they called it) use a gel bead with 2 oligos. One of them is the RT primer, the other one is like a PCR primer to amplify the Tn5 tagged DNA. They called the 3' end of it: "Spacer: An 8-bp sequence on the Gel Bead ATAC Barcode oligo that enables barcode attachment to transposed DNA fragments."
I thought they first Tn5 tagged the nuclei, then generate droplet containing the nuclei and gel bead (with RT mix, also DTT to desolve the beads). The reaction was carried out inside the droplet at 37 C for 45 min and 25 C for 30 min. That looks like an RT. But I just dont understand how can they add the barcode to the 5' end of Tn5 adapter. Normally, we need to PCR it with primer with barcode. But their reaction 37/25 degree C doesn't seems to be able to achieve let the "spacer" to server as a PCR primer. So how can the spacer be linked to the Tn5 adapter DNA?
Or is it done by some kind of splint ligation?
Best
silin
the manual link, and i also attached the image of the 2nd oligo.
The 10x genomics join scATAC-seq and scRNA-seq (Single Cell Multiome ATAC + Gene Expression, as they called it) use a gel bead with 2 oligos. One of them is the RT primer, the other one is like a PCR primer to amplify the Tn5 tagged DNA. They called the 3' end of it: "Spacer: An 8-bp sequence on the Gel Bead ATAC Barcode oligo that enables barcode attachment to transposed DNA fragments."
I thought they first Tn5 tagged the nuclei, then generate droplet containing the nuclei and gel bead (with RT mix, also DTT to desolve the beads). The reaction was carried out inside the droplet at 37 C for 45 min and 25 C for 30 min. That looks like an RT. But I just dont understand how can they add the barcode to the 5' end of Tn5 adapter. Normally, we need to PCR it with primer with barcode. But their reaction 37/25 degree C doesn't seems to be able to achieve let the "spacer" to server as a PCR primer. So how can the spacer be linked to the Tn5 adapter DNA?
Or is it done by some kind of splint ligation?
Best
silin
the manual link, and i also attached the image of the 2nd oligo.
