Hi,
In an effort to utilize single-cell RNAseq, I'm following the general outline of SmartSeq2 (thank you Picelli et al!) but with a slightly modified workflow in order to incorporate Unique Molecular Identifiers and cell-specific barcodes. I'm basing my strategy on the bioRxiv manuscript from Soumillon et al (2014) which was then used by a comparative methods bioRxiv manuscript a few months ago. My central question is about the library prep stage, however.
To the template-switch oligo, I've added UMI and cell-specific barcodes to the 3' end of the gene. I want to let the Nextera Tn5 add in the P7 adapter. But I redesigned the i5 adapter to add the P5 adapter sequence and also permit enrichment for the fragments that contain my UMI & barcode information. Since I do not have the final Nextera i5 sequences, I'm utilizing the TruSeq sequences. It's left me with an uncomfortably long i5 primer, but one that encodes P5, sequencing primer binding sequence, and a sequence to bring it all in.
I've attached a schematic (see pdf). My questions are:
1. Has anyone tried this strategy of a libraries with a Nextera end and a TruSeq end? Do you think the library prep step will succeed or will this fail during the enrichment PCR?
2. Have I made a mistake in understanding how the Tn5 inserts sequences?
3. Should I avoid the Tn5 and instead try something like NEBNext for library prep in order to make it easier to use a custom i5 primer?
Thank you!
Ramin
In an effort to utilize single-cell RNAseq, I'm following the general outline of SmartSeq2 (thank you Picelli et al!) but with a slightly modified workflow in order to incorporate Unique Molecular Identifiers and cell-specific barcodes. I'm basing my strategy on the bioRxiv manuscript from Soumillon et al (2014) which was then used by a comparative methods bioRxiv manuscript a few months ago. My central question is about the library prep stage, however.
To the template-switch oligo, I've added UMI and cell-specific barcodes to the 3' end of the gene. I want to let the Nextera Tn5 add in the P7 adapter. But I redesigned the i5 adapter to add the P5 adapter sequence and also permit enrichment for the fragments that contain my UMI & barcode information. Since I do not have the final Nextera i5 sequences, I'm utilizing the TruSeq sequences. It's left me with an uncomfortably long i5 primer, but one that encodes P5, sequencing primer binding sequence, and a sequence to bring it all in.
I've attached a schematic (see pdf). My questions are:
1. Has anyone tried this strategy of a libraries with a Nextera end and a TruSeq end? Do you think the library prep step will succeed or will this fail during the enrichment PCR?
2. Have I made a mistake in understanding how the Tn5 inserts sequences?
3. Should I avoid the Tn5 and instead try something like NEBNext for library prep in order to make it easier to use a custom i5 primer?
Thank you!
Ramin