Molecular biology protocols:
The most recent protocol for Smart-seq3 (protocol version3; updated Feb 19,2020) is available at protocols.io, here.
We will continously link to newer protocol verions, should we make further improvements.
We have integrated Smart-seq3 data processing capabilities into zUMIs (Parekh et al. 2018) to correctly parse molecular and cellular barcodes in an efficient analysis pipeline.
The computational pipeline to process Smart-seq3 data, reconstruct RNAs, assign RNAs to alleles and transcript isoforms is available in our github repository smart-seq3.
Frequently asked questions:
When making smart-seq3 libraries, I get lower yield then with Smart-seq2 TSO?
It is mentioned in the paper, and also protocol. The yield is lower in Smartseq3 because we cant use the semi suppresive TSO/PCR design as Smartseq2. That is also why we suggest to add 1-2 more PCR cycles for Smartseq3 to reach similar cDNA yields. The complexity within the libraries with Smart-seq3 are however more complex!
I have been making lots of Smart-seq2 libraries, but for me Smart-seq3 does not work?
There are more differences with Smart-seq3 that simply the chemistry, and users have to change their habits away from Smartseq2 to Smartseq3. Users that makes libraries as in Smart-sqeq2 but with Smart-seq3 reagents will run into problems. Please look at the protocol closely, and it should work as a breeze. If not, please email us and we will help you get going!