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Old 11-01-2017, 09:24 AM   #1
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Location: Stanford

Join Date: Jun 2009
Posts: 181
Post Smart-3SEQ: a cheap, fast protocol that is sensitive enough for FFPE single cells

Our lab has just posted a preprint about our new RNA-seq protocol, Smart-3SEQ. It combines the previous 3SEQ method for 3' digital gene expression and Smart-seq/Smart-seq2 methods for working with very low amounts of input. The resulting protocol is actually simpler and faster than any previous one (~3 hours) and it uses very cheap off-the-shelf reagents (~$5). Combined with relatively inexpensive sequencing conditions (we use fewer than 10M 1x76 nt reads per human sample) it can be very useful for large-scale studies or just for saving money.

On the small scale, Smart-3SEQ is sensitive and accurate even down to single-cell inputs. Like 3SEQ but unlike Smart-seq/Smart-seq2, it is robust to fragmented RNA, such as from FFPE tissue. In the manuscript we demonstrate this by using Smart-3SEQ to profile single cells extracted from clinical archival tissue, which was not previously possible. Thus Smart-3SEQ enables a range of new studies in molecular pathology and other preclinical research.

The protocol itself is included with the preprint as Supplemental File 2. It includes special instructions for working with the Arcturus XT laser-capture microdissection system on either fresh or FFPE tissue. I've also attached two spreadsheets for ordering the oligonucleotides from IDT: one that you can copy and paste into the "Bulk Input" tool (the 2S primer must be ordered as Custom RNA and the rest as Custom DNA) and one that you can upload to order the barcoded PCR primers in a 96-well plate (you still need the 1S and 2S from the other file). Note that it may be possible to save more money by ordering the PCR primers with IDT's "Ultramer" synthesis, and then you don't need HPLC purification, plus you might only need one phosphorothioate instead of two - but we haven't tested that yet. The current PCR primers are for 48-plex single-indexing but we're now testing 96-plex unique dual indexes as well.

Another way to save money on the protocol is to make your own SPRI bead mix.

I'm happy to answer any questions here about the manuscript, the protocol, LCM, RNA-seq, etc.
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