Smart-3SEQ

Attached is a set of spreadsheets to automate your calculations for diluting the TS-RT and PCR

The Smart-3SEQ paper has been posted online as an Advance Article in the next issue of Genome

How many PCR cycles did you apply? perhaps try 20? The laser capture material protocols should

These oligonucleotides are relatively expensive and sequencing experiments even moreso, so I wouldn

Thank you so much for the analysis!! That's great to hear it is still sequenceable and I'll

Our genome centre has been doing a 1% PhiX spike-in, should they be using more? No, the low signal

Thanks. What's the paper that compared the stains? On 6/27/22 6:29 AM, Adam Passman wrote: Thanks

The QC metric that will be really informative from this is the proportion of alignable reads after

That's a very good question and I don't have a very good answer. The short version is: if you

I've never set up a NovaSeq myself, but the MiSeq Denature & Dilute protocol calls for 5 μL @

in fact, this may help our core avoiding waiting for libraries to load in the other lanes. On this

These configuration files add support for i5-only indexing on the MiSeq and remove a few extraneous

Thanks for sharing all this information. I would say these samples look great for FFPE, though I'

Generally that sounds normal. There's always a little bit of variation in yield from one library

Thanks for confirming. Here we go...:) On Thursday, 5 August 2021 at 10:18:17 UTC-6 Joe Foley wrote:

Sweet. Thanks. j On Tuesday, 20 July 2021 at 15:45:31 UTC-6 Joe Foley wrote: Yes, in the second SPRI

I haven't looked much at different RNA qualities, since the protocol works even on very degraded

Right. On 4/24/21 1:19 AM, Erin Wu wrote: OK, Thanks very much! You mean the main problem is that

What we saw with low-quality libraries (FFPE LCM) was a lot of poly(A) artifacts in the i7 index read

Hi, For our last set of experiments we did 30% on the NovaSeq, with the intention of optimizing down

This library appears severely overamplified. The wide peaks around 500 and 10000 bp are almost

You can stain the slides immediately after completely dry. The tissue can fall off from slides

In the earliest stages of developing the protocol, we did an extra cleanup before PCR, but we found

There are several possible reasons why we see read alignments in negative-control samples. One

Ok, I see. Sincerely thanks for your reply! 在2020年8月18日星期二 UTC+8 上午1:12:32<Joe Foley> 写道： It

Thanks! It is good to know that the mapping percentage we get is not too low for this method. Thank

Yep, the yield was better than expected, likely because we started with almost perfect RNA samples

We've just posted yet another revision (possibly the last?) of the Smart-3SEQ manuscript on

Thanks for the comprehensive reply! If it is of interest to anyone I will post updates on how this

It depends on your input material. In the dataset from human reference RNAs (in the paper) across the

I don't favor deduplication for this protocol. With any variant of 3SEQ we expect a lot of

For LCM FFPE libraries we always use 76-base single-end reads (the shortest option) on the NextSeq,