Question: RNA-strandness of single cell library prep methods
0
gravatar for piyushjo
5 weeks ago by
piyushjo100
piyushjo100 wrote:

EDIT: I TALKED TO THE REPRESENTATIVE AND SHE EXPLAINED ME THAT THE SMARTER ULTRA LOW + NEXETRA LIBRARIES ARE UNSTRANDED. I GUESS ITS THE SAME FOR FLUIDIGM USING THE SMARTER PROTOCOL. THANKS.

Hi,

I am little confused with what option to use for rna-stradness when aligning scRNA-seq data. I have two different dataset, one prepared using SMARTer ultralow RNA protocol and second using Fludigm-mRNA seq method. In both method, I see first strand being synthesized first and than the second strand, but as an input for Nextera it suggest the double stranded cDNA is used. Could you help me figure out what rna-stradness opton shall I use for HISAT2:RF (firststrand) or FR?

Smarter protocol research-pasteur-fr-smarter-ultra-low

Fluidigm mRNA seq protocol Screenshot-from-2018-12-09-13-29-51

ADD COMMENTlink modified 21 days ago by RamRS20k • written 5 weeks ago by piyushjo100

I guess yes firststrand (RF) sounds the one to use then.

ADD REPLYlink modified 5 weeks ago • written 5 weeks ago by Juke-341.7k

So I thought the same, but then I went on this website and thre SMARTer seq protocol is listed under FR. https://chipster.csc.fi/manual/library-type-summary.html

ADD REPLYlink written 5 weeks ago by piyushjo100

Good job contacting them was the thing to do. So it is not the same library as the one described by chipster ( SMARTer Stranded Total RNA )

ADD REPLYlink written 5 weeks ago by Juke-341.7k

Yeah. The tech mentioned that "Stranded" libraries preps have "stranded" mention in the kit name. I was confused by the description "maintain the strand information". She explained that refers to making cDNA properly without any strand information.

ADD REPLYlink written 5 weeks ago by piyushjo100

If you are not sure, you can process RNA-seq data using both stand options. Based on the results, it should be obvious which is the correct strand (if stranded, usually over 90% of the reads are on one strand).

See also this previous discussion: How to tell is sequencing data is unstranded, firststrand, or secondstrand

ADD REPLYlink modified 5 weeks ago • written 5 weeks ago by igor7.2k

I did that too. Both ways it was >90%.

ADD REPLYlink written 5 weeks ago by piyushjo100

@ piyushjo , in the embed text you pasted, there was an additional spammy link to a movie-related website that I removed just now. Please clean up embed links before you post them here. Usually, these image hosting providers add some stuff to redirect to their website and then additionally garnish it with some spam if we're not careful, and we need to remove this extraneous stuff before sharing.

ADD REPLYlink modified 21 days ago • written 21 days ago by RamRS20k

Sorry! I used the imagedb website.

ADD REPLYlink written 21 days ago by piyushjo100

Your links are from imgbb, so I'm not sure what happened. Maybe the service you used uses imgbb and just adds spam on top?

ADD REPLYlink written 20 days ago by RamRS20k
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