<p>Hi there,<br />
I know that this is probably a common and newbie question but I can&#39;t find the solution. I have a strand specific RNA-seq data, specifically first-strand. Which is the correct way to specify this in trinity, RF or FR? I&#39;ve read the manual but is not clear for me...<br />
<br />
Thanks in advance.</p>

The Trinity documentation says:

If you have strand-specific data, specify the library type. There are four library types:

 - Paired reads:
  - **RF**: first read (/1) of fragment pair is sequenced as anti-sense (reverse(**R**)), and second read (/2) is in the sense strand (forward(**F**)); typical of the dUTP/UDG sequencing method.
  - **FR**: first read (/1) of fragment pair is sequenced as sense (forward), and second read (/2) is in the antisense strand (reverse)
 - Unpaired (single) reads:
  - **F**: the single read is in the sense (forward) orientation
  - **R**: the single read is in the antisense (reverse) orientation

The [TopHat manual](https://ccb.jhu.edu/software/tophat/manual.shtml) says:

    Library Type        Examples                    Description
    fr-unstranded       Standard Illumina           Reads from the left-most end of the fragment (in transcript coordinates) map to the transcript strand, and the right-most end maps to the opposite strand.
    fr-firststrand      dUTP, NSR, NNSR             Same as above except we enforce the rule that the right-most end of the fragment (in transcript coordinates) is the first sequenced (or only sequenced for single-end reads). Equivalently, it is assumed that only the strand generated during first strand synthesis is sequenced.
    fr-secondstrand     Ligation, Standard SOLiD    Same as above except we enforce the rule that the left-most end of the fragment (in transcript coordinates) is the first sequenced (or only sequenced for single-end reads). Equivalently, it is assumed that only the strand generated during second strand synthesis is sequenced.

To complete that, in Trinity `fr-firststrand` corresponds to `RF` and `fr-secondstrand` corresponds to `FR`.

You can have a look here: http://rnaseq.uoregon.edu. They well explain the difference between first-strand and second-strand synthesis.

You can also have look to this publication: Sequencing technologies - the next generation. Nature reviews. Genetics. 2010. doi:10.1038/nrg2626

Briefly, if you know the technology used for the sequencing you should be able to guess.