Experimental set up for mapping mutations using RNA-seq data.

Below are the methods we used to maintain fish, set up crosses and pool embryos, extract RNA from those pools, create cDNA and Illumina libraries, and perform RNA-Seq.

Detailed methods: pdf

Stock Maintenance
We have been able to map mutations generated in one strain and maintained through backcrosses to that same line (e.g. mutagenize AB and cross to AB). However, we have found that outcrossing to a different "mapping" strain (e.g. WIK) increases the number of SNPs identified, thereby increasing the confidence when identifying a mapping interval. As we have done more mapping it is clear that outcrossing really increases the power of mapping, with little expense in terms of candidate identification; we therefore highly recommend an outcross.

Crosses
We have performed all mapping experiments using single-pair crosses of known mutant carriers. From this cross we identify and sort mutant and wildtype sibling embryos into separate pools. We have pooled anywhere from 10 to 120 mutant embryos and successfully mapped the mutation. Using small numbers of animals results in large regions of linkage, but using large numbers of animals results in diminishing returns in regards to reducing the linked region. We suggest collecting ~50 mutant embryos for mapping. You must also collect wildtype siblings to use this method, and we have always collected an equal number of mutant and wildtype embryos.

RNA extraction
High-quality full-length RNA is key. Any method that results in non-contaminated, non-degraded messenger RNA will work. In our hands, Trizol (Invitrogen) results in the best results. Note that any leftover guanidinium thiocyanate or phenol has the potential to inhibit downstream reactions creating cDNA and sequencing libraries.

cDNA and Illumina library preparation
We  used the Illumina TruSeq kit preparation method to create sequencing libraries from our RNA samples.

---