MRNA Decay

To define candidate loci that may have been targeted by selection, we first calculated nS_L for all SNPs and then established a cutoff based on the 1st and 99th percentile of the empirical distributions for each population independently. In addition, we assigned P values to each SNP using parametric simulations, as described in the section Estimation of P values. Results are presented by ranking the absolute normalized nS_L scores in each population. For the nS_L scores in the 1st and 99th percentiles, we performed a hierarchical cluster analysis of the fragment lengths between pairwise differences. Based on this analysis, we determined the number of different haplotypes backgrounds for each allele. This information was used for plotting purposes (visualization of the haplotypes carrying the derived and the ancestral allele).
We used RefSeqGenes (http://www.ncbi.nlm.nih.gov/refseq/rsg/, last accessed March 3, 2014) to assign SNPs to genes, when applicable. For this purpose, we focused solely on genes with transcript products, that is, with mature mRNA. When more than one transcript was available we used the longest transcript product. We then assigned SNPs into genes according to the transcript coordinates. We kept the most extreme score per gene to rank all genes according to this score and performed the Gene Ontology enrichment analysis using the GOrilla tool Eden et al. (2009) with two list of genes; the list of candidates (top 3% of genes) and the list of all genes in the study (from 14,269 to 14,279 genes, depending on the population). Any category belonging to GO process, GO function and GO component was considered significant if having a corrected P value ≤ 0.05. The corrected P value was computed as the P value provided by GOrilla times the number of tested GO terms.
iHS was computed using the code released by Voight et al. (2006) (link). iHS scores were normalized similarly to nS_L. The EHH and rEHH were computed with our own code according to the description given by Sabeti (2005) , where the EHH and rEHH of a particular core haplotype t are calculated as follows:

where c is the number of samples of a particular core haplotype, e is the number of samples of a particular extended haplotype, and S is the number of unique extended haplotypes (Sabeti 2005 ).
The rEHH is . And, is the decay of EHH on all other core haplotypes combined:

where n is the number of different core haplotypes (Sabeti 2005 ).
For the purpose of this article, the core haplotypes of interest are defined by the presence or absence of a single SNP.

NT-SMG-9 cDNA was subcloned between the EcoRI and NcoI sites on modified N-terminal HisTag pRAT4, pRHO and pGEX-6P-2 plasmids (GE Healthcare Bio-Sciences, Buckinghamshire, UK). The initial GST fusion construct was made comprising amino acids 1–180. After the observation of spontaneous self-cleavage in the initial GST-fusion construct, the site of cleavage was identified by mass spectroscopy and the construct re-cloned comprising amino acids 12–180. All proteins were expressed in Escherichia coli strain BL21(DE3) and the expression detected in a soluble fraction after cell lysis by sonication. GST-fusion proteins were purified with GST-Trap columns (20 ml, GE Healthcare Bio-Sciences) and NT-SMG-9 was isolated from GST after digestion with the 3C protease. A second GST-trap column followed by a cation exchange chromatography in SP-sepharose HiTrap colum (5 ml, GE Healthcare Bio-Sciences) followed by a final step of gel-filtration chromatography (Sephacryl S-100, GE Healthcare Bio-Sciences) yielded a highly pure preparation as judged by SDS–PAGE after Coomassie brilliant blue staining. Protein concentration was determined by UV absorption at 280 nm. The protein solutions were concentrated with an Amicon Ultra device (Millipore, Bedford, MA, USA). Mass spectroscopy was used to assess the identity as well as the purity of final preparations.