The effect of initial conditions and RANKL treatment on long-term osteoclast dynamics We next assessed the effects of the plating density and RANKL

.6% of the contigs down-regulated at fc #0.5 and 10.3% up-regulated at fc $2. In red muscle, 1,366 contigs appeared in Kenpaullone web swimmers only and thus indicated exercise specific transcripts, while 1,503 contigs appeared in resters only. In white muscle, 1,361 contigs appeared in swimmers only and 1,185 contigs appeared in resters only. However, all of these contigs that were specific for swimmers or resters were small except for the mentioned interferon-induced very large GTPase 1-like contig of 418 nt with a RPKM value of 20.2 that was only found in the white muscle of resters. Because RPKM values are determined on 25833960 basis of 1000 nt, decreases in contig size are associated with increased noise, which can be defined as the occurrence of false differentially expressed contigs due to their small size. Because of this, contig size thresholds need to be applied for justifying the quantification of expression. In the present study, although abundance was positively correlated to size, as also reported by Hegedus et al., we have applied a size Red muscle Resters Sequence reads 17.885,503 Mapped reads unique non-unique Unmapped reads 8.097,376 7.809,917 287,459 9.788,127 White muscle Swimmers Resters 17.415,589 7.642,409 7.394,390 248,019 9.773,180 15.082,988 6.743,174 6.483,635 259,539 8.339,814 Swimmers 16.588,952 6.992,219 6.739,483 252,736 9.596,733 The number of 51 nucleotides sequence reads, mapped reads and unmapped reads for each individual group are indicated. doi:10.1371/journal.pone.0053171.t001 Deep RNA Sequencing of Trout Muscle RED MUSCLE Number Total numbers Contigs Contigs $200 nt Contigs $500 nt Maximum contig length Number of contigs differentially expressed by RPKM Contigs down-regulated in swimmers vs. resters 14,932 Contigs up-regulated in swimmers vs. resters Exercise specific contigs Non exercise specific contigs Contigs annotation accuracy and origin Sum of well annotated contigs Annotation from SIGENAE salmonid ESTs Annotation from zebrafish RefSeq proteins Annotation from Metazoa RefSeq proteins Contigs $500 nt Contigs down-regulated in swimmers vs. resters 118 Contigs up-regulated in swimmers vs. resters 51 1.8 0.8 66,035 46,785 8,649 10,601 44.3 31.4 5.8 7.1 21,172 1,366 1,503 10.0 14.2 0.9 1.0 149,159 31,609 6,512 15,779 21.2 4.4 % of total WHITE MUSCLE Number % of total 118,572 32,061 5,977 16,748 27.0 5.0 14,928 12,167 1,361 1,185 12.6 10.3 1.1 1.0 61,437 41,696 9,104 10,639 51.8 35.2 7.7 9.0 71 29 1.2 0.5 Numbers and maximum size of assembled contigs, differential contig expression by Reads Per Kilobase per Million mapped reads values, iterative BLAST annotation results and differentially expressed contigs $500 nt are indicated. doi:10.1371/journal.pone.0053171.t002 6 Deep RNA Sequencing of Trout Muscle threshold of $500 nt since this size group was virtually without noise as visualized by MA plots. Contigs that were smaller than 500 nt showed increasing false differential expression noise with smaller size. The larger contigs showed less noise in differential expression as determined by RPKM. In red muscle, the expression of 118 large contigs was down-regulated at fc #0.5 and 51 were up-regulated at fc $2 in swimmers. In 23838678 the white muscle of swimmers, the expression of 71 large contigs was down-regulated at fc #0.5 and 29 were up-regulated at fc $2. Differentially Expressed Genes Common in Red and White Muscle Red and white muscle shared only seven contigs that were differentially expressed in response to exercise on the

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