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1 Molecular Systems iology Evolutionary divergence of mouse P Mei-Sheng Xiao et al Expanded View Figures Nucleotide percentage (%) T G Nucleotide percentage (%) T G Frequency of PS motif (%) filtered_ps raw_ps pseudo_ps Position to cleavage site 124bp L SP leavage site bp Position to cleavage site D E F L_ps( ) L_SP_ps( ) SP_ps( ) Log2 (read counts) U UU UUU UU GU U u UU GU UG G U UG L_ps( ) L_SP_ps( ) SP_ps( ) Log2 (read counts) Figure EV1. Features of identified ps clusters., The nucleotide composition around the cleavage sites identified in 57L/6J and SPRET/EiJ, respectively. The frequency of 13 known PS motifs within 100 nt upstream of ps identified in SPRET/EiJ. pseudo_ps represents ps identified by using reads without nongenomic T (Materials and Methods). raw_ps and filtered_ps represent the ps determined by the PSS reads with and without further filtering, respectively (Materials and Methods). X-axis is different types of PS motifs, and y-axis shows the percentage of ps with the specific motif in the upstream region. D Up: We retained only the ps clusters, of which the region ( 124 nt to 24 nt) flanking their cleavage sites could be reciprocally aligned between the genomes of 57L/6J and SPRET/EiJ using LiftOver. Down: the Venn diagram shows the number of ps that were identified in both strains (shared) or only in one strain. E, F The cumulative distribution function (DF) of the number of 3 0 mrn-seq reads mapped to shared, 57L/6J-specific, and SPRET/EiJ-specific ps from 57L/6J- and SPRET/EiJ-derived sample, respectively. EV1 Molecular Systems iology 12: ª 2016 The uthors
2 Mei-Sheng Xiao et al Evolutionary divergence of mouse P Molecular Systems iology Rep2 (log2 read counts) 20 r = 0.98 n = Rep2 (log2 read counts) 20 r = 0.98 n = Rep1 (log2 read counts) Rep1 (log2 read counts) D 3 mrn-seq (log2 read counts) r = 0.72 n = mrn-seq (log2 read counts) r = 0.76 n = RN seq (log2 RPKM) RN seq (log2 RPKM) E F 3 mrn-seq (log2 read counts) r = 0.79 n = mrn-seq (log2 read counts) r = 0.82 n = RN Seq (log2 RPKM) RN Seq (log2 RPKM) Figure EV2. Quality control of 3 0 REDS and 3 0 mrn-seq., Reproducibility of 3 0 mrn-seq results between two replicates in 57L/6J and SPRET/EiJ, respectively. The scatterplots compare the number of reads mapped to each ps between the two replicates., D The correlation of gene expression level estimated by RN-Seq (x-axis) and 3 0 mrn-seq (y-axis) in 57L/6J and SPRET/EiJ samples, respectively. E, F The correlation of gene expression level estimated by RN-Seq (x-axis) and 3 0 mrn-seq (y-axis) in two mouse strains, but only for genes with single ps isoform. ª 2016 The uthors Molecular Systems iology 12: EV2
3 Molecular Systems iology Evolutionary divergence of mouse P Mei-Sheng Xiao et al Rep2 (allelic ps usage difference) r = 0.90 n = Figure EV3. Scatterplot comparing the allelic difference of ps usages measured in the two independent replicate experiments Rep1 (allelic ps usage difference) EV3 Molecular Systems iology 12: ª 2016 The uthors
4 Mei-Sheng Xiao et al Evolutionary divergence of mouse P Molecular Systems iology L>SP_ps (940) P value = 0.21 SP>L_ps (936) P value = 0.73 control_ps (2502) L>SP_ps (940) P value = 0.01 SP>L_ps (936) P value = 0.08 control_ps (2502) Difference of MFE (L SP) Difference of MFE (L SP) L>SP_ps (940) P value = 0.72 SP>L_ps (936) P value = 0.38 control_ps (2502) Difference of MFE (L SP) Figure EV4. Predicted RN secondary structure in the flanking region of ps. The cumulative distribution function (DF) of allelic difference in MFE of distal upstream region (: UE), proximal downstream region (: DE), distal downstream region (: DE) from ps with usage biased toward 57L/6J (L, blue line), SPRET/EiJ (SP, red line), or without biases (control, gray). The statistical significance of the difference between biased group and control group was determined by Kolmogorov Smirnov test. ª 2016 The uthors Molecular Systems iology 12: EV4
5 Molecular Systems iology Evolutionary divergence of mouse P Mei-Sheng Xiao et al PRS score TSS(15939) Start codon(5934) Stop codon(10628) ps(27514) all (27514) distal (1047) ase ase ase ase PRS score ase ase ase ase Figure EV5. RN secondary structure in TSS, start codon, stop codon and ps. PRS scores (y-axis) were plotted on each base (x-axis) within the ps flanking region. lack and red lines represent the PRS scores calculated based on all ps and only the most distal ps, respectively. The gray bar marks the upstream region ( 30 nt to 15 nt) known to contain ps signal. Figure EV6. Motifs impact ps usage. The same as Fig 4, but after removing all the ps containing canonical ps motif U in the 100 nt upstream region. Scatterplot comparing the allelic difference in hexamer frequency in downstream region (0 100 nt) between two groups of ps, one with usage biased toward 57L/6J (L, x-axis) and SPRET/EiJ (SP, y-axis), respectively. Each gray dot represents one specific hexamer, and the dot size represents the frequency of the hexamer. E oxplots showing the repressive effect of poly(u) tract on ps usage is dependent of its length. g indicates both alleles have the same length of poly(u) tract (6 Us) in the 100 nt upstream region. U 5 /U 4 /U 3 indicates only one allele has an intact 6 Us, whereas another allele has only 5/4/3 Us (). Y-axis represents the allelic difference in ps usage. In panels (D and E), the length of poly(u) tract increases to 7 (D) and 8 (E), respectively. Mann Whitney U-test was used to determine the statistical significance (P < 0.05). The solid horizontal bars, box ranges, the upper and lower bar represent median, 75 th percentile, 25 th percentile, maximum and minimum value, respectively. F Scatterplot comparing the frequency of all hexamers in the 100-nt region upstream of cleavage sites between trans-regulatory (x-axis) and control ps without parental divergence (y-axis). EV5 Molecular Systems iology 12: ª 2016 The uthors
6 Mei-Sheng Xiao et al Evolutionary divergence of mouse P Molecular Systems iology Hexamer frequency difference (SP L) UUUUUU UU Hexamer frequency difference (SP L) Hexamer frequency difference (L SP) Hexamer frequency difference (L SP) D E ps usage change ps usage change Hexamer frequency (ontrol) ps usage change g(u 6 ) U 5 U 4 U 3 g(u 7 ) U 6 U 5 U 4 U 3 g(u 8 ) U 7 U 6 U 5 U 4 F Hexamer frequency (Trans) Figure EV6. ª 2016 The uthors Molecular Systems iology 12: EV6
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