SUPPLEMENTAL FIGURE 1
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1 SUPPLEMENTL FIGURE 1 C Supplemental Figure 1. pproach for removal of snorns from Rpl13a gene. () Wild type Rpl13a exonintron structure is shown, with exo in black and intronic snorns in red rectangles. Numbering in black reflects the limits of the exonic sequences, relative to the tralation start of the gene. () Enlarged detail of snorncontaining intro is displayed. Numbering in red reflects the limits of the intronic sequences and the snorns, relative to the tralation start of the gene. (C) Targeting cotruct was generated by removing nucleotides encoding U3a, nucleotides encoding U33, nucleotides encoding U34, and nucleotides encoding U3a.
2 SUPPLEMENTL FIGURE body weight (g) 1 1 female age (weeks) / 3 1 male age (weeks) body fat (%) 1 1 / / male female C RN:Rplp (rel units) U33 U1 U U7 SNOR8 SNOR73 / Supplemental Figure. ody weight and composition of Rpl13asnoless mice. () Mean (±SE) serial weights for (filled circles) and / (open circles) mice. n = 11 mice/genotype/ sex. () Mean percent body fat (SE) for (filled bars) and / (open bars) mice at 7 to 8 weeks of age. n 4 mice/genotype/sex. (C) qpcr quantified expression (relative to Rplp) of Rpl13a snorn U33 and unrelated snorns in and / liver tissue. Mean standard error (SE) for a minimum of n = mice/genotype., P <.1 for / vs. ;, noignificant determined by unpaired ttest
3 SUPPLEMENTL FIGURE 3 glucose KCl mm mm 8 mm 16 mm mm fluorescence inteity (rel units) time (sec) mm / glucose KCl mm 8 mm 16 mm mm fluorescence inteity (rel units) time (sec)
4 Supplemental Figure 3. Representative calcium oscillatio. Traces of calcium oscillatio at,, 8 and 16 mm glucose, or with addition of mm KCl in representative single βcell within whole islet from () and / () mouse. s expected, higher glucose concentratio trigger larger calcium oscillatio in islets of both genotypes, and addition of KCl triggers fast and maximal influx of calcium into the cytoplasm, resulting in greater spike and coequent decay of fluorescence inteity. Note higher relative change in fluorescence inteity in / vs..
5 SUPPLEMENTL FIGURE 4 / mtdn : ndn / / C Trolox equivalent (µm/µg proteinmin) 1 1 / Supplemental Figure 4. Mitochondrial morphology and antioxidant capacity are unchanged in Rpl13asnoless islets. () Representative thin section electron micrographs of isolated and / islets. Scale bar, µm. () Quantification of (filled bars) and / (open bars) islet mitochondrial DN (mtdn) relative to nuclear DN (ndn). Mea (SE) for n = 4 mice/genotype. (C) Trolox equivalent antioxidant capacity of lysates from and / islets for n = 4 mice/genotype
6 1 1 / IL1β expression (rel units) CD68 8 / / / STZ 1 / / / / sno STZ / control STZ / STZ 4 1 / / C F4/8 TNFα / Ki67 nuclei (%) GTT UC (mg/dlmin x 13) SUPPLEMENTL FIGURE / / sno STZ Supplemental Figure. STZinduced inflammation and βcell replication is indistinguishable between and Rpl13asnoless islets. () UC during GTT in 16week old and / mice. n = 9 mice/genotype. () qpcr quantified expression of inflammatory markers (relative to Rplp) in wild type (filled bars) and /(open bars) islets days after PS (black) or STZ (red) treatment. Mea (SE) for n = 3 mice per genotype/treatment. (C) Representative pancreas sectio stained for Ki67 days after STZ. Scale bar, µm. Graph shows quantification of mea (SE) for n = 4 mice per genotype/treatment., p <. for compariso indicated;, not significant determined by unpaired ttest
7 Supplemental Table 1. Quality Control for RNseq Samples Sample Total reads Total aligned % ligned % Uniquely aligned verage length FRET (average quality) %GC Rpl13asnoless1 43,134,384 34,8, % 6.16% % Rpl13asnoless,,11 43,147,48 76.% 66.87% % Rpl13asnoless3 9,38,49 47,91,43 7.9% 61.% % Rpl13asnoless4,9,748 4,93, % 64.69% % 1 76,68,177 61,733,9 7.66% 6.8% % 3,914,668 46,34, % 68.3% % 3 7,69,914 47,486, % 64.1% % 4 9,76,1,11, % 6.33% %
8 Supplemental Table. Differentially expressed genes between Rpl13asnoless and islets Fold change Gene ID Gene name ( / vs. ) Pvalue FDR step up LSMean(/) LSMean() rhgef6 Rho guanine nucleotide exchange factor E 4.7E Itih4 Interalphatrypsin inhibitor heavy chain H E6.8E Mir64 MiR E.6E Rgs Regulator of Gprotein signaling E7 4.E Slc14a Urea traporter E6.8E Upp Uridine phosphorylase E7 4.E
9 Supplemental Table 3. Primer Sequences; related to Experimental Procedures PCR for Genotyping sequence ' to 3' forward (F) GC GG TTG CTG CTC GG G T TG G reverse (R) CC GC CTG CTT TC GC TTT GC CTG qrtpcr for mrns Rplp F Rplp R Rpl13a F Rpl13a R Ucp F Ucp R qrtpcr for snorns universal stem loop reverse primer U3a F U3a SLRT U33 F U33 SLRT U34 F U34 SLRT U3a F U3a SLRT U1 F U1 SLRT U F U SLRT U7 F U7 SLRT SNOR73 F SNOR73 SLRT SNOR8 F SNOR8 SLRT TC CCT GC GC CCG CCG TG TGC TC TGC TTG GG CCC CG TT TG GGT CGG GTG G T CC GGC CTT TTC CTT GCG TTT CT C G CC TTG CC GG G GGT TGG CTT TC GG GG TT C TCC CG CC CC CG CC GG TCC TG TC GC C CT CC C GCG TGG TCC CG CC CC CG CCG CC CG CC CGC CG GTC TC GC TTG TG TG GC TC TCC CC T GCG TGG TCC CG CC CC CG CCG CC CG CC CGC C GCC TC CGT CTG TG TGT TCT GCT TT CC TC TT GTT GCG TGG TCC CG CC CC CG CCG CC CG CC CGC GC GTC TC CTC CG TG GTC TTC GG TG GCG TGG TCC CG CC CC CG CCG CC CG CC CGC TCC TGG C GCC T GT GC CC C CT CT G GCG TGG TCC CG CC CC CG CCG C TCT TG TG TC CT TCC CG C GCG TGG TCC CG CC CC CG CCG C GT G CG CT TGG CCT GC CTT C GCG TGG TCC CG CC CC CG CCG C C GGG GC TT GGG CT T GCG TGG TCC CG CC CC CG CCG CC CG CC CGC TTT TGT CCG TCT GC C TT TG GC GCG TGG TCC CG CC CC CG CCG CC CG CC CGC TTT GTT G
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