Supplementary MaterialsSupplemental Number 1: The tdTomato expression in Compact disc4+ T cells from R26tdTomato/Ox40Cre mice. in Compact disc4+Compact disc25? T cells (Still left), YM348 and Compact disc44+Compact disc62L? cell percentage in tdTomato? and tdTomato+ typical T cells (Best). (C) Consultant FACS plots of tdTomato+ cells in Tconv and Tregs from different lymph organs. (D) YM348 Figures for (C). The info is normally represent of three unbiased mice. Data_Sheet_1.PDF (9.8M) GUID:?3465519E-0666-42E6-A1D8-591EA0E29313 Supplemental Figure 3: Saturation analysis of sequencing depth. (A) Rarefaction evaluation of clonotype amount. To be able to assess whether sequencing depth was saturated or not really, rarefaction evaluation was performed. Random reads of raising amount had been subsampled in the fresh reads dataset, YM348 and the real amounts of clonotypes had been computed from each group of subsampled reads. Similar analysis was carried out for both mice. This representative number was from mouse 1. The inside figure is for sample M1T7 and M1T8. (B) Rarefaction Analysis on Bhattacharyya Similarity Index. Rarefaction analysis was used to study the relationship between sampling depth and Bhattacharyya similarity index estimation (46). Subsampling was performed on a level of RNA molecules. Hundred percentage corresponded to all the RNA molecules obtained at the highest sequencing depth for each sample. Increasing percentage of RNA molecules was randomly subsampled from both target RNA molecule datasets, then similarity index between the two subsets were determined. The similarity index raises with increasing subsampling depth in the beginning then reaches a plateau. The dash collection represents the sampling depth we used to calculate similarity index. This is one representative of two mice. Data_Sheet_1.PDF (9.8M) GUID:?3465519E-0666-42E6-A1D8-591EA0E29313 Supplemental Number 4: TCR repertoire coverage and V gene section utilization analysis for conventional and regulatory T cells. (A) Summary of diversity coverage in all repertoires. The diversity coverage is YM348 calculated as the number of unique clonotype divided by the number of cells. Clonotype is defined on different levels: unique CLG4B RNA sequence and unique CDR3 nucleotide sequence (A combination of V and J segments at nucleotide level). (B,C) CDR3 Amino Acid length distribution for repertoires within each mouse. These two figures show no significant difference. (D,E) Frequencies of V beta gene segment usage within all the samples of each mouse. Data_Sheet_1.PDF (9.8M) GUID:?3465519E-0666-42E6-A1D8-591EA0E29313 Supplemental Figure 5: Clonal frequency of shared clones among different fraction of T cells in the Peyer’s patch. Pie charts illustrate clonal frequencies of shared clones between indicated populations. Major populations that are shared between different cell fractions are labeled in the corresponding slices and are indicated with the same color. The frequencies of non-overlapping clones are shown in the gray slices. The population size for each cell fraction is indicated in the parentheses underneath each pie chart. Data_Sheet_1.PDF (9.8M) GUID:?3465519E-0666-42E6-A1D8-591EA0E29313 Supplemental Table 1: Percentage of conventional T cells in each divided generations. The experiment was described in Method and Figure 3. The average percentage of Tcon cells in each generation was shown in the table. * 0.05, ** 0.01, and *** 0.001. This data is representative of three independent experiments. Data_Sheet_1.PDF (9.8M) GUID:?3465519E-0666-42E6-A1D8-591EA0E29313 YM348 Supplemental Table 2: Similarity index variance estimation based on bootstrap. Bootstrap method was introduced to estimate the similarity index variance. Similarity index was calculated from each bootstrapped sample, which is randomly resampled (with replacement) from total RNA molecules until reach the same size of the original dataset. On average, ~60% of distinct RNA molecules in the original dataset will be covered in each new sample (47). After repeating this procedure for 100 times, mean and standard deviation were estimated. Data_Sheet_1.PDF (9.8M) GUID:?3465519E-0666-42E6-A1D8-591EA0E29313 Supplemental Table 3: Bhattacharyya similarity index between different samples. The similarity between 16 samples from two mice was compared by calculating Bhattacharyya similarity index. The value of similarity index between all pairs was shown in the table. The highlight indicated the similarity index within the same animal. Data_Sheet_1.PDF (9.8M) GUID:?3465519E-0666-42E6-A1D8-591EA0E29313 Supplemental.