Supplemental Fig. 1

A DN DN C Dβ1XJβ1 Dβ2XJβ2 125 125 Norm. count Norm. count

100 100 ± SD 75 ± SD 75 CD2 CD5

B DP 50 50

DP alleles alleles β 25 β 25 TCR TCR % of unrecombined Norm. count Norm. count 0 % of unrecombined 0

DN1 DN2 DN3 DN4 DN1 DN2 DN3 DN4 S5fl/fl CD2 CD5 S5fl/flhCD2iCre S5fl/fl S5fl/fl hCD2iCre C Obrázek: Vložit do supplementary - zjistit co je ta negativní populace

fl/fl flox/flox flox/flox D S5 E Thymocytes: Smarca5 Smarca5 hCD2-iCre Trbv1 fl/fl DP DP Trbv12-1 S5 hCD2iCre DN1 DN2 DN3 DN4 o Mode

T iTCRβ iTCRβ Trbv12-2 99.0% 97.7% Trbv12-3 Trbv13-1 Normalized

Trbv13-2 Norm. count Trbv13-3 Trbv14 Trbv15 Trbv16 Tcrβ Trbv17 S5fl/fl Trbv19 fl/fl Trbv2 S5 hCD2iCre Trbv20 Smarca5 isotype control Trbv21 Trbv23 Trbv24 0 50 100 150 DP CD4 CD8 Trbv26 TPM value Trbv29 Trbc1 Trbv3

Trbc2 Norm. count Trbv30 Trbv31 0 5000 10000 15000 Trbv4 TPM value Trbv5 Trbv6 Tcrβ 0 500 1000 1500 2000 TPM value

Supplemental Figure 1 Flow cytometry analysis of CD2 (A) and CD5 (B) molecules on CD4 and CD8 double negative (DN) and double positive (DP) cell populations in thymi of control and S5fl/flhCD2iCre mice. Similar results were obtained in at least 3 repeat experiments. (C) Quantitative detection of the D-J recombination of the Tcrb . qPCR determines relative loss of the intact allele after the TCR recombination. Graphs show relative proportion ± SD of non-recombinated Tcrb gene (measured here as presence of intact Dβ1-Jβ1 and Dβ2-Jβ2 segments, respectively) in individual DN populations. Data are representative at least of three control and three S5fl/flhCD2iCre animals. (D) RNA-seq analysis of Tcrb gene rearrangement associated transcripts expressed in control (white bars) and S5fl/flhCD2iCre (black bars) DP stage thymocytes. The transcript abundance of Smarca5, Tcrb constant regions (both shown in rectangle) and all expressed Tcrb variable regions with BaseMean value higher than 10 were quantified as transcripts per kilobase million (TPM). (E) Surface expression of Tcrβ on individual thymic subpopulations of control and S5fl/flhCD2iCre animals analyzed by flow cytometry. As an isotype control to H57-597 clone (ani-Tcrβ) was used fluorescently labeled Armenian Hamster IgG (dark histograms). Data are representative of three experiments. B - Log Adjusted P A 10 50 −5.0 0 Log S5 t w c in w upr do Genes 1 corresponds gated within 2013). DN3a is the DN3a Supplemental ( o ompar A 180 86 56 t DN3ac wnr DP 2 fl/fl equal Ankrd6 Supplemental Fig.2 ) Fmnl2 FoldChange Cacna1e t DN3a Smoc1 expression The egula Tdrd5 e

thymic and cells hCD2-iCre d Numbers log t egula ompar to top Rasgrp4 o DP t ed in 2 DP FC p as adj −2.5 to t 200 ed Cd81

e suspension. thymocytes

compared of d = in the Figur

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from v14n-2, Sqr Medag ock1, vs

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P5 DP -/-

Pmaip1/Noxa log2 Fold Change Cdkn1a Sac3d1 Rnf19b Phlda3 Rps27l Epha2 Zmat3 Mast4 Fuca1 Dyrk3 Vwce Padi4 Bbc3 Enc1 Ninj1 Rrad Plk2 Aen Bax Fas S5 fl/fl F ACS-sorted DP T rp53

-/- Thymocytes hCD2-iCre S5 fl/fl

hCD2-iCre S5 T rp53 fl/fl -/-

-2 -1 0 1 2