Fate challenge purges the regulatory T cell repertoire of unstable clones - supplementary tables S.Junius et al

paper: "Fate challenge purges the regulatory T cell repertoire of unstable clones" Steffie Junius, Adamantios V. Mavrogiannis, Pierre Lemaitre, Margaux Gerbaux, Frederik Staels, Vanshika Malviya, Oliver Burton, Raul Yhossef Tito Tadeo, Jeroen Raes, Joost P. M. van Meerwijk, Stephanie Humblet-Baron, Adrian Liston*, Susan M. Schlenner* Folder single-cell RNA sequencing - Fig. 4: Purpose: identify differentially expressed genes between ex-Treg obtained 5, 10, or 15 days post-adoptive transfer into a lymphopenic environment compared to ex vivo Treg in order to identify Treg subset with heightened instability. Method: Treg from Foxp3YFP-Cre Rosa26RFP (PMID: 18387831, PMID: 17171761) were co-injected with congenically-disparated naive T cells in a 1:1 ratio into Rag1KO mice and ex-Treg were sorted at 15 days, 10 days or 5 days post-transfer. As a control, ex vivo Treg was sorted from Foxp3YFP-Cre Rosa26RFP (d0 sample). Next, samples were stained with multiplexing antibodies using the BD Single-Cell Multiplexing Kit (BD biosciences), then pooled and stained with the following oligonucleotide-conjugated antibodies: anti‑CD25 (PC61), anti-CD44 (IM7), anti-CD62L (MEL-14), anti-GITR (DTA-1), anti-CD4 (RM4-5), anti‑TCRb (H57-597), anti‑CD69 (H1.2F3), anti‑TIGIT (1G9), anti‑CD95 (Jo2), anti‑CD122 (TM‑1β), anti‑Tim-3 (5D12), anti‑CCR7 (4B12), anti‑CD103 (M290), anti‑CD279 (J43), anti‑CD274 (MIH5), anti‑CD233 (C9B7W), anti‑CD71 (C2), anti‑CD278 (7E7G9), anti‑ITGB7 (M293), anti‑CD137 (1AH2), anti‑CD40 (3/23), anti‑CD3e (145-2C) from BD Biosciences. Single-cell capture and cDNA library preparation was performed using the BD Rhapsody Single-cell analysis system (BD Biosciences), according to the manufacturer’s instructions, using a custom gene panel (591 genes). Sequencing was performed on an Illumina NextSeq500 instrument using a Mid‑Output kit v2.5 (150 cycles, paired-end). Sample demultiplexing, barcode processing, alignment, filtering, UMI counting were done using the standard BD Biosciences Rhapsody analysis pipeline on Seven Bridges (www.sevenbridges.com). Result and conclusion: In this study, we showed that ex-Treg downregulate Treg core-specific genes immediately upon adoptive transfer into a lymphopenic environment. Further, we identified a naïve-like Treg population enriched for unstable Treg clones. Folder microbiome sequencing- Fig S4: Purpose: Analyze diversity in microbial communities within fecal samples of SPF housed mice and SPF mice co-housed with pet stor