title: colonTreg_2-3_GvHD_Tcrb_R2

description:
re-isolated
mTRB_Tx198-1-296-C-ra_AB.RunAB.clonotypes.TRB.txt

data_processing:
base calling: Base-calling and demultiplexing based on Illumina barcodes was carried out using the bcl2fastq Conversion Software (v1.8.4) provided by Illumina (Illumina INc., San Diego, California, USA).
paired-end read alignment: UMI-barcoded and paired TCR-sequencing reads were joined using PEAR (v0.9.11).
UMI-based generation of MIGs: Demultiplexing on the basis of UMI-barcodes and adaptor trimming was done using MIGEC (v1.2.9) resulting in consensus sequences of molecular identifier groups (MIGs).
MIG mapping: MIGs were mapped using MIXCR (v3.0.13).
Assembly: RepSeq.IO library v1.6
Supplementary files format and content: Mapping output of MIXCR
Library strategy: TCR-seq

source_name: CD4+CD25+ regulatory T cells

extract_protocol:
Total cellular RNA was isolated from Treg (freshly isolated, in vitro expanded, or re-isolated after BMT) using the RNeasy Mini Kit or the RNeasy Micro Kit (Qiagen). Following the manufacturer’s recommendations, samples originating from 500 to 500k cells were processed with the Micro Kit whereas the Mini Kit was applied to all samples exceeding 500k cells. RNA concentration and quality were simultaneously assessed using RNA Screentape Analysis or High Sensitivity RNA Screentape Analysis (Agilent), depending on the expected yield.
Total cellular RNA was obtained as described in section “RNA-seq library preparation”. TCR repertoire sequencing libraries for Illumina sequencing were prepared on the basis of {Mamedov, 2013 #7} and the protocol was adapted, as follows. For the generation of 5’RACE-ready cDNA total RNA ranging from 10 - 200 ng (low input) or 200 - 1000 ng (high input) in a total of 10 µl RNAse-free water was combined with 1 µL of 12 µM or 25 µM 5’-CDS Primer A in a total volume of 11 µL, respectively. Reactions were incubated at 72°C for 3 min followed by 42°C for 2 min for poly-dT primer annealing. Next, 1 µL of 10 µM (low input) or 50 µM (high input) TCR_UMI_Smarter template-switching oligo (TSO) was added at room temperature. RT Buffer Mix (5x First-Strand-Buffer Mix, 20mM dNTP, 100 mM DTT) was freshly prepared on ice before it was supplemented with RNaseOUT™ Recombinant Ribonuclease Inhibitor (40 U/µL) (Thermo Fisher Scientific) and SMARTScribe Reverse Transcriptase (100 U/µL) (Takara) at room temperature. A volume of 8 µL of the resulting reverse transcription master mix was added to each reaction. After homogenization, the 20 µL reactions were incubated at 42°C for 90 min followed by 70°C for 10 min and put on hold at 10°C in a hot-lid thermal cycler. Following reverse transcription, excess TSO was digested using Uracil-DNA Glycosylase (UDG) (New England Biolabs) by adding 4 µL of UDG master mix adjusted for TSO input amount and successive incubation at 37°C for 60 min. The master mix for PCR1 was prepared on ice using the Advantage 2 PCR Kit (Takara) along with primers specific for the constant regions of the Trac and Trbc loci as well as a universal forward primer specific for a binding sequence introduced via the TSO. 24 µL of RACE-ready cDNA were combined with 26 µL of PCR1 master mix and subjected to a two-step PCR program (95°C, 1min; 95°C, 30 s & 68°C, 70 s (x times); 68°C, 7 min; 8°C, hold). Depending on the RNA input amount, PCR cycle numbers ranged from 16 to 28. The 50 µL PCR product was purified with 27.5 µL of AMPure XP beads (Beckman Coulter) according to the manufacturer’s instructions. After elution in 23.5 µL of Monarch® DNA Elution Buffer (New England Biolabs), reactions were split into 10 µL technical replicates for successive extension PCR (PCR2). PCR2 master mix was assembled using the Advantage 2 PCR Kit (Takara) and gene-specific nested primers (Trac, Trbc loci) introducing Illumina Nextera XT i5 indices (S501, S502, S503, S508, S517, S520, S507), distinguishing Tcra from Tcrb libraries within each reaction. After addition of 36 µl PCR2 master mix, 4 µL of Nextera XT Index Kit v2 (Illumina) adapters were used for indexing libraries individually. Libraries were amplified using the same program as in PCR1 with 18 PCR cycles. Library purification was carried out using  AMPure XP beads (Beckman Coulter) at a volume ratio of 0.8 according to the manufacturer’s instructions. After final elution in 17 µL of Monarch® DNA Elution Buffer (New England Biolabs), concentration was measured with the Qubit™ dsDNA HS Assay Kit (Thermo Fisher Scientific) and fragment size profile was assessed using the High Sensitivity D1000 or D5000 ScreenTape Assay (Agilent). Libraries were stored at -20°C until sequencing (paired-end 300bp) on the Illumina MiSeq system. Oligonucleotide sequences are summarized in Supplemental Table 3, Libraries are summarized in Supplemental Tables 4-7.
poly-dT-primed, gene-specifically enriched TRA/TRB library preparation

value: C57BL/6 Foxp3GFP+

tag: sampleID

value: allo

value: GvHD

value: colon

tag: pcr2 cy. no.

tag: chain


title: HK-2_CTL_3

status: Public on Jan 01 2024

type: SRA

city: Shanghai

first: Yutong

last: Hou

address: 227 South Chongqing Road

country: China

institute: SJTU

library_source: transcriptomic

data_processing:
Sequence reads were trimmed for adaptor sequence/low-quality sequence using fastp.
Trimmed sequence reads were mapped to GRCm38.102/hg38 or GRCh38.102/mm10 using Hisat2.
Read count extraction and normalization were performed using Stringtie.
Assembly: hg38; mm10
Supplementary files format and content: Tab-delimited text files include raw counts and FPKM values for each Sample.

molecule: total RNA

organism: Homo sapiens

extract_protocol:
RNA was harvested using TRNzol Universal Reagent (TIANGEN). 2ug of total RNA was used for the construction of sequencing libraries.
RNA libraries for RNA-seq were prepared using mRNA-seq Lib Prep Kit for Illumina (ABclonal, China) following Sample Preparation Guide.

tag: cell line

tag: genotype

value: WT


title: scTCR-seq_Colon recipient 3

city: Regensburg

first: Michael

last: Rehli

address: F.-J.-Strauss-Allee 11

department: Internal Med III

country: Germany

institute: University Hospital Regensburg

data_processing:
ScRNA:Sequencing reads were demultiplexed using cellranger mkfastq (version 4.0.0),scTCR:Fastq files were processed using cellranger (version 5.0.0)
ScRNA:The reads were subsequently mapped to the mouse genome using cellranger count (version 4.0.0).
ScRNA: Quality control and analysis was performed with the Seurat package (v4.0.0) in R.
scTCR:Fastq files were processed using cellranger (version 5.0.0)
ScTCR:The reads were subsequently mapped to the mouse genome using cellranger count (version 5.0.0).
ScTCR: Quality control and analysis was performed with the Seurat package (v5.0.0) in R
Assembly: ScRNA: Mouse reference, mm10 (GENCODE vM23/Ensembl98),scTCR: mm10 reference genome (refdata-cellranger-vdj-GRCm38-alts-ensembl-5.0.0
Supplementary files format and content: cellranger output, rds (R.4.1.1), loom (python 3.8.13), H5ad (python 3.8.13)
Library strategy: scTCR-seq

organism: Mus musculus

source_name: Donor allo reisolated from Recipient 3

growth_protocol: Donor Treg Expansion: Splenocytes from transgenic FoxP3EGFP mice (B6; CD45.2; H-2b) were stained with anti-CD25-PE and anti-PE UltraPure MicroBeads and enriched for CD25+ cells by MACS® (Miltenyi Biotec). The CD25+ fraction was further stained for CD4, CD8 and CD62L and Treg were sorted on a FACSAriaTM IIu or FACSAriaTM Fusion (Becton Dickinson) as CD4+CD25highCD62L+ cells; purity of sorted cells was >98%. For allospecific in vitro expansion, sorted Treg were seeded at 5x104/well in cDMEM together with 2,5x104/well (d0) or 1x104/well (restimulation on d7) anti-CD11c MicroBead-enriched (Miltenyi Biotec) and irradiated (30 Gy) CD11c+ DC from BALB/c mice. Cells received fresh medium on d4 and d10 and were harvested on d12.

extract_protocol:
For re-isolation of donor Treg on day 7 after transplantation, single cell suspensions from spleen, liver and colon were prepared, stained for H-2Kb, CD45.1, CD45.2, TCRβ and CD4 and sorted on a FACSAria IIuTM or a FACSAriaTM Fusion.
ScRNA-seq libraries were constructed using 13-16 cycles of PCR. Products were purified using Ampure XP beads and quality was controlled using Agilent Tapestation.  ScTCR-seq libraries were generated from the same cDNAs using the Single Cell V(D)J Enrichment Kit, Mouse T Cell (10x Genomics).
ScRNA-libraries were sequenced using single-read sequencing (S1 flow cell, 100 bp) on the Illumina NovaSeq™. scTCR-libraries were sequenced on the Illumina NextSeq 550 with 150 cycles single-read sequencing.

treatment_protocol: Prophylactic treatment of GvHD: Single cell suspensions from BM (femora and tibiae of both hind legs) and spleen were prepared. T cells were depleted from BM (T cell depleted bone marrow; TCDBM) by using anti-CD90.2 MicroBeads with the MidiMACS® system (Miltenyi Biotec). CD4+CD25- conventional T cells (Tconv) were isolated from splenocytes by depletion of CD25+ cells using anti-CD25-PE and anti-PE UltraPure MicroBeads (Miltenyi Biotec), followed by enrichment of CD4+ cells from the CD25- fraction through labeling with anti-CD4 MicroBeads (Miltenyi Biotec) using the MidiMACS® system. For short-term (d7) FACS and RNA-seq experiments BALB/c recipients were lethally irradiated (8 Gy) on the day of BMT and transplanted i.v. with 2.5 x106 TCDBM cells and 1x106 Tconv from B6-CD45.1 mice with or without in vitro expanded alloTreg from Foxp3gfp mice at a 1:1 ratio.

tag: phenotype

tag: Sex

value: female

tag: cell type

value: Donor Treg allo reisolated from Colon recipient 3

tag: cell markers


title: colonTreg_2-2_GvHD_Tcrb_R2

description:
re-isolated
mTRB_Tx198-1-294-C-ra_AB.RunAB.clonotypes.TRB.txt

growth_protocol: Splenocytes were stained with anti-CD25-PE and anti-PE UltraPure MicroBeads and enriched for CD25+ cells by MACS® (Miltenyi Biotec). The CD25+ fraction was further stained for CD8, CD4 and CD62L and Treg were sorted on a FACSAria IIuTM or a FACSAriaTM Fusion (BD Biosciences) as CD4+CD25highCD62L+ cells; purity of sorted cells was >98%. Polyclonal in vitro expansion of Treg was performed as recently described {Riegel, 2020 #61}. In brief, sorted Treg were cultured in DMEM (high glucose, Gibco/Invitrogen) with 10% FCS, 2mM L-glutamine, 10mM HEPES, 1% NEAA (PAN Biotech), 50U/ml penicillin, 50µg/ml streptomycin and 5x10-5 M 2-mercaptoethanol  (Gibco/Invitrogen) (cDMEM) in 96-U-well plates (1x104/well) and stimulated with CD3/CD28-beads (Treg-Expansion Kit, Miltenyi Biotec, 4 beads/cell) and rhIL-2 (2000 U/ml; Proleukin®, Chiron). Cultures received 100 µl/well fresh medium on d4, were restimulated on d7 with 1 bead/cell after transfer into 24-well-plates (1x106 cells/ml/well), fed and split on d10 as needed and harvested on d12. For allospecific in vitro expansion sorted Treg were seeded at 5x104/well in cDMEM together with 2,5x104/well (d0) or 1x104/well (restimulation on d7) anti-CD11c MicroBead-enriched (Miltenyi Biotec) and irradiated (30 Gy) CD11c+ DC  from BALB/c mice. Cells received fresh medium on d4 and d10 and were harvested on d12.

treatment_protocol: Single cell suspensions from BM (femora and tibiae of both hind legs) and spleen were prepared. T cells were depleted from BM (T cell depleted bone marrow; TCDBM) by using anti-CD90.2 MicroBeads with the MidiMACS® system (Miltenyi Biotec). CD4+CD25- conventional T cells (Tconv) were isolated from splenocytes by depletion of CD25+ cells using anti-CD25-PE (all antibodies used in this study are listed in Supplemental Table 2)  and anti-PE UltraPure MicroBeads (Miltenyi Biotec), followed by enrichment of CD4+ cells from the CD25- fraction through labeling with anti-CD4 MicroBeads (Miltenyi Biotec) using the MidiMACS® system. For short-term (d7) FACS and RNA-seq experiments BALB/c recipients were lethally irradiated (8 Gy) on the day of BMT and transplanted i.v. with 2.5 x106 TCDBM cells and 1x106 Tconv from B6-CD45.1 mice with or without in vitro expanded allo or polyTreg from Foxp3gfp mice at a 1:1 ratio. Recipients of the ‘no GvHD’ group received TCDBM and Treg only. In long-term survival experiments lethally irradiated BALB/c recipients were transplanted with 2.5 x106 TCDBM on d0 and 0.25 x106 Tconv were administered on d2 for GvHD induction. Mice of the GvHD prophylaxis groups (allo or poly) received additional 0.25x106 in vitro expanded Treg from B6 WT mice on d0. Recipients were monitored daily, body weight and GvHD symptoms assessed twice weekly by non-blinded investigators applying a standardized scoring system (Cooke et al., 1996). Single cell suspensions from BM, spleen and mesenteric lymph nodes were prepared. Erythrocytes were lysed in splenic and BM cell suspensions. For leukocyte isolation from the liver, the organ was flushed with PBS through the portal vein and the gallbladder was removed before it was excised, transferred to a petri dish and dissected into small pieces using a scalpel. Fragments were suspended in RPMI (Lonza/Biozym)/5% FCS, strained and washed in RPMI w/o FCS before Percoll centrifugation (40%/80%; 800 x g/20 min/20°C, w/o break). Leukocytes were resuspended in RPMI/5% FCS, erythrocytes were lysed, cells were washed in RPMI/10% FCS and kept on ice until further use. For intestinal leukocyte isolation, large intestine was excised, cut into 0.5 cm pieces, incubated twice for 20 min at 37 °C in HBSS/5mM EDTA/1mM DTT (Sigma-Aldrich) followed by vigorous shaking to isolate intraepithelial leukocytes (IEL). For lamina propria leukocytes (LPL), the fragments were transferred to HBSS without phenol red containing calcium and magnesium with 5% FCS together with enzymes of the Lamina propria dissociation kit (Miltenyi Biotec) and incubated for 30 min at 37°C. The fragments were further mechanically dissociated using the GentleMACS system (protocol: m_intestine_01; Miltenyi Biotec), strained, washed and pooled together with IEL before Percoll (Sigma-Aldrich)  centrifugation (30%; 512 xg/15 min/20°C). For baseline (no BMT) colon Treg sorts IEL were not included (no Tregs in untreated animals) and colonic LPL were further enriched using anti-CD45 MicroBeads (Miltenyi Biotec) and the AutoMACS® system (Miltenyi Biotec). Splenocytes were enriched for CD25+ cells by MACS® using anti-CD25-PE and anti-PE UltraPure MicroBeads before sorting. Cells were further stained for CD8, CD4 and CD62L (also CD45 for colon-derived cells) and Treg were sorted on a FACSAriaTM IIu or a FACSAriaTM Fusion (Becton Dickinson). From spleen and liver, CD62L+ and CD62L– subpopulations were isolated, from colon, only CD62L–- cells were obtained (sorting strategy is shown in detail in Supplemental Fig.2a). For re-isolation of donor Treg on day 7 after transplantation, single cell suspensions from spleen, liver and colon were prepared as described, stained for H-2Kb, CD45.1, CD45.2, TCRβ and CD4 and sorted on a FACSAria IIuTM or a FACSAriaTM Fusion (for detailed sorting strategy see Supplemental Fig.2b). Staining was performed in PBS/2%FCS with anti-CD16/CD32 antibodies to block FcR-binding and DAPI (Sigma-Aldrich) or Fixable Viability Dye (Thermo Fisher) to exclude dead cells. Detailed information on all antibodies used in this study is listed in Supplemental Table 2. For staining of intracellular markers, the Foxp3/Transcription Factor Staining Buffer Set® (eBioscience) was used.  Multicolour FACS staining for surface markers was performed using Brilliant Stain Buffer (BD Biosciences) where applicable. Data was acquired on a FACSymphony® A5 SORP (Becton Dickinson) and analyzed using FlowJo® software (v10.7.1 or v10.8.0; Treestar Inc). For clustering and embedding of multiparameter flow cytometry data (as presented in Supplemental Fig.2a) FlowJo® plugins DownSample_v3.3 and UMAP_v3.1 were used. The UMAP plot in Supplemental Fig. 2a was created on the basis of 27 samples from spleen, liver and colon (all n=3) from 3 experimental groups: alloTreg prophylaxis, polyTreg prophylaxis and GvHD control and 3 independent experiments, with every sample representing a cell pool of 2-3 mice. Cells were stained for CD45.1, CD45.2, H-2kb, TER119, TCRβ, CD8α, CD4, CD19, Nkp46, CD11b, Ly6C, Gr-1 and CD25. Dead cells were excluded with DAPI. Samples were gated on live single CD45.1+TER119- and CD45.2+TER119- cells, downsampled to 15.000 cells each and assembled into one file.

tag: strain

tag: mouse (pool/culture no.)

tag: type

tag: model

tag: tissue

tag: treg donor

tag: recipient

tag: pcr1 cy. no.

value: Beta

tag: techn. replicate


title: HK-2_CTL_1


title: scTCR-seq_Spleen recipient 2

source_name: Donor allo reisolated from Recipient 2

value: transgenic FoxP3EGFP mice (B6; CD45.2; H-2b)

value: Donor Treg allo reisolated from Spleen recipient 2


title: colonTreg_3-1_GvHD_Tcrb_R2

description:
re-isolated
mTRB_Tx198-2-341-C-ra_A.RunAB.clonotypes.TRB.txt

tag: sampleID

tag: pcr2 cy. no.


title: HK-2_KO_1

tag: cell line

value: PCMT1 KO


title: spleenTreg_7-6_noGvHD_Tcrb_R2

description:
re-isolated
mTRB_251_Tx198-12-4216_S_rp_A.RunAB.clonotypes.TRB.txt

value: poly

value: BMT control

value: spleen

tag: chain


title: scTCR-seq_Colon recipient 2

tag: phenotype

value: Donor Treg allo reisolated from Colon recipient 2


title: scTCR-seq_Liver recipient 2

tag: Sex

value: Donor Treg allo reisolated from Liver recipient 2


title: spleenTreg_7-5_noGvHD_Tcrb_R2

description:
re-isolated
mTRB_248_Tx198-12-4214_S_rp_A.RunAB.clonotypes.TRB.txt


title: Mus musulus 770261

city: Durham

first: Vincent

middle: Michael

last: Perez

state: NC

address: 25 Alexandria Wy

department: Translational reasearch

country: USA

institute: Tempus AI

data_processing:
All FASTQ files were processed using a combination of STAR v2.7.10a and Salmon v1.2.0 with the mouse build GRCh38 - mm10 as a reference). FASTQ files from the same sample, but different lanes, were merged with STAR’s fastq_channel_merged_paired function. Reads were mapped using STAR’s fastq_channel_merged_paired_star function. Picard and QC, count matrix assembly, and bam files were generated for each sample using both Picard v2.27.4, STAR v2.7.10a, and Salmon v1.2.0. TRUST4 v1.0.12 (Tcr Repertoire Utilities for Solid Tissue) were used to reconstruct TCR and BCR sequences from BAM files generated from the STAR/Salmon outputs. All parameters for TRUST4 were set to default. The final output from TRUST4 was summary matrices for each sample that included hypervariable complementarity-determining region 3 (CDR3) nucleotide and amino acid sequences, counts, frequencies, and V, D, and J chain names.
Assembly: GRCm38
Supplementary files format and content: Complete gene count matrix (expression_raw_annotated.csv), gene count matrix normalized with Deseq2 and converted to log2 scale (expression_normalized_log2.csv), output from TRUST4 algorithm (processed_trust4.zip).

source_name: p53/MCA sarcoma

extract_protocol:
Tumors were dissected from mice, minced, and digested using the Miltenyi Biotec tumor dissociation kit. Tumor specimens and matched muscle control were harvested and stored in RNALater (Ambion) at −80°C until all samples were collected. RNA extractions from each sample were performed using RNeasy Fibrous Tissue Mini Kit (Qiagen). Extracted total RNA quality and concentration were assessed on a NanoDrop Spectrophomoter (Thermo Fisher Scientific).
RNA-seq libraries were prepared using TruSeq Small RNA Library Preparation Kits (Illumina) following the manufacturer’s protocol. RNA sequencing was performed on an Illumina Novoseq in 151 base pair, paired-end configuration. Greater than 50,000 reads per cell were collected per the manufacturer's recommendation.

value: p53/MCA sarcoma

tag: genotype

value: 129/SVJ WT

tag: treatment

value: Grp 4-NO RT+Iso for OX40+Ctl CpG+Iso PD-1


title: spleenTreg_7-4_noGvHD_Tcrb_R2

description:
re-isolated
mTRB_254_Tx198-12-4205_S_rp_A.RunAB.clonotypes.TRB.txt

tag: strain

value: C57BL/6 Foxp3GFP+


title: scTCR-seq_Donor allo_2

source_name: Donor allo

tag: cell type

value: Donor Treg allo

tag: cell markers


title: mESC Tbxt delE6/+ rep1

city: Cambridge

first: Bo

last: Xia

state: MA

address: 415 Main Street

institute: Broad institute

data_processing:
Raw sequencing reads were mapped to the mouse genome (mm10) with STAR 2.7.2a aligner. The resultant strand-specific read counts of all samples were integrated into a matrix for downstream analysis. Differentially expressed genes (DEGs) were detected by DESeq2 1.40.2 with the cutoff of log2 fold expression change >0.5 and p.adj < 0.05. The top 500 variable genes from DESeq2 across all samples were used to perform principal component analysis. The Tbxt-target genes were collected from Lolas et al. (2014), defined by significant Tbxt ChIP-seq signal in in vitro differentiated mESCs. The set of Tbxt-target genes was intersected with the significant DE genes identified in each mutant sample compared to the wild type controls, and these were aggregated to generate the overall set of differentially expressed Tbxt-target genes across the analyzed mESC lines. These differentially expressed Tbxt-target genes were visualized through a heatmap, with the log10-transformed normalized transcript matrix followed by z-score standardization across samples.
Assembly: mm10
Supplementary files format and content: *.tab: The processed data files generated by STAR2 after mapping to mm10 reference genome contain 4 columns: The first column corresponds to the Ensembl gene ID. Columns 2, 3, and 4 correspond to the number of reads mapped to each gene. The fourth column from each file was merged to a new datasheet and used for downstream analysis. The difference between columns 2, 3, and 4 is the strandedness. If an unstranded library prep kit was used, column 2 should be selected. If a stranded library prep kit was used, we must choose from column 3 or 4. The information from which strand the mRNA originated from is retained. Column 3 corresponds to the first read strand aligned, and column 4 corresponds to the second read strand aligned.

molecule: polyA RNA

source_name: mESC

extract_protocol:
Total RNA from each genotype was extracted using RNeasy Plus Mini Kit (QIAGEN, ID: 74134). mRNA was enriched using NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB, Cat. No. E7490L).
RNA-seq libraries were prepared using NEBNext Ultra II Directional RNA Library Prep Kit (NEB, Cat. No. 759 E7765L) and sequenced on one lane of NovaSeq X Plus 10B 2*150.

value: mESC

value: Tbxt delE6/+


title: mESC Tbxt Insert ASAY rep2

value: Tbxt Insert ASAY


title: mESC Tbxt Insert ASAY rep1


title: spleenTreg_6-6_noGvHD_Tcrb_R2

description:
re-isolated
mTRB_234_Tx198-10-4196_S_rp_A.RunAB.clonotypes.TRB.txt

tag: mouse (pool/culture no.)

tag: type

tag: model

value: Beta


title: k562, WTHYPBcentrip, rep10

city: Saint Louis

first: Juanru

last: Guo

state: MO

address: 4515 McKinley Ave

department: Department of Genetics

institute: Washington University in St. Louis  School of Medicine

data_processing:
*library strategy: Calling Cards
nf-callingcards
Reads are prepared by extracting barcodes (UMItools) and trimming adapters (Trimmomatic)
If not already done, genome is prepared (Samtools faidx and aligner indicies)
Reads were aligned (bwa)
Alignment QC metrics were extracted (samtools) and reads were mapped to identify Calling Cards insertions (custom scripts).
Peak calling and significance statistics were determined (pycallingcards)
Assembly: hg38
Supplementary files format and content: Analyzed Calling Cards data is outputted as qBED (formerly CCF) files. Each row is a discrete SRT insertion and has six columns: CHROM,START,END,VALUE,STRAND,ANNOTATION. The VALUE represents the number of sequencing reads associated with that insertion. The ANNOTATION column stores sample-specific information such as i5/i7/SRT barcode.

growth_protocol: The K562 cell line was cultured in RPMI medium containing 10% FBS and penicillin-streptomycin. 2x10^6 cells were transfected with 5ug of donor Barcoded-PBase-SRT-Puro and 5ug helper (hyPBase or hyPBase-centrip) using the Neon electroporation system for a total of 30 replicates. For a negative control, one replicate of K562 cells was transfected with 5ug Barcoded-PBase-SRT-Puro plasmid only. We used the following settings-pulse voltage: 1450V; pulse width: 10ms; pulse number: 3. After transfection, three replicates were plated into a 10cm dish. Cells were grown under 2ug/ml puromycin selection for 11 days, by which time almost all negative control transfectants were dead. After 11 days, each replicate was dissociated into a single-cell suspension with 0.25% trypsin-EDTA and resuspended in PBS. Aliquots of each replicate were cryopreserved in culture media supplemented with 5% DMSO. The remaining cells were pelleted by centrifugation at 300g for 5 minutes. Cell pellets were either processed immediately or kept at -80C in RNAProtect Cell Reagent.

extract_protocol:
Total RNA was isolated from every three replicates using the RNEasy Plus Mini Kit following manufacturer's instructions. RNA was eluted in 40ul RNase-free water and quantified using the Qubit RNA HS Assay Kit. We performed first strand synthesis on each replicate with Maxima H Minus Reverse Transcriptase. We mixed 2ug of total RNA with 1ul of 10mM dNTPs and 1ul of 50uM SMART_dT18VN primer, brought the total volume up to 14ul, and incubated it at 65C for 5 minutes. After a 1 minute incubation on ice, we added 4ul RT Buffer, 1ul RNaseOUT, and 1ul of Maxima H Minus Reverse Transcriptase. The solution was mixed by pipetting and incubated at 50C for 1 hour followed by heat inactivation at 85C for 10 minutes.
First strand cDNA synthesis was performed by reverse transcribing polyA mRNAs using an oligo(dT) primer that also adds on a common primer sequence. Self-reporting transcripts (SRTs) were amplified by PCR. PCR products were tagmented and indexed using the Illumina Nextera XT library preparation kit. For detailed protocol, see bioRvix (https://www.biorxiv.org/content/10.1101/2023.06.07.544098v1)

tag: transposase

value: WTHYPBcentrip


title: spleenTreg_6-5_noGvHD_Tcrb_R2

description:
re-isolated
mTRB_231_Tx198-10-4193_S_rp_A.RunAB.clonotypes.TRB.txt

tag: tissue

tag: treg donor

tag: recipient

tag: pcr1 cy. no.


title: k562, WTHYPBcentrip, rep9

tag: cell line


title: colonTreg_2-1_GvHD_Tcrb_R2

description:
re-isolated
mTRB_Tx198-1-293-C-ra_AB.RunAB.clonotypes.TRB.txt

tag: sampleID

value: allo

value: GvHD

value: colon

tag: pcr2 cy. no.

tag: chain

tag: techn. replicate


title: scTCR-seq_Spleen recipient 1

source_name: Donor allo reisolated from Recipient 1

tag: phenotype

value: transgenic FoxP3EGFP mice (B6; CD45.2; H-2b)

tag: Sex

value: female

value: Donor Treg allo reisolated from Spleen recipient 1


title: mESC Tbxt delE6/delE6 rep2

tag: cell type

tag: genotype

value: Tbxt delE6/delE6


title: scTCR-seq_Liver recipient 1

value: Donor Treg allo reisolated from Liver recipient 1

tag: cell markers


title: mESC Tbxt delE6/+ rep2


title: k562, WTHYPBcentrip, rep7


title: mESC Tbxt delE6/delE6 rep1


title: k562, WTHYPBcentrip, rep8


title: spleenTreg_7-3_noGvHD_Tcrb_R2

description:
re-isolated
mTRB_242_Tx198-12-4209_S_ra_AB.RunAB.clonotypes.TRB.txt

tag: strain

tag: mouse (pool/culture no.)

tag: type

value: BMT control

value: spleen


title: spleenTreg_6-4_noGvHD_Tcrb_R2

description:
re-isolated
mTRB_228_Tx198-10-4192_S_rp_AB.RunAB.clonotypes.TRB.txt

value: C57BL/6 Foxp3GFP+

value: poly

tag: model

tag: tissue


title: k562, WTHYPBcentrip, rep6

tag: transposase


title: scTCR-seq_Colon recipient 1

value: Donor Treg allo reisolated from Colon recipient 1


title: spleenTreg_7-2_noGvHD_Tcrb_R2

description:
re-isolated
mTRB_239_Tx198-12-4208_S_ra_A.RunAB.clonotypes.TRB.txt

tag: treg donor

tag: recipient

tag: pcr1 cy. no.


title: k562, WTHYPBcentrip-rep2

tag: cell line


title: k562, WTHYPBcentrip-rep1


title: mESC WT rep2

value: mESC

value: WT


title: k562, WTHYPBcentrip, rep5


title: spleenTreg_7-1_noGvHD_Tcrb_R2

description:
re-isolated
mTRB_245_Tx198-12-400_S_ra_A.RunAB.clonotypes.TRB.txt

tag: sampleID

tag: pcr2 cy. no.

tag: chain

value: Beta


title: scTCR-seq_Donor allo

tag: phenotype

tag: Sex

value: Donor Treg allo reisolated from Donor allo


title: spleenTreg_6-3_noGvHD_Tcrb_R2

description:
re-isolated
mTRB_225_Tx198-10-4190_S_ra_A.RunAB.clonotypes.TRB.txt

value: allo

tag: techn. replicate


title: k562, WTHYPBcentrip, rep4

value: WTHYPBcentrip


title: polyTreg_6_donor

description:
expanded
218-241_d12_rp

data_processing:
base calling: Base-calling and demultiplexing of sequencing reads was carried out using the bcl2fastq Conversion Software (v1.8.4) provided by Illumina (Illumina Inc., San Diego, California, USA).
mapping: Sequencing reads were mapped to the mouse genome (GRCm38.p5) using STAR v2.5.3a. The genome index incorporated gene annotation from GENCODE Mouse release M16 to aid in spliced alignment.
generation of unique raw reads per geneTables of raw uniquely mapped read counts per gene were generated during mapping using the built-in --quantMode GeneCounts option in STAR.
Differential expression analysis was carried out on raw gene counts using edgeR (v3.34.0) in R (v4.1.0)
Assembly: Gencode Release M16 (GRCm38.p5); https://www.gencodegenes.org/mouse/release_M16.html
Supplementary files format and content: raw reads:column 1: gene ID (Ensemble); other columns: counts for the 2nd read strand aligned with RNA (column 4 of STAR output) for individual samples
Supplementary files format and content: normalized reads: column 1-5: Ensembl ID, Gene Symbol,Transcript Length, Gene Type; other columns: log2 transformed, batch-corrected and normalized read counts for individual samples (edgeR output).

extract_protocol:
Total cellular RNA was isolated from Treg (freshly isolated, in vitro expanded, or re-isolated after BMT) using the RNeasy Mini Kit or the RNeasy Micro Kit (Qiagen). Following the manufacturer’s recommendations, samples originating from 500 to 500k cells were processed with the Micro Kit whereas the Mini Kit was applied to all samples exceeding 500k cells. RNA concentration and quality were simultaneously assessed using RNA Screentape Analysis or High Sensitivity RNA Screentape Analysis (Agilent), depending on the expected yield.
Preparation of dsDNA libraries for Illumina sequencing was facilitated using the SMART-Seq® Stranded Kit (Takara) according to the manufacturer’s instructions. Depending on sample quality and abundance, fragmentation times and second PCR cycle numbers were adjusted, respectively. For some samples, first PCR products were pooled to minimize carry-over losses. The concentration of dsDNA libraries was measured with the Qubit™ dsDNA HS Assay Kit (Thermo Fisher Scientific). DNA fragment size distribution was assessed using the High Sensitivity D1000 ScreenTape Assay (Agilent). Libraries were stored at -20°C until sequencing was performed using the Illumina NextSeq550 sequencer and libraries are summarized in Supplemental Tables 4-7.

value: in vitro

value: donor


title: ILC1_KO_3

city: St. Louis

last: Peng

address: 660 S. Euclid Ave.

institute: Washington University School of Medicine in St. Louis

data_processing:
Base calls and demultiplexing were performed with Illumina’s bcl2fastq software and a custom python demultiplexing program with a maximum of one mismatch in the indexing read. RNA-seq reads were then aligned to the Ensembl release 76 primary assembly with STAR version 2.5.1a1. Gene counts were derived from the number of uniquely aligned unambiguous reads by Subread:featureCount version 1.4.6-p52. Sequencing of RNA isolated from spheroid lines: Total RNA was extracted using RNeasy Micro Kit (Qiagen) per manufacturer’s instructions. Samples were prepared according to library kit manufacturer’s protocol (Dynal mRNA Direct Invitrogen) and sequencing was conducted on an Illumina HiSeq. Basecalls and demultiplexing were performed with Illumina’s bcl2fastq software and a custom python demultiplexing program with a maximum of one mismatch in the indexing read.
Gene count tables were processed and analyzed using the DESeq2 package with R 4.1. Prior to differential comparison, genes with fewer than 10 counts among all of the samples were excluded. Differentially expressed genes (DEG) were considered significant when padj<0.05.
Assembly: mm10
Supplementary files format and content: aligned raw count table

extract_protocol:
Total RNA was extracted using RNeasy Micro Kit (Qiagen) per manufacturer’s instructions
. Samples were prepared according to library kit manufacturer’s protocol (SMARTer Low Input RNA Kit, Clontech Takara Bio), indexed, pooled, and sequenced on an Illumina NovaSeq 6000.

tag: cell type

tag: genotype

value: Ncr1-iCre Inpp4b flox/flox


title: k562, WTHYPBcentrip, rep3


title: spleenTreg_6-2_noGvHD_Tcrb_R2

description:
re-isolated
mTRB_222_Tx198-10-4183_S_ra_A.RunAB.clonotypes.TRB.txt

tag: strain

tag: mouse (pool/culture no.)


title: polyTreg_5_donor

description:
expanded
138-210_d11_rp

tag: type

tag: model


title: spleenTreg_6-1_noGvHD_Tcrb_R2

description:
re-isolated
mTRB_219_Tx198-10-4181_S_ra_A.RunAB.clonotypes.TRB.txt

tag: tissue


title: ILC1_WT_2

value: Inpp4b flox/flox


title: polyTreg_4_donor

description:
expanded
103-208_d11_rp

tag: treg donor


title: k562, WTHYPBcentrip, rep2

tag: transposase


title: liverTreg_7-6_noGvHD_Tcrb_R2

description:
re-isolated
mTRB_252_Tx198-12-4216_L_rp_AB.RunAB.clonotypes.TRB.txt

value: liver

tag: recipient

tag: pcr1 cy. no.


title: ILC1_WT_1

source_name: liver


title: polyTreg_3_donor

description:
expanded
65-199_d12_rp


title: KP Lung tumor, 10 weeks, rep 2

city: Wuhan, China

first: Ci

last: Yang

address: 115 East Lake Road

institute: Wuhan University

data_processing:
CutadaptData filteringAt least 10 bp overlap (agatcggaag), allowing a base error rate of 20%
Library quality inspection (2100 detection of library size, fluorescence quantitative detection of total library concentration < quantifluor st fluorometer, Promega, e6090; quant it PicoGreen dsDNA assay kit, Invitrogen, p7589) >, qPCR quantitative detection of effective library concentration < Thermo Scientific steponeplus real time PCR systems >)
All RNA-seq data wrer aligned to Mus musculus GRCm38 genome with Ensembl byTophat2 microexon-search and library-type=fr-firststrand
Illumina NovaSeq 6000
Quantitative gene expression using union scheme in HTSeq
Assembly: Mus_musculus.GRCm39.dna.toplevel.fa
Supplementary files format and content: excel with FPKM for every gene and every sample

source_name: lung

growth_protocol: KP and KP25 mice were intranasally injected with Ad-Cre for 10 weeks.Tumor-burdened lungs from KL and KL25 mice were perfused through alveolar lavage and cardiac lavage with PBS and the lung tumors were collected. Single-cell suspensions were obtained from the lung tumors with the gentleMACS followed by incubation with CD45 magnetic beads for 15 min.

extract_protocol:
The cells were homogenized in 2 ml of TRIzol (Invitrogen). Total RNAs were prepared and the quality of RNAs was determined by agarose gel electrophoresis and spectrophotometer analysis.
TruSeq Stranded mRNA LT Sample Prep Kit was used to synthesized the first strand cDNA through the action of random primers and reverse transcriptase, the second strand cDNA is synthesized with the first strand cDNA as the template, cDNA library construction followed by sequencing on an Illumina NovaSeq 6000 platform.  RNA-sequencing reads were first trimmed to remove poly(A) and unqualified reads with cutadapt, then aligned to Mus musculus GRCm38 genome with Ensembl. The numbers of counts were summarized at the gene level using HTseq.

value: lung

value: CD45- lung tumor cells

tag: treatment

value: 10 weeks after  intranasal injection of Ad-Cre


title: polyTreg_7_donor

description:
expanded
238-245_d12_rp

value: C57BL/6 Foxp3GFP+

tag: pcr2 cy. no.


title: polyTreg_2_donor

description:
expanded
27-198_d13_rp

value: poly


title: KP Lung tumor, 10 weeks, rep 1


title: k562, WTHYPB, rep10

tag: cell line

value: WTHYPB


title: liverTreg_7-5_noGvHD_Tcrb_R2

description:
re-isolated
mTRB_249_Tx198-12-4214_L_rp_AB.RunAB.clonotypes.TRB.txt

tag: sampleID

value: BMT control

tag: chain


title: mESC WT rep1

value: mESC

value: WT


title: alloTreg_7_donor

description:
expanded
237-245_d12_ra


title: liverTreg_7-4_noGvHD_Tcrb_R2

description:
re-isolated
mTRB_255_Tx198-12-4205_L_rp_AB.RunAB.clonotypes.TRB.txt

value: Beta

tag: techn. replicate


title: ILC1_KO_4

tag: cell type


title: k562, WTHYPB, rep9


title: KP25 Lung tumor, 10 weeks, rep 2

tag: genotype


title: alloTreg_6_donor

description:
expanded
217-241_d12_ra

tag: strain

value: allo


title: liverTreg_6-6_noGvHD_Tcrb_R2

description:
re-isolated
mTRB_235_Tx198-10-4196_L_rp_AB.RunAB.clonotypes.TRB.txt

tag: mouse (pool/culture no.)


title: alloTreg_5_donor

description:
expanded
137-210_d11_ra

tag: type


title: liverTreg_6-5_noGvHD_Tcrb_R2

description:
re-isolated
mTRB_232_Tx198-10-4193_L_rp_AB.RunAB.clonotypes.TRB.txt

tag: model


title: KP25 Lung tumor, 10 weeks, rep 1

tag: tissue


title: k562, WTHYPB, rep7


title: Blood_2_TCR

city: shanghai

first: yifan

last: zhang

address: no

institute: fudan university

library_source: transcriptomic single cell

data_processing:
Sequencing reads from gene expression and V(D)J libraries were aligned to mm10 mouse reference genome using cellranger 7.0.0 with “multi” mode. The generated count matrices and V(D)J contig annotations were used for downstream analysis performed with Seurat(v4.1.1) and Scirpy(v0.11.2). All samples were aggregated into a single SeuratObject. Cells with gene number (<300 & >97.5% quantile) or high mitochondrial transcript ratio (>25%), and genes expressed in less than 3 cells were excluded. After removing unwanted cells from the dataset, all samples were combined with function "merge". Next, we employed a global-scaling normalization method "LogNormalize" to normalize the feature expression measurements (UMI counts) for each cell by the total expression, then data integration was performed by canonical correlation analysis according to shared sources of variation across multiple datasets using SelectIntegrationFeatures, FindIntegrationAnchors and IntegrateData functions. Highly variable genes (top 3000) were extracted to perform the principal component analysis (PCA) and top 30 of significant principle components were used for cluster analysis. Clusters were visualized using the Uniform Manifold Approximation and Projection (UMAP). Marker genes for each cluster and subgroup were identified by contrasting gene expression of cells from certain cluster or subgroup to that of others using the Seurat FindMarkers function.
Assembly: mm10
Supplementary files format and content: Matrix table with raw gene counts

source_name: Blood

extract_protocol:
Bone marrow, lymph node and peripheral blood cells isolated from C57BL/6J female mice were stained with Live/Dead dye and lymphocytes were isolated using a BDFACSAria™ IIISorter. Cells were manually counted by Trypan blue and AO-PI after each centrifugation and resuspension.
By using Chromium Next GEM Single Cell 5' Kit v2 (10x Genomics, 1000263) and Chromium Next GEM Chip K Single Cell Kit (10x Genomics, 1000287), we performed single cell TCR/BCR-seq and 5’ gene expression profiling. The cell suspension was loaded onto the Chromium single cell controller (10x Genomics) to generate single-cell gel beads in the emulsion according to the manufacturer’s protocol. Captured cells were lysed and the released RNA were barcoded through reverse transcription in individual GEMs. Cell-barcoded 5’ gene expression libraries and V(D)J enriched TCR/BCR libraries were sequenced on an Illumina NovaSeq6000 system.

value: Blood

tag: Sex

value: Female


title: k562, WTHYPB, rep6

tag: transposase


title: alloTreg_3_donor

description:
expanded
64-199_d12_ra

value: in vitro

value: donor

tag: treg donor

tag: recipient


title: liverTreg_7-3_noGvHD_Tcrb_R2

description:
re-isolated
mTRB_243_Tx198-12-4209_L_ra_AB.RunAB.clonotypes.TRB.txt

tag: pcr1 cy. no.


title: k562, WTHYPB, rep5


title: Blood_2_BCR


title: alloTreg_2_donor

description:
expanded
26-198_d13_ra

tag: pcr2 cy. no.


title: k562, WTHYPBcentrip, rep1

tag: cell line

value: WTHYPBcentrip


title: k562, WTHYPB, rep8


title: spleenCD62LnegTreg_4_resident

description:
isolated
258-010_S_rn_neg

value: homeostatic control

value: spleen


title: liverTreg_6-4_noGvHD_Tcrb_R2

description:
re-isolated
mTRB_229_Tx198-10-4192_L_rp_AB.RunAB.clonotypes.TRB.txt

tag: sampleID

value: liver


title: k562, WTHYPB, rep4


title: liverTreg_7-2_noGvHD_Tcrb_R2

description:
re-isolated
mTRB_240_Tx198-12-4208_L_ra_AB.RunAB.clonotypes.TRB.txt

tag: chain


title: k562, WTHYPB, rep3


title: k562, WTHYPB, rep2


title: liverTreg_7-1_noGvHD_Tcrb_R2

description:
re-isolated
mTRB_246_Tx198-12-400_L_ra_AB.RunAB.clonotypes.TRB.txt

tag: techn. replicate


title: spleenCD62LposTreg_4_resident

description:
isolated
257-010_S_rn_pos


title: liverTreg_6-3_noGvHD_Tcrb_R2

description:
re-isolated
mTRB_226_Tx198-10-4190_L_ra_AB.RunAB.clonotypes.TRB.txt


title: LN_2_ScRNAseq

source_name: lymph node

value: lymph node


title: Blood_2_ScRNAseq


title: LN_2_TCR


title: alloTreg_4_donor

description:
expanded
102-208_d11_ra

value: C57BL/6 Foxp3GFP+


title: spleenCD62LnegTreg_3_resident

description:
isolated
206-004_S_rn_neg


title: liverTreg_6-2_noGvHD_Tcrb_R2

description:
re-isolated
mTRB_223_Tx198-10-4183_L_ra_AB.RunAB.clonotypes.TRB.txt


title: k562, WTHYPB, rep1


title: spleenCD62LposTreg_3_resident

description:
isolated
205-004_S_rn_pos


title: LN_1_TCR


title: spleenCD62LnegTreg_2_resident

description:
isolated
204-003_S_rn_neg


title: OVCAR5 cells, 18:1 50uM + CAY10566 1uM, 24h,3

city: Kyoto

first: Jumpei

last: Ogura

state: Kyoto

address: 54 Shogoin Kawaharacho, Sakyo-ku

department: Department of Gynecology and Obstetrics

country: Japan

institute: Kyoto University Graduate School of Medicine

data_processing:
Raw reads were processed using the Trim Galore software (RRID: SCR_011847). The cleaned reads were aligned to the human genome GRCh38 (ENSEMBL release 99) using the STAR aligner (v2.7.1a; RRID: SCR_004463).
Assembly: hg38
Supplementary files format and content: Tab-separatedvalues file includes TPM values for each sample

growth_protocol: Cells were cultured in RPMI-1640 medium (Catalog# R8758; Sigma) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS; Catalog# FB-1061; Biosera, Cholet, France) and penicillin-streptomycin (Catalog# P4458; Sigma). Cells were cultured at 37 °C in an incubator supplied with 5% CO2.

extract_protocol:
Total RNA from cells was extracted using an RNeasy Micro kit (Catalog#74004, Qiagen, Valencia, CA, USA)  according to the supplier’s instructions.
TruSeq stranded mRNA Library was used  according to the supplier’s instructions.

treatment_protocol: OVCAR5 cells were seeded into six-well plate and cultured for 24 h under standard conditions. Following the initial 24-hour culture period, the cells were subjected to different treatment conditions for an additional 24 h in the following media: (1) medium containing 5% FFA-free BSA with DMSO as the control vehicle (2) medium containing 5% FFA-free BSA with 1 µM CAY10566 (3) medium containing 5% FFA-free BSA with 50 µM stearate and DMSO (4) medium containing 5% FFA-free BSA with 50 µM stearate and 1 µM CAY10566 (5) medium containing 5% FFA-free BSA with 50 µM oleate and DMSO (6) medium containing 5% FFA-free BSA with 50 µM oleate and 1 µM CAY10566.

tag: cell type

value: high grade ovarian serous adenocarcinoma

tag: treatment


title: liverTreg_6-1_noGvHD_Tcrb_R2

description:
re-isolated
mTRB_220_Tx198-10-4181_L_ra_AB.RunAB.clonotypes.TRB.txt

tag: strain


title: OVCAR5 cells, 18:1 50uM + CAY10566 1uM, 24h,2

tag: genotype


title: LN-1-BCR


title: OVCAR5 cells, 18:1 50uM + CAY10566 1uM, 24h,1


title: colonTreg_7-6_noGvHD_Tcrb_R2

description:
re-isolated
mTRB_253_Tx198-12-4216_C_rp_A.RunAB.clonotypes.TRB.txt

tag: mouse (pool/culture no.)

tag: type

value: poly

value: colon


title: spleenCD62LposTreg_2_resident

description:
isolated
203-003_S_rn_pos

tag: model

tag: tissue


title: OVCAR5 cells, 18:1 50uM + DMSO, 24h,3


title: BM_2_TCR

source_name: Bone marrow

value: Bone marrow

tag: Sex


title: spleenCD62LnegTreg_1_resident

description:
isolated
202-002_S_rn_neg

tag: treg donor


title: colonTreg_7-5_noGvHD_Tcrb_R2

description:
re-isolated
mTRB_250_Tx198-12-4214_C_rp_A.RunAB.clonotypes.TRB.txt

value: BMT control

tag: recipient


title: BM_2_ScRNAseq


title: LN_2_BCR


title: colonTreg_6-6_noGvHD_Tcrb_R2

description:
re-isolated
mTRB_236_Tx198-10-4196_C_rp_A.RunAB.clonotypes.TRB.txt

tag: pcr1 cy. no.

value: Beta


title: colonTreg_7-4_noGvHD_Tcrb_R2

description:
re-isolated
mTRB_256_Tx198-12-4205_C_rp_A.RunAB.clonotypes.TRB.txt

tag: pcr2 cy. no.


title: OVCAR5 cells, 18:1 50uM + DMSO, 24h,1

tag: cell line

value: WT


title: BM_2_BCR


title: OVCAR5 cells, 18:1 50uM + DMSO, 24h,2


title: liverCD62LposTreg_5_resident

description:
isolated
259-010_L_rn_pos


title: BM_1_TCR


title: liverCD62LnegTreg_4_resident

description:
isolated
260-010_L_rn_neg


title: LN_1_ScRNAseq


title: OVCAR5 cells, 18:0 50uM + CAY10566 1uM, 24h,2


title: colonTreg_6-4_noGvHD_Tcrb_R2

description:
re-isolated
mTRB_230_Tx198-10-4192_C_rp_A.RunAB.clonotypes.TRB.txt

tag: sampleID


title: BM_1_ScRNAseq


title: colonTreg_7-3_noGvHD_Tcrb_R2

description:
re-isolated
mTRB_244_Tx198-12-4209_C_ra_A.RunAB.clonotypes.TRB.txt

value: allo

tag: chain


title: BM-1-BCR

value: Female


title: OVCAR5 cells, 18:0 50uM + CAY10566 1uM, 24h,1


title: spleenCD62LposTreg_1_resident

description:
isolated
201-002_S_rn_pos


title: liverCD62LnegTreg_3_resident

description:
isolated
214-004_L_rn_neg


title: colonTreg_7-2_noGvHD_Tcrb_R2

description:
re-isolated
mTRB_241_Tx198-12-4208_C_ra_A.RunAB.clonotypes.TRB.txt

tag: techn. replicate


title: OVCAR5 cells, 18:0 50uM + DMSO, 24h,3

tag: cell type


title: liverCD62LposTreg_3_resident

description:
isolated
211-003_L_rn_pos


title: Ms1402_BEC_Sox9cKO

city: Augusta

first: Sergei

last: Bombin

institute: Augusta University

data_processing:
The demultiplexing, barcoded processing, and gene counting were made using Parse Biosciences pipeline v0.9.6
Assembly: GRCm39
Supplementary files format and content: Tab-separated values files and matrix files

extract_protocol:
fixed cells processed in Evercode Whole Transcriptome kit (Parse Biosciences), per manufacturer protocol
Evercode Whole Transcriptome kit (Parse Biosciences), 100K cells
split-pool combinatorial barcoding on fixed/frozen whole cells; random hexamer and oligo (dT) primers for reverse transcription

value: biliary epithelial cells


title: liverCD62LnegTreg_2_resident

description:
isolated
210-002_L_rn_neg


title: colonTreg_7-1_noGvHD_Tcrb_R2

description:
re-isolated
mTRB_247_Tx198-12-400_C_ra_A.RunAB.clonotypes.TRB.txt


title: Ms1405_BEC_Sox9con


title: colonTreg_6-3_noGvHD_Tcrb_R2

description:
re-isolated
mTRB_227_Tx198-10-4190_C_ra_AB.RunAB.clonotypes.TRB.txt


title: OVCAR5 cells, 18:0 50uM + CAY10566 1uM, 24h,3

tag: treatment


title: OVCAR5 cells, 18:0 50uM + DMSO, 24h,1


title: colonTreg_6-2_noGvHD_Tcrb_R2

description:
re-isolated
mTRB_224_Tx198-10-4183_C_ra_AB.RunAB.clonotypes.TRB.txt

tag: strain


title: NK cells, Ctr, scRNAseq

city: Beijing

first: Chen

last: Donglin

state: 北京Beijing

address: Tsinghua

institute: Tsinghua

data_processing:
They demultiplexing, barcoded processing, gene counting and aggregation were made using the Cell Ranger software, v2.1.1.
Supplementary files format and content: mm10
Supplementary files format and content: Tab-separared values and matrix files

source_name: spleen

extract_protocol:
Spleen NK cells were isolated from  normal and dietary restriction mice by flow cytometry
Library was constructed using the state-of-the-art Chromium Next GEM Single Cell 3' GEM Kit v3.1 (10× Genomics, 1000130) and Library Construction Kit (10× Genomics, 1000190), following established protocols. Briefly, a cell suspension with a density of 300-600 living cells per microliter, as determined by Count Star, was loaded onto the Chromium single-cell controller (10×Genomics). This process generated single-cell gel beads in emulsion, facilitating the isolation of individual cells for downstream analysis. To ensure cell viability, single cells were first centrifuged at 2000 rpm at 4°C to remove the supernatant. The cells were then re-suspended in PBS containing 0.04% BSA and stained with trypan blue. A microscopic evaluation was performed to confirm the viability of the cells. Subsequently, the cells were added to each channel, and the target cell population was carefully recovered. Captured cells were lysed, and the released RNA molecules were barcoded through reverse transcription within individual gel beads. Reverse transcription was conducted using a C1000 Touch TM Thermal Cycler (Bio Rad) at 53°C for 45 minutes, followed by 85°C for 5 minutes, and then held at 4°C. The generated cDNA was amplified, and its quality was assessed using an Agilent 4200 instrument.

value: NK cells

value: control


title: colonTreg_6-5_noGvHD_Tcrb_R2

description:
re-isolated
mTRB_233_Tx198-10-4193_C_rp_A.RunAB.clonotypes.TRB.txt


title: liverCD62LnegTreg_1_resident

description:
isolated
208-001_L_rn_neg

value: homeostatic control


title: NK cells, DR, scRNAseq

value: spleen

value: dietary restriction


title: liverCD62LposTreg_2_resident

description:
isolated
209-002_L_rn_pos

value: liver


title: OVCAR5 cells, CAY10566 1uM, 24h,3

tag: genotype


title: liverCD62LposTreg_1_resident

description:
isolated
207-001_L_rn_pos

value: C57BL/6 Foxp3GFP+

tag: mouse (pool/culture no.)


title: hi-GelMA

city: Irvine

last: Butenko

state: CA

address: Irvine

institute: University of California, Irvine

data_processing:
10X Chromium sequencing FASTQ output files were aligned using Cell Ranger
Assembly: mm10
Supplementary files format and content: tab separated values and matrix

source_name: skin wound+hydrogel

extract_protocol:
PWD5 wound tissue was dissected from the surrounding edge and healthy tissue, minced with scissors, and then dissociated with 10 ml solution of 2.7 mg/mL collagenase P (Sigma), 1mM pyruvate, 10 mM HEPES, in RPMI basal media at 37°C for 2 hours at 37C, triturating every 15 minutes. Digestion was halted with the addition of 2ml 2% FBS and live cells were isolated using Miltenyi dead cell separation kit and MS columns, per manufacturer’s instructions. Cells were finally suspended in 1ml 0.04% BSA and kept on ice until processed for library preparation.
RNA library was prepared using a 10X Chromium V3.1 kit and sequenced with NovaSeq on an S4 flow cell (UCI Genomic High Throughput Facility).

value: skin wound+hydrogel

value: hi-GelMA


title: colonTreg_6-1_noGvHD_Tcrb_R2

description:
re-isolated
mTRB_221_Tx198-10-4181_C_ra_A.RunAB.clonotypes.TRB.txt

tag: type

value: colon


title: polyTreg_7_donor_Tcrb_R2

description:
expanded
mTRB_238_Tx198-12-245_d12_rp_A.RunAB.clonotypes.TRB.txt

value: poly

tag: model

value: in vitro

value: donor


title: lo-GelMA

tag: tissue

value: lo-GelMA


title: OVCAR5 cells, CAY10566 1uM, 24h,2

value: high grade ovarian serous adenocarcinoma

value: WT


title: OVCAR5 cells, 18:0 50uM + DMSO, 24h,2


title: OVCAR5 cells, CAY10566 1uM, 24h,1


title: NonTreated

value: Non treated


title: polyTreg_6_donor_Tcrb_R2

description:
expanded
mTRB_218_Tx198-10-241_d12_rp_A.RunAB.clonotypes.TRB.txt

value: Beta


title: polyTreg_4_donor_Tcrb_R2

description:
expanded
mTRB_103_Tx198-4-208_d11_rp_A.RunAB.clonotypes.TRB.txt


title: OVCAR5 cells, DMSO, 24h,3

value: DMSO, 24h


title: colonTreg_4_resident

description:
isolated
263-009_C_rn_neg

value: homeostatic control


title: Kidney_KO_4

source_name: Kidney

value: Kidney

value: Pcmt1 KO


title: Kidney_KO_3


title: colonTreg_3_resident

description:
isolated
262-008_C_rn_neg


title: liverCD62LposTreg_4_resident

description:
isolated
213-004_L_rn_pos

value: liver


title: colonTreg_2_resident

description:
isolated
261-007_C_rn_neg

value: C57BL/6 Foxp3GFP+


title: Ari-3

city: Hangzhou

first: Yuhong

last: Cao

address: pingle road 325

institute: ZheJiang Institute For Drug Control

description: taotal RNA from iPSC-CMs

data_processing:
CLC Genomics Workbench v 11.0.1
Assembly: mm 10
Supplementary files format and content: tab-delimited text files include counts and RPKM values for each sample

source_name: iPSC-CM

extract_protocol:
RNA was harvested using Rneasy mini plus kit of iPSC-CMs was extracted using RNeasy Mini Kit (Qiagen).1.3 μg of total RNA was used for the construction of sequencing libraries.
RNA libraries for RNA-seq were prepared using SMARTER mRNA-Seq Library Prep Kit following manufacturer's protocols.

value: iPSC-CM

value: Cardiomyocytes

value: 1microM Ari


title: OVCAR5 cells, DMSO, 24h,2


title: polyTreg_3_donor_Tcrb_R2

description:
expanded
mTRB_Tx198-2-d12-rp_AB.RunAB.clonotypes.TRB.txt

value: poly


title: colonTreg_1_resident

description:
isolated
215-005_C_rn_neg

value: colon


title: Kidney_KO_1


title: Ari-2

description: total RNA  from iPSC-CMs


title: alloTreg_7_donor_Tcrb_R2

description:
expanded
mTRB_237_Tx198-12-245_d12_ra_A.RunAB.clonotypes.TRB.txt

value: allo

value: in vitro

value: donor


title: spleenTreg_3-6_GvHD

description:
re-isolated
83-349_S_rp

value: GvHD

value: spleen


title: polyTreg_2_donor_Tcrb_R2

description:
expanded
mTRB_Tx198-1-d13-rp_AB.RunAB.clonotypes.TRB.txt

value: Beta


title: Kidney_WT_4

value: WT


title: Kidney_KO_2


title: polyTreg_1_donor_Tcrb_R2

description:
expanded
mTRB_176_Treg_d14_poly_A.RunAB.clonotypes.TRB.txt

value: C57BL/6 WT


title: colonTreg_5_resident

description:
isolated
264-010_C_rn_neg

value: homeostatic control


title: alloTreg_6_donor_Tcrb_R2

description:
expanded
mTRB_217_Tx198-10-241_d12_ra_A.RunAB.clonotypes.TRB.txt


title: spleenTreg_3-5_GvHD

description:
re-isolated
80-347_S_rp


title: Kidney_WT_3

value: Kidney


title: spleenTreg_3-4_GvHD

description:
re-isolated
77-343_S_rp


title: Kidney_WT_2


title: alloTreg_4_donor_Tcrb_R2

description:
expanded
mTRB_102_Tx198-4-208_d11_ra_A.RunAB.clonotypes.TRB.txt


title: OVCAR5 cells, DMSO, 24h,1

value: high grade ovarian serous adenocarcinoma

value: DMSO, 24h


title: spleenTreg_3-3_GvHD

description:
re-isolated
74-348_S_ra

value: C57BL/6 Foxp3GFP+


title: Con-2

description: taotal RNA from iPSC-CMs

value: iPSC-CM

value: Cardiomyocytes

value: 0.1percent DMSO


title: alloTreg_3_donor_Tcrb_R2

description:
expanded
mTRB_Tx198-2-d12-ra_AB.RunAB.clonotypes.TRB.txt


title: spleenTreg_3-2_GvHD

description:
re-isolated
71-346_S_ra


title: Kidney_WT_1


title: Con-1

description: total RNA  from iPSC-CMs


title: Lung, AT2, EGFR, Tp53 and Pten loss, Myc transgene, pooled

city: NEW YORK

first: Ashley

middle: M

last: Laughney

state: New York

address: 413 East 69th Street

department: Physiology, Biophysics, and Systems Biology

institute: Weill Cornell Medicine

data_processing:
Single cell RNA-seq reads were aligned, demultiplexed, and processed using  CellRanger (v5.0.1). Single cells libraries were filtered for high quality cells based on cell size, mitochondrial content, doublet score, and library complexity.
Assembly: mm10, plus TdTom and Tg.EGFR transgenes
Supplementary files format and content: .h5ad: anndata object for single cell RNA-seq experiments. Contains normalize,  log-transformed, and imputed counts, along with cell and gene level feature information.

source_name: Lung

growth_protocol: all cells were freshly isolated from lungs of normal or tumorigenic mice

extract_protocol:
10X Chromium 5' single cell RNA v3 chemistry protocol
10X Genomics Chromium Next GEM Single Cell 3' Reagent Kits, v3.1  Dual Index Chemistry

treatment_protocol: vehicle or osimertinib

value: Lung


title: alloTreg_2_donor_Tcrb_R2

description:
expanded
mTRB_Tx198-1-d13-ra_AB.RunAB.clonotypes.TRB.txt

source_name: CD4+CD25+ regulatory T cells

value: allo


title: spleenTreg_2-6_GvHD

description:
re-isolated
45-299_S_rp

value: poly


title: myotubes,D8, DupExon8-9, edited,rep3

city: EVRY

first: Corre

last: Guillaume

address: 1 rue de l'internationale

country: France

institute: GENETHON

data_processing:
paired-end reads were aligned to the reference transcriptome using SALMON 1.9.0 using default settings and  -l A and --validateMappings arguments
Read count extraction was performed using R 4.3.0 and the tximport package (v1.28.0)
Differential gene expression was performed using R and the package DESeq2 (v1.40.1) on genes with more than 10 reads across samples and using arguments lfcThreshold = 0, pAdjustMethod = "fdr", independentFiltering = T
Assembly: GRCh38.p13 / transcriptome gencode.v43
Supplementary files format and content: tab-delimited file containing counts per gene (row) and sample (columns) from tximport function
Supplementary files format and content: tab-delimited file containing effective length per gene (row) and sample (columns) from tximport function
Supplementary files format and content: tab-delimited file containing abundance per gene (row) and sample (columns) from tximport function
Supplementary files format and content: tab-delimited file containing the differential gene expression analysis for each biological sample between edited VS un-edited samples

source_name: immortalized myoblast

growth_protocol: Myoblasts were cultured in Skeletal Muscle Cell Growth Medium (PromoCell, C-23060)

extract_protocol:
Total RNA extraction was performed from myotubes (at day 8 of differentiation) lysates using the RNeasy Plus kit (Qiagen).
library construction performed by Azenta
sequencing performed by Azenta using NovaSeq 2x150

treatment_protocol: Myoblasts were differenciated into myotubes for eight days in Skeletal Muscle Differentiation Medium (PromoCell, C-23061)

value: immortalized myoblast

tag: mutation

tag: replicate

value: edited


title: myotubes,D8, DupExon8-9, untreated,rep3

value: untreated


title: Lung, Normal AT2, Pten loss, Myc transgene, pooled


title: spleenTreg_2-5_GvHD

description:
re-isolated
42-298_S_rp

value: GvHD

value: spleen

tag: treg donor

tag: recipient


title: Lung, Normal PNEC, Myc transgene, pooled

value: PNEC-Myc


title: Ari-1

description: taotal RNA from iPSC-CMs

value: WT

value: 1microM Ari


title: spleenCD62LposTreg_4_resident_Tcra_R2

description:
isolated
mTRA_257-010-S-rn-pos_A.RunAB.clonotypes.TRA.txt

data_processing:
base calling: Base-calling and demultiplexing based on Illumina barcodes was carried out using the bcl2fastq Conversion Software (v1.8.4) provided by Illumina (Illumina INc., San Diego, California, USA).
paired-end read alignment: UMI-barcoded and paired TCR-sequencing reads were joined using PEAR (v0.9.11).
UMI-based generation of MIGs: Demultiplexing on the basis of UMI-barcodes and adaptor trimming was done using MIGEC (v1.2.9) resulting in consensus sequences of molecular identifier groups (MIGs).
MIG mapping: MIGs were mapped using MIXCR (v3.0.13).
Assembly: RepSeq.IO library v1.6
Supplementary files format and content: Mapping output of MIXCR
Library strategy: TCR-seq

extract_protocol:
Total cellular RNA was isolated from Treg (freshly isolated, in vitro expanded, or re-isolated after BMT) using the RNeasy Mini Kit or the RNeasy Micro Kit (Qiagen). Following the manufacturer’s recommendations, samples originating from 500 to 500k cells were processed with the Micro Kit whereas the Mini Kit was applied to all samples exceeding 500k cells. RNA concentration and quality were simultaneously assessed using RNA Screentape Analysis or High Sensitivity RNA Screentape Analysis (Agilent), depending on the expected yield.
Total cellular RNA was obtained as described in section “RNA-seq library preparation”. TCR repertoire sequencing libraries for Illumina sequencing were prepared on the basis of {Mamedov, 2013 #7} and the protocol was adapted, as follows. For the generation of 5’RACE-ready cDNA total RNA ranging from 10 - 200 ng (low input) or 200 - 1000 ng (high input) in a total of 10 µl RNAse-free water was combined with 1 µL of 12 µM or 25 µM 5’-CDS Primer A in a total volume of 11 µL, respectively. Reactions were incubated at 72°C for 3 min followed by 42°C for 2 min for poly-dT primer annealing. Next, 1 µL of 10 µM (low input) or 50 µM (high input) TCR_UMI_Smarter template-switching oligo (TSO) was added at room temperature. RT Buffer Mix (5x First-Strand-Buffer Mix, 20mM dNTP, 100 mM DTT) was freshly prepared on ice before it was supplemented with RNaseOUT™ Recombinant Ribonuclease Inhibitor (40 U/µL) (Thermo Fisher Scientific) and SMARTScribe Reverse Transcriptase (100 U/µL) (Takara) at room temperature. A volume of 8 µL of the resulting reverse transcription master mix was added to each reaction. After homogenization, the 20 µL reactions were incubated at 42°C for 90 min followed by 70°C for 10 min and put on hold at 10°C in a hot-lid thermal cycler. Following reverse transcription, excess TSO was digested using Uracil-DNA Glycosylase (UDG) (New England Biolabs) by adding 4 µL of UDG master mix adjusted for TSO input amount and successive incubation at 37°C for 60 min. The master mix for PCR1 was prepared on ice using the Advantage 2 PCR Kit (Takara) along with primers specific for the constant regions of the Trac and Trbc loci as well as a universal forward primer specific for a binding sequence introduced via the TSO. 24 µL of RACE-ready cDNA were combined with 26 µL of PCR1 master mix and subjected to a two-step PCR program (95°C, 1min; 95°C, 30 s & 68°C, 70 s (x times); 68°C, 7 min; 8°C, hold). Depending on the RNA input amount, PCR cycle numbers ranged from 16 to 28. The 50 µL PCR product was purified with 27.5 µL of AMPure XP beads (Beckman Coulter) according to the manufacturer’s instructions. After elution in 23.5 µL of Monarch® DNA Elution Buffer (New England Biolabs), reactions were split into 10 µL technical replicates for successive extension PCR (PCR2). PCR2 master mix was assembled using the Advantage 2 PCR Kit (Takara) and gene-specific nested primers (Trac, Trbc loci) introducing Illumina Nextera XT i5 indices (S501, S502, S503, S508, S517, S520, S507), distinguishing Tcra from Tcrb libraries within each reaction. After addition of 36 µl PCR2 master mix, 4 µL of Nextera XT Index Kit v2 (Illumina) adapters were used for indexing libraries individually. Libraries were amplified using the same program as in PCR1 with 18 PCR cycles. Library purification was carried out using  AMPure XP beads (Beckman Coulter) at a volume ratio of 0.8 according to the manufacturer’s instructions. After final elution in 17 µL of Monarch® DNA Elution Buffer (New England Biolabs), concentration was measured with the Qubit™ dsDNA HS Assay Kit (Thermo Fisher Scientific) and fragment size profile was assessed using the High Sensitivity D1000 or D5000 ScreenTape Assay (Agilent). Libraries were stored at -20°C until sequencing (paired-end 300bp) on the Illumina MiSeq system. Oligonucleotide sequences are summarized in Supplemental Table 3, Libraries are summarized in Supplemental Tables 4-7.
poly-dT-primed, gene-specifically enriched TRA/TRB library preparation

value: homeostatic control

tag: pcr1 cy. no.

tag: pcr2 cy. no.

value: alpha


title: spleenTreg_2-4_GvHD

description:
re-isolated
39-297_S_rp

value: C57BL/6 Foxp3GFP+


title: spleenCD62LposTreg_5_resident_Tcra_R2

description:
isolated
mTRA_176_Treg_d0_poly_A.RunAB.clonotypes.TRA.txt

value: C57BL/6 WT

tag: sampleID


title: Con-3

description: total RNA  from iPSC-CMs

tag: cell line

value: iPSC-CM

value: Cardiomyocytes


title: spleenCD62LposTreg_3_resident_Tcra_R2

description:
isolated
mTRA_205_d0_S_Cd62lpos_A.RunAB.clonotypes.TRA.txt

tag: chain

tag: techn. replicate


title: myotubes,D8, DupExon2-9, edited,rep3

tag: treatment


title: spleenTreg_2-3_GvHD

description:
re-isolated
36-296_S_ra

tag: strain


title: Lung, Normal AT2, Myc transgene, pooled

value: Lung

tag: genotype


title: spleenTreg_2-2_GvHD

description:
re-isolated
33-294_S_ra

tag: mouse (pool/culture no.)

value: allo


title: myotubes,D8, DupExon2-9, untreated,rep3


title: spleenCD62LposTreg_2_resident_Tcra_R2

description:
isolated
mTRA_203_d0_S_Cd62lpos_A.RunAB.clonotypes.TRA.txt

tag: type


title: myotubes,D8, DupExon2, untreated,rep3


title: Lung, Normal AT2, Pten loss, pooled


title: alloTreg_1_donor_Tcrb_R2

description:
expanded
mTRB_196_Treg_d14_allo_A.RunAB.clonotypes.TRB.txt

tag: model

value: in vitro

value: donor

value: Beta


title: spleenTreg_3-1_GvHD

description:
re-isolated
68-341_S_ra

tag: tissue


title: spleenTreg_2-1_GvHD

description:
re-isolated
30-293_S_ra


title: Lung, Normal PNEC, pooled

value: PNEC-wt


title: myotubes,D8, DupExon8-9, edited,rep2

value: immortalized myoblast

tag: mutation

value: edited


title: spleenCD62LposTreg_1_resident_Tcra_R2

description:
isolated
mTRA_201_d0_S_Cd62lpos_A.RunAB.clonotypes.TRA.txt

value: spleen


title: spleenCD62LnegTreg_4_resident_Tcra_R2

description:
isolated
mTRA_258-010-S-rn-neg_A.RunAB.clonotypes.TRA.txt


title: liverTreg_3-6_GvHD

description:
re-isolated
81-349_L_rp

value: poly

value: GvHD

value: liver


title: myotubes,D8, DupExon8-9, untreated,rep2

tag: replicate

value: untreated


title: liverTreg_3-5_GvHD

description:
re-isolated
78-347_L_rp

value: C57BL/6 Foxp3GFP+

tag: treg donor


title: Lung, de novo LUAD, Myc wt, pooled


title: spleenCD62LnegTreg_3_resident_Tcra_R2

description:
isolated
mTRA_206-004-S-rn-neg_A.RunAB.clonotypes.TRA.txt

value: homeostatic control

tag: recipient

value: alpha


title: myotubes,D8, DupExon2, edited,rep3


title: Lung, de novo LUAD, Rb1 wt, off DOX, pooled


title: liverTreg_3-4_GvHD

description:
re-isolated
72-348_L_ra


title: myotubes,D8, DupExon2-9, edited,rep2


title: spleenCD62LnegTreg_2_resident_Tcra_R2

description:
isolated
mTRA_204-003-S-rn-neg_A.RunAB.clonotypes.TRA.txt

tag: pcr1 cy. no.


title: myotubes,D8, DupExon2-9, untreated,rep2


title: liverTreg_3-2_GvHD

description:
re-isolated
69-346_L_ra

tag: pcr2 cy. no.


title: Lung, Normal AT2, pooled


title: Lung, LUAD off DOX, time 3, pooled


title: myotubes,D8, DupExon2, edited,rep2


title: spleenCD62LnegTreg_1_resident_Tcra_R2

description:
isolated
mTRA_202-002-S-rn-neg_A.RunAB.clonotypes.TRA.txt

tag: sampleID


title: Lung, LUAD restarted on DOX, pooled


title: liverCD62LposTreg_5_resident_Tcra_R2

description:
isolated
mTRA_259-010-L-rn-pos_A.RunAB.clonotypes.TRA.txt


title: Lung, de novo LUAD, Rb1 wt, pooled


title: liverTreg_3-1_GvHD

description:
re-isolated
66-341_L_ra


title: liverTreg_2-6_GvHD

description:
re-isolated
43-299_L_rp


title: myotubes,D8, DupExon8-9, edited,rep1

tag: cell line


title: myotubes,D8, DupExon2, untreated,rep2


title: liverCD62LposTreg_4_resident_Tcra_R2

description:
isolated
mTRA_213-004-L-rn-pos_A.RunAB.clonotypes.TRA.txt

tag: chain


title: Lung, LUAD off DOX, time 2, pooled


title: liverTreg_2-5_GvHD

description:
re-isolated
40-298_L_rp


title: Lung, SCLC genotype, Pten loss, AT2 origin, time 2, pooled


title: liverTreg_2-4_GvHD

description:
re-isolated
37-297_L_rp


title: liverCD62LposTreg_1_resident_Tcra_R2

description:
isolated
mTRA_207-001-L-rn-pos_A.RunAB.clonotypes.TRA.txt

tag: techn. replicate


title: myotubes,D8, DupExon2-9, edited,rep1

tag: treatment


title: liverCD62LposTreg_2_resident_Tcra_R2

description:
isolated
mTRA_209-002-L-rn-pos_A.RunAB.clonotypes.TRA.txt

tag: strain


title: Lung, SCLC genotype, Pten loss, AT2 origin, time 1, pooled

tag: genotype


title: liverTreg_2-3_GvHD

description:
re-isolated
34-296_L_ra

tag: mouse (pool/culture no.)


title: liverCD62LnegTreg_4_resident_Tcra_R2

description:
isolated
mTRA_260-010-L-rn-neg_A.RunAB.clonotypes.TRA.txt

tag: type


title: liverTreg_2-2_GvHD

description:
re-isolated
31-294_L_ra

tag: model


title: liverCD62LnegTreg_3_resident_Tcra_R2

description:
isolated
mTRA_214-004-L-rn-neg_A.RunAB.clonotypes.TRA.txt

tag: tissue


title: Lung, AT2, EGFR, Tp53 and Pten loss, pooled


title: liverTreg_2-1_GvHD

description:
re-isolated
28-293_L_ra


title: liverCD62LnegTreg_2_resident_Tcra_R2

description:
isolated
mTRA_210-002-L-rn-neg_A.RunAB.clonotypes.TRA.txt


title: myotubes,D8, DupExon2, untreated,rep1

tag: mutation


title: Lung, LUAD off DOX, time 1, pooled


title: colonTreg_3-6_GvHD

description:
re-isolated
82-349_C_rp

value: colon


title: human multilineage differentiating stress-enduring enriched cell_3

city: Xinxiang

first: Han

last: Li

address: 601 Jinsui Road, Hongqi District, Xinxiang City, Henan Province

institute: Xinxiang Medical University

data_processing:
Cutadapt was used to trim adapter sequences/low-quality reads, allowing 20% base mismatch.
The filtered reads were aligned to the reference genome using HISAT2 software.
HTSeq statistics were used to align the Read Count value of each gene as the original gene expression.
Read counts was normalized to log2-counts per million with the functions calcNormFactors and voom from the R package
Assembly: hg38
Supplementary files format and content: tab-delimited text file includes normalized data for each sample

source_name: mesenchymal stem cell

extract_protocol:
RNA was extracted using TRIzol reagent (DP424, Tiangen) according to the manufacturer’s instruction. 0.5 µg of total RNA was used for the construction of sequencing libraries.
RNA-seg libraries were prepared by Personal Biotechnology Co., Ltd.

tag: cell type

value: mesenchymal stem cell


title: myotubes,D8, DupExon8-9, untreated,rep1

tag: replicate


title: Lung, de novo LUAD, pooled

value: Lung


title: liverCD62LnegTreg_1_resident_Tcra_R2

description:
isolated
mTRA_208-001-L-rn-neg_A.RunAB.clonotypes.TRA.txt

tag: treg donor

tag: recipient


title: human multilineage differentiating stress-enduring enriched cell_2


title: Lung, LUAD treated with osi, time1,  pooled

value: osimertinib


title: colonTreg_5_resident_Tcra_R2

description:
isolated
mTRA_264-010-C-rn-neg_A.RunAB.clonotypes.TRA.txt

tag: pcr1 cy. no.

tag: pcr2 cy. no.


title: colonTreg_3-4_GvHD

description:
re-isolated
76-343_C_rp


title: colonTreg_3-5_GvHD

description:
re-isolated
79-347_C_rp


title: colonTreg_4_resident_Tcra_R2

description:
isolated
mTRA_263-009-C-rn-neg_A.RunAB.clonotypes.TRA.txt

tag: sampleID


title: human multilineage differentiating stress-enduring enriched cell_1


title: Lung, de novo SCLC, pooled

value: PNEC-RPMT


title: colonTreg_3-3_GvHD

description:
re-isolated
73-348_C_ra

value: allo


title: myotubes,D8, DupExon2-9, untreated,rep1

tag: cell line


title: Lung, de novo LUAD, rep 3


title: myotubes,D8, DupExon2, edited,rep1


title: Lung, SCLC genotype, AT2 origin, pooled


title: human menstrual blood-derived endometrium stem cell_3


title: Lung, de novo LUAD, rep 2


title: colonTreg_3-2_GvHD

description:
re-isolated
70-346_C_ra


title: colonTreg_3_resident_Tcra_R2

description:
isolated
mTRA_262-008-C-rn-neg_A.RunAB.clonotypes.TRA.txt

tag: chain

tag: techn. replicate


title: colonTreg_3-1_GvHD

description:
re-isolated
67-341_C_ra

tag: strain


title: human menstrual blood-derived endometrium stem cell_2


title: colonTreg_2_resident_Tcra_R2

description:
isolated
mTRA_261-007-C-rn-neg_A.RunAB.clonotypes.TRA.txt


title: Lung, de novo LUAD, rep 1

tag: genotype


title: spleenTreg_3-6_GvHD_Tcra_R2

description:
re-isolated
mTRA_Tx198-2-349-S-rp_A.RunAB.clonotypes.TRA.txt

tag: mouse (pool/culture no.)


title: human menstrual blood-derived endometrium stem cell_1


title: Lung, de novo SCLC, rep 3


title: spleenCD62LposTreg_6_resident_Tcra_R2

description:
isolated
mTRA_196_Treg_d0_allo_A.RunAB.clonotypes.TRA.txt

value: C57BL/6 WT

tag: type


title: colonTreg_2-6_GvHD

description:
re-isolated
44-299_C_rp

tag: model


title: spleenTreg_3-5_GvHD_Tcra_R2

description:
re-isolated
mTRA_Tx198-2-347-S-rp_A.RunAB.clonotypes.TRA.txt

tag: tissue


title: spleenTreg_3-4_GvHD_Tcra_R2

description:
re-isolated
mTRA_Tx198-2-343-S-rp_A.RunAB.clonotypes.TRA.txt


title: HBsAg high group FNA10

city: Rotterdam

first: Andre

last: Boonstra

address: Dr. Molewaterplein 40

department: Dept. of Gastroenterology and Hepatology

country: Netherlands

institute: Erasmus MC

data_processing:
To process raw files to count matrices, 10x Genomics Cell Ranger 6.1.2 with the human reference genome GRCh38 was used with default settings.
Cell Ranger-processed filtered feature matrices were analyzed using R version 4.2.2 and Seurat version 4.3.0.
Assembly: GRCh38-1.2.0
Supplementary files format and content: Processed files with output of the 10X cellranger pipeline (filtered_feature_bc_matrix, .tsv and .matrix files).
Supplementary files format and content: Seurat objects (RDS files) with R scripts (Rmd and html files).
Supplementary files format and content: Processed data files FNAs (Cell Ranger-processed filtered feature matrices)
Supplementary files format and content: Processed data files PBMCs (Cell Ranger-processed filtered feature matrices)

source_name: FNAs

extract_protocol:
10X Genomics Single Cell 3’ v3 Reagent Kit, or 10x Genomics Single Cell 5’ v2 Reagent Kit
10X Genomics Single Cell 3’ v3 Reagent Kit, and 10x Genomics Single Cell 5’ v2 Reagent Kit

value: FNAs

tag: disease state

value: chronic hepatitis B patient

value: liver fine-needle aspirates

tag: group

value: HBsAg high group

tag: treatment

value: nucleoside analogues


title: HBsAg low group FNA9

tag: cell type

value: HBsAg low group


title: colonTreg_2-5_GvHD

description:
re-isolated
41-298_C_rp

value: poly

value: GvHD

tag: treg donor

tag: recipient


title: Lung, de novo SCLC, rep 1


title: colonTreg_2-4_GvHD

description:
re-isolated
38-297_C_rp

value: C57BL/6 Foxp3GFP+

tag: pcr2 cy. no.


title: HBsAg high group FNA8


title: colonTreg_2-3_GvHD

description:
re-isolated
35-296_C_ra


title: C5

city: West Hollywood

first: Simon

last: Knott

state: California

address: 8687 Melrose Ave

department: BFG

institute: Cedars Sinai

description: Triple-receptor negative breast cancer

data_processing:
Demultiplexed fastq files for gene expression libraries were processed using Cellranger (version 4.0.0, GRCh38-3.0.0 transcriptome reference). Doublet cells were identified using Scrublet (Wolock et al., 2019) (version 0.2.1) from the filtered feature barcode matrices produced by Cellranger. Scrublet was run using the 10% highest variable genes, identified using the Scanpy2 scanpy.pp.highly_variable_genes function (scanpy version 1.5.1), with an expected doublet rate of 10%. Cells were scored as candidate doublets by Scrublet and removed if their doublet score exceeded 0.25. In addition, the top 1% of cells by UMI count per biopsy were filtered as presumed doublets. Remaining cells were finally filtered to have less than 10% of their UMI’s mapped to mitochondrial genes and to express greater than 100 genes.
Assembly: mm10
Supplementary files format and content: H5AD file containing celltype, subtype,batch, treatment and other metadata columns for each cell

source_name: Breast

extract_protocol:
Murine TNBC tumors were minced and dissociated using the Miltenyi Biotec Mouse Tumor Dissociation kit, then CD45-positive cells were enriched using the Miltenyi Biotec anti-mouse CD45 microbeads
Single-cell gene expression librarieswere prepared according to the 10X Genomics Single Cell 3’ v2 protocol.

value: Breast

value: Female

value: Radiation + Pembrolizumab


title: HBsAg low group FNA7


title: spleenTreg_2-5_GvHD_Tcra_R2

city: Regensburg

last: Rehli

institute: University Hospital Regensburg

description:
re-isolated
mTRA_Tx198-1-298-S-rp_A.RunAB.clonotypes.TRA.txt

tag: sampleID

value: spleen

tag: pcr1 cy. no.

value: alpha


title: colonTreg_2-2_GvHD

description:
re-isolated
32-294_C_ra

value: colon


title: C4

description: Triple-receptor negative breast cancer

value: Radiation + IgG


title: spleenTreg_2-4_GvHD_Tcra_R2

description:
re-isolated
mTRA_Tx198-1-297-S-rp_A.RunAB.clonotypes.TRA.txt

tag: chain


title: colonTreg_2-1_GvHD

description:
re-isolated
29-293_C_ra

value: allo


title: B6

description: Triple-receptor negative breast cancer


title: HBsAg low group FNA6

value: FNAs


title: Lung, de novo SCLC, rep 2

value: Lung

value: PNEC-RPMT


title: spleenTreg_7-4_noGvHD

description:
re-isolated
254-4205_S_rp

value: BMT control


title: B5

description: Triple-receptor negative breast cancer


title: HBsAg high group FNA5

value: chronic hepatitis B patient

value: liver fine-needle aspirates

value: HBsAg high group

value: nucleoside analogues


title: spleenTreg_3-2_GvHD_Tcra_R2

description:
re-isolated
mTRA_Tx198-2-346-S-ra_A.RunAB.clonotypes.TRA.txt

value: C57BL/6 Foxp3GFP+

value: GvHD

tag: techn. replicate


title: spleenTreg_7-6_noGvHD

description:
re-isolated
251-4216_S_rp

tag: strain

value: poly


title: spleenTreg_2-6_GvHD_Tcra_R2

description:
re-isolated
mTRA_Tx198-1-299-S-rp_A.RunAB.clonotypes.TRA.txt


title: HBsAg high group FNA4


title: B4

description: Triple-receptor negative breast cancer

value: Breast

tag: genotype

value: Female


title: spleenTreg_7-5_noGvHD

description:
re-isolated
248-4214_S_rp

tag: mouse (pool/culture no.)

value: spleen


title: spleenTreg_3-1_GvHD_Tcra_R2

description:
re-isolated
mTRA_Tx198-2-341-S-ra_A.RunAB.clonotypes.TRA.txt

tag: type

value: alpha


title: HBsAg high group FNA1


title: spleenTreg_7-1_noGvHD

description:
re-isolated
245-400_S_ra

tag: model


title: HBsAg low group HBV_X983 (PBMC20)

source_name: PBMCs

tag: tissue

value: PBMCs

value: peripheral blood mononuclear cells

value: HBsAg low group


title: B3

description: Triple-receptor negative breast cancer

value: Pembrolizumab


title: HBsAg low group HBV_X980 (PBMC19)

tag: disease state


title: spleenTreg_3-3_GvHD_Tcra_R2

description:
re-isolated
mTRA_Tx198-2-348-S-ra_A.RunAB.clonotypes.TRA.txt

value: allo


title: spleenTreg_2-3_GvHD_Tcra_R2

description:
re-isolated
mTRA_Tx198-1-296-S-ra_A.RunAB.clonotypes.TRA.txt


title: spleenTreg_7-3_noGvHD

description:
re-isolated
242-4209_S_ra

value: BMT control


title: spleenTreg_2-2_GvHD_Tcra_R2

description:
re-isolated
mTRA_Tx198-1-294-S-ra_A.RunAB.clonotypes.TRA.txt


title: spleenTreg_2-1_GvHD_Tcra_R2

description:
re-isolated
mTRA_Tx198-1-293-S-ra_A.RunAB.clonotypes.TRA.txt


title: spleenTreg_7-2_noGvHD

description:
re-isolated
239-4208_S_ra


title: HBsAg high group HBV_X846 (PBMC17)

value: chronic hepatitis B patient

tag: group


title: mF1

description: Triple-receptor negative breast cancer

tag: treatment

value: Radiation


title: liverTreg_3-6_GvHD_Tcra_R2

description:
re-isolated
mTRA_Tx198-2-349-L-rp_A.RunAB.clonotypes.TRA.txt

value: liver


title: HBsAg high group HBV_X861 (PBMC18)

tag: cell type

value: HBsAg high group

value: nucleoside analogues


title: mE1

description: Triple-receptor negative breast cancer


title: mD1

description: Triple-receptor negative breast cancer

value: Radiation + IgG


title: liverTreg_3-5_GvHD_Tcra_R2

description:
re-isolated
mTRA_Tx198-2-347-L-rp_A.RunAB.clonotypes.TRA.txt

value: C57BL/6 Foxp3GFP+

value: poly

value: GvHD

tag: treg donor


title: spleenTreg_6-6_noGvHD

description:
re-isolated
234-4196_S_rp

value: spleen

tag: recipient


title: HBsAg low group HBV_X842 (PBMC16)


title: spleenTreg_6-5_noGvHD

description:
re-isolated
231-4193_S_rp

tag: pcr2 cy. no.


title: liverTreg_3-4_GvHD_Tcra_R2

description:
re-isolated
mTRA_Tx198-2-343-L-rp_AB.RunAB.clonotypes.TRA.txt

tag: sampleID

value: alpha


title: spleenTreg_6-4_noGvHD

description:
re-isolated
228-4192_S_rp


title: B1

description: Triple-receptor negative breast cancer

value: Breast

value: Female

value: IgG


title: mB1

description: Triple-receptor negative breast cancer

value: Pembrolizumab


title: spleenTreg_6-3_noGvHD

description:
re-isolated
225-4190_S_ra

value: allo


title: B2

description: Triple-receptor negative breast cancer


title: liverTreg_2-6_GvHD_Tcra_R2

description:
re-isolated
mTRA_Tx198-1-299-L-rp_A.RunAB.clonotypes.TRA.txt

tag: pcr1 cy. no.


title: HBsAg high group HBV_X680 (PBMC14)

value: PBMCs

value: peripheral blood mononuclear cells


title: HBsAg low group HBV_X670 (PBMC13)

value: chronic hepatitis B patient

value: HBsAg low group


title: spleenTreg_6-2_noGvHD

description:
re-isolated
222-4183_S_ra

value: BMT control


title: mA1

description: Triple-receptor negative breast cancer


title: spleenTreg_6-1_noGvHD

description:
re-isolated
219-4181_S_ra


title: HBsAg low group HBV_X726 (PBMC15)

value: nucleoside analogues


title: liverTreg_2-4_GvHD_Tcra_R2

description:
re-isolated
mTRA_Tx198-1-297-L-rp_A.RunAB.clonotypes.TRA.txt

value: liver

tag: chain


title: liverTreg_3-3_GvHD_Tcra_R2

description:
re-isolated
mTRA_Tx198-2-348-L-ra_A.RunAB.clonotypes.TRA.txt

value: C57BL/6 Foxp3GFP+

tag: techn. replicate


title: HBsAg high group HBV_X610 (PBMC12)

value: HBsAg high group


title: liverTreg_7-4_noGvHD

description:
re-isolated
255-4205_L_rp

tag: strain

value: poly


title: mC1

description: Triple-receptor negative breast cancer

tag: genotype


title: liverTreg_7-6_noGvHD

description:
re-isolated
252-4216_L_rp

tag: mouse (pool/culture no.)


title: liverTreg_3-2_GvHD_Tcra_R2

description:
re-isolated
mTRA_Tx198-2-346-L-ra_ABC.RunAB.clonotypes.TRA.txt

tag: type

value: GvHD


title: Late relapse KP, rep2

city: Philadelphia

first: Jingya

last: Qiu

state: PA

address: 421 Curie Blvd, 532

department: Radiation Oncology

institute: University of Pennsylvania

data_processing:
Adapter sequences trimmed using cutadapt v2.9 (parameters -m 30 -e 0.2 -q 10 -O 3)
Reads were pseudo-aligned and genes were quantified using salmon v1.8.0 (parameters --validateMappings --rangeFactorizationBins 4 --numBootstraps 3 --seqBias --gcBias)
Salmon counts were imported into R with tximport v1.22.0 and a count matrix summarized at the gene-level was generated.
Counts were vst normalized using DESeq2 v1.34.0
Assembly: GRCm38/mm10
Supplementary files format and content: vst-normalized RNA count matrix (.csv)

source_name: CD45- cancer cells

growth_protocol: We utilized two syngeneic mouse lung cancer models: the Kraslox-stop-lox(lsl)-G12D/+; p53flox/flox (KP) genetically engineered mouse model and the Lewis lung carcinoma (LLC1). Mice were injected with tumor cells. On day 15, CD45- tumor cells were harvested and sorted.

extract_protocol:
On day 15 tumors were harvested, red blood cell lysis was performed, and a single cell suspension was created. Tumor cells were stained with Live/Dead Aqua and CD45. Samples were sorted on an Aria (BD) by gating on live, CD45 negative cells. Total RNA was isolated and purified from the cells using Isol-RNA Lysis Reagent (Fisher) and treated with DNase I (Fisher).
RNA-seq libraries were prepared using the TruSeq Stranded Total RNA Library Prep Kit (Illumina), samples were pooled and sequenced on Illumina Novaseq 6000 with 100 cycles (50 bp paired-end reads)

value: CD45- cancer cells

tag: cell line

value: KP

value: Late relapse KP


title: HBsAg high group PBMC10

tag: tissue


title: liverTreg_2-5_GvHD_Tcra_R2

address: F.-J.-Strauss-Allee 11

description:
re-isolated
mTRA_Tx198-1-298-L-rp_A.RunAB.clonotypes.TRA.txt

tag: model

value: alpha


title: Late relapse KP, rep1

tag: group


title: 2nd relapse KP, rep3

value: 2nd relapse KP

tag: treatment


title: liverTreg_7-5_noGvHD

description:
re-isolated
249-4214_L_rp


title: liverTreg_3-1_GvHD_Tcra_R2

description:
re-isolated
mTRA_Tx198-2-341-L-ra_A.RunAB.clonotypes.TRA.txt

value: allo


title: Late relapse KP, rep3


title: liverTreg_2-3_GvHD_Tcra_R2

description:
re-isolated
mTRA_Tx198-1-296-L-ra_A.RunAB.clonotypes.TRA.txt


title: HBsAg low group PBMC9

value: PBMCs

tag: disease state

tag: cell type


title: HBsAg high group PBMC8

value: peripheral blood mononuclear cells


title: 2nd relapse KP, rep2


title: liverTreg_2-2_GvHD_Tcra_R2

description:
re-isolated
mTRA_Tx198-1-294-L-ra_A.RunAB.clonotypes.TRA.txt

tag: treg donor

tag: recipient


title: liverTreg_7-3_noGvHD

description:
re-isolated
243-4209_L_ra

tag: pcr2 cy. no.


title: HBsAg low group PBMC7

value: chronic hepatitis B patient


title: 2nd relapse KP, rep1


title: liverTreg_7-1_noGvHD

description:
re-isolated
246-400_L_ra


title: liverTreg_2-1_GvHD_Tcra_R2

description:
re-isolated
mTRA_Tx198-1-293-L-ra_A.RunAB.clonotypes.TRA.txt

tag: sampleID

tag: pcr1 cy. no.


title: HBsAg high group HBV_X210 (PBMC11)

description: Sample was excluded in this study after clustering of PBMCs


title: HBsAg low group PBMC6

value: HBsAg low group


title: colonTreg_3-6_GvHD_Tcra_R2

description:
re-isolated
mTRA_Tx198-2-349-C-rp_A.RunAB.clonotypes.TRA.txt

value: colon

tag: chain

tag: techn. replicate


title: liverTreg_6-6_noGvHD

description:
re-isolated
235-4196_L_rp

tag: strain

value: BMT control


title: 1st relapse KP, rep2

value: 1st relapse KP


title: liverTreg_6-5_noGvHD

description:
re-isolated
232-4193_L_rp

value: C57BL/6 Foxp3GFP+

tag: mouse (pool/culture no.)

value: liver


title: colonTreg_3-5_GvHD_Tcra_R2

description:
re-isolated
mTRA_Tx198-2-347-C-rp_A.RunAB.clonotypes.TRA.txt

tag: type

value: poly


title: HBsAg high group PBMC4

value: HBsAg high group

value: nucleoside analogues


title: 1st relapse KP, rep1

value: CD45- cancer cells

tag: cell line

value: KP


title: HBsAg high group PBMC5

tag: tissue


title: HBsAg low group PBMC3


title: Chronic IFNG KP, rep3

value: Chronic IFNG KP


title: colonTreg_3-4_GvHD_Tcra_R2

description:
re-isolated
mTRA_Tx198-2-343-C-rp_A.RunAB.clonotypes.TRA.txt

tag: model

value: GvHD

value: alpha


title: colonTreg_2-6_GvHD_Tcra_R2

description:
re-isolated
mTRA_Tx198-1-299-C-rp_AB.RunAB.clonotypes.TRA.txt


title: liverTreg_6-4_noGvHD

description:
re-isolated
229-4192_L_rp


title: HBsAg high group PBMC1

value: PBMCs

tag: group


title: Chronic IFNG KP, rep2

tag: treatment


title: liverTreg_6-3_noGvHD

description:
re-isolated
226-4190_L_ra

value: allo


title: P4

city: Duarte

first: Zhenlong

address: 1500 East Duarte Road

institute: City of hope

data_processing:
The Seurat R package(Stuart et al., 2019) was employed to facilitate various critical data processing and analysis tasks, including gene and cell filtration, data normalization, principal component analysis (PCA), identification of variable genes, clustering analysis, and Uniform Manifold Approximation and Projection (UMAP) dimension reduction
Sequencing was conducted using the Illumina NovaSeq 6000 platform with the paired-end setting, involving 28 cycles of read1, 101 cycles of R2, 10 cycles of index i7, and 10 cycles of index i5 read

source_name: ILCs freshly isolated from the blood of patients with AML

growth_protocol: The medium was supplemented with 10% human AB serum (Sigma-Aldrich), IL-2 (500 IU/ml), IL-7 (20 ng/ml), and IL-15 (20 ng/ml). Medium and cytokines were refreshed every two days by replacing half of the media containing 1× concentration of cytokines. Every four days, ILC2s were re-cultured onto new plates with fresh OP9. Two weeks later, the ILC2s stained with lineage (anti-CD3, anti-CD4, anti-CD8, anti-CD14, anti-CD15, anti-CD16, anti-CD19, anti-CD20, anti-CD33, anti-CD34, anti-CD123, anti-CD203c, anti- FcεRIα, and anti-CD56), anti-CD161, anti-CRTH2, and anti-c-Kit (anti-CD117) antibodies were sorted using a BD FACSAriaTM Fusion (BD Biosciences). FACS-sortedILC2s were plated and cultured onto fresh OP9 cells in the fresh MEM GlutaMAX media with cytokines

extract_protocol:
Ex ILC2s, ILCs from the blood of healthy donors (HD ILCs), ILCs from the blood of patients with AML (AML ILCs), and human CD45+ cells from the bone marrow of NSG mice (Traced ILC2s) were sorted using BD FACSAria
Cell samples containing 3,000-6,000 cells were captured using a 10 × Genomics Chromium controller and the 10 × V3.1 Single Cell 3’ Solution kit (Chromium Next GEM Single Cell 3’ Regent kits V3.1, Cat# PN-1000268). All protocols were followed by the manufacturer's instructions. The final sequencing libraries were assessed for library size using a High Sensitivity DNA Chip (Agilent, Cat# 5067-4626). Library concentration was determined using a Qubit High Sensitivity DNA assay Kit (ThermoFisher Scientific, Cat# Q32854). Sequencing was conducted using the Illumina NovaSeq 6000 platform with the paired-end setting, involving 28 cycles of read1, 101 cycles of R2, 10 cycles of index i7, and 10 cycles of index i5 read. The sequencing kit used was S4 Reagent kit v1.5 (Illumina, Cat# 20028313) at Tgen. Demultiplexing, genome alignment, and feature-barcode matrix generation were performed using the 10 × Genetics Cell Ranger software pipeline.
10x genomics Single Cell RNA-seq

treatment_protocol: Ex ILC2s, ILCs from the blood of healthy donors (HD ILCs), ILCs from the blood of patients with AML (AML ILCs), and human CD45+ cells from the bone marrow of NSG mice (Traced ILC2s) were sorted using BD FACSAria. Cell samples from three donors in each of the Ex ILC2s, HD ILCs, and Traced ILC2s groups were pooled into single samples for single-cell sequencing (ScRNAseq).

value: PBMC-derived ILC2s


title: 1st relapse KP, rep3


title: liverTreg_7-2_noGvHD

first: Michael

description:
re-isolated
240-4208_L_ra

data_processing:
base calling: Base-calling and demultiplexing of sequencing reads was carried out using the bcl2fastq Conversion Software (v1.8.4) provided by Illumina (Illumina Inc., San Diego, California, USA).
mapping: Sequencing reads were mapped to the mouse genome (GRCm38.p5) using STAR v2.5.3a. The genome index incorporated gene annotation from GENCODE Mouse release M16 to aid in spliced alignment.
generation of unique raw reads per geneTables of raw uniquely mapped read counts per gene were generated during mapping using the built-in --quantMode GeneCounts option in STAR.
Differential expression analysis was carried out on raw gene counts using edgeR (v3.34.0) in R (v4.1.0)
Assembly: Gencode Release M16 (GRCm38.p5); https://www.gencodegenes.org/mouse/release_M16.html
Supplementary files format and content: raw reads:column 1: gene ID (Ensemble); other columns: counts for the 2nd read strand aligned with RNA (column 4 of STAR output) for individual samples
Supplementary files format and content: normalized reads: column 1-5: Ensembl ID, Gene Symbol,Transcript Length, Gene Type; other columns: log2 transformed, batch-corrected and normalized read counts for individual samples (edgeR output).

extract_protocol:
Total cellular RNA was isolated from Treg (freshly isolated, in vitro expanded, or re-isolated after BMT) using the RNeasy Mini Kit or the RNeasy Micro Kit (Qiagen). Following the manufacturer’s recommendations, samples originating from 500 to 500k cells were processed with the Micro Kit whereas the Mini Kit was applied to all samples exceeding 500k cells. RNA concentration and quality were simultaneously assessed using RNA Screentape Analysis or High Sensitivity RNA Screentape Analysis (Agilent), depending on the expected yield.
Preparation of dsDNA libraries for Illumina sequencing was facilitated using the SMART-Seq® Stranded Kit (Takara) according to the manufacturer’s instructions. Depending on sample quality and abundance, fragmentation times and second PCR cycle numbers were adjusted, respectively. For some samples, first PCR products were pooled to minimize carry-over losses. The concentration of dsDNA libraries was measured with the Qubit™ dsDNA HS Assay Kit (Thermo Fisher Scientific). DNA fragment size distribution was assessed using the High Sensitivity D1000 ScreenTape Assay (Agilent). Libraries were stored at -20°C until sequencing was performed using the Illumina NextSeq550 sequencer and libraries are summarized in Supplemental Tables 4-7.


title: Chronic IFNG KP, rep1


title: colonTreg_2-5_GvHD_Tcra_R2

description:
re-isolated
mTRA_Tx198-1-298-C-rp_AB.RunAB.clonotypes.TRA.txt


title: colonTreg_2-4_GvHD_Tcra_R2

description:
re-isolated
mTRA_Tx198-1-297-C-rp_A.RunAB.clonotypes.TRA.txt


title: colonTreg_3-3_GvHD_Tcra_R2

description:
re-isolated
mTRA_Tx198-2-348-C-ra_A.RunAB.clonotypes.TRA.txt


title: P3


title: Parental KP, rep3

value: Parental KP


title: Parental KP, rep2


title: liverTreg_6-2_noGvHD

description:
re-isolated
223-4183_L_ra


title: P2


title: liverTreg_6-1_noGvHD

description:
re-isolated
220-4181_L_ra


title: P


title: colonTreg_3-2_GvHD_Tcra_R2

description:
re-isolated
mTRA_Tx198-2-346-C-ra_ABC.RunAB.clonotypes.TRA.txt

value: colon


title: colonTreg_7-4_noGvHD

description:
re-isolated
256-4205_C_rp

value: BMT control


title: colonTreg_7-6_noGvHD

description:
re-isolated
253-4216_C_rp


title: Parental KP, rep1


title: colonTreg_7-5_noGvHD

description:
re-isolated
250-4214_C_rp


title: H2

source_name: ILCs freshly isolated from the blood of healthy door


title: Relapsed LLC1, rep3

value: Relapsed LLC1


title: H


title: colonTreg_7-1_noGvHD

description:
re-isolated
247-400_C_ra

value: C57BL/6 Foxp3GFP+


title: colonTreg_3-1_GvHD_Tcra_R2

description:
re-isolated
mTRA_Tx198-2-341-C-ra_A.RunAB.clonotypes.TRA.txt


title: B

source_name: Transfered ILC2s


title: colonTreg_2-3_GvHD_Tcra_R2

description:
re-isolated
mTRA_Tx198-1-296-C-ra_AB.RunAB.clonotypes.TRA.txt


title: colonTreg_2-1_GvHD_Tcra_R2

description:
re-isolated
mTRA_Tx198-1-293-C-ra_AB.RunAB.clonotypes.TRA.txt


title: Relapsed LLC1, rep2


title: spleenTreg_7-6_noGvHD_Tcra_R2

description:
re-isolated
mTRA_251_Tx198-12-4216_S_rp_A.RunAB.clonotypes.TRA.txt

value: spleen


title: colonTreg_2-2_GvHD_Tcra_R2

description:
re-isolated
mTRA_Tx198-1-294-C-ra_AB.RunAB.clonotypes.TRA.txt


title: Chronic IFNG LLC1, rep2

value: Chronic IFNG LLC1


title: spleenTreg_7-5_noGvHD_Tcra_R2

description:
re-isolated
mTRA_248_Tx198-12-4214_S_rp_A.RunAB.clonotypes.TRA.txt

value: poly


title: colonTreg_6-6_noGvHD

description:
re-isolated
236-4196_C_rp


title: colonTreg_7-3_noGvHD

description:
re-isolated
244-4209_C_ra


title: E2

source_name: Expanded ILC2s


title: colonTreg_7-2_noGvHD

description:
re-isolated
241-4208_C_ra


title: Relapsed LLC1, rep1


title: h03C1_TCR

data_processing:
All immune cells following the single cell pipeline outlined in Methods
All non-immune cells following the single cell pipeline outlined in Methods
All TCR data
Assembly: hg38
Supplementary files format and content: H5AD file containing celltype, subtype, patient, batch, pCR and other metadata columns for each immune cell
Supplementary files format and content: H5AD file containing celltype, subtype, patient, batch, pCR and other metadata columns for each non-immune cell
Supplementary files format and content: CSV file containing TCR data

extract_protocol:
Human TNBC core needle biopsies were dissociated using the Miltenyi BiotecHuman Tumor Dissociation Kit, then CD45-positive cells were enriched using the Miltenyi Biotec anti-human CD45 microbeads and CD45-negative cells were obtained from the non-enriched portion
Single-cell gene expression and paired TCR (CD45-positive only) librarieswere prepared according to the 10X Genomics Single Cell 5’ v1 protocol.

value: Breast

tag: age

value: adult

tag: Sex

value: female

value: Pembrolizumab + Radiation

tag: disease state

value: Triple-receptor negative breast cancer


title: E

tag: cell type


title: spleenTreg_7-4_noGvHD_Tcra_R2

description:
re-isolated
mTRA_254_Tx198-12-4205_S_rp_A.RunAB.clonotypes.TRA.txt

tag: treg donor

tag: recipient

tag: pcr2 cy. no.

value: alpha


title: h03C1_P


title: colonTreg_6-5_noGvHD

description:
re-isolated
233-4193_C_rp


title: h66C_TCR


title: spleenTreg_6-6_noGvHD_Tcra_R2

description:
re-isolated
mTRA_234_Tx198-10-4196_S_rp_A.RunAB.clonotypes.TRA.txt

tag: sampleID

tag: pcr1 cy. no.

tag: chain


title: Parental LLC1, rep3

value: CD45- cancer cells

value: Parental LLC1


title: spleenTreg_6-5_noGvHD_Tcra_R2

description:
re-isolated
mTRA_231_Tx198-10-4193_S_rp_A.RunAB.clonotypes.TRA.txt

tag: strain

tag: mouse (pool/culture no.)

tag: techn. replicate


title: colonTreg_6-4_noGvHD

description:
re-isolated
230-4192_C_rp

tag: type

tag: tissue


title: Parental LLC1, rep2

tag: cell line

tag: group


title: h66C_N

tag: age

tag: treatment


title: colonTreg_6-3_noGvHD

description:
re-isolated
227-4190_C_ra

value: allo

tag: model


title: spleenTreg_6-4_noGvHD_Tcra_R2

description:
re-isolated
mTRA_228_Tx198-10-4192_S_rp_AB.RunAB.clonotypes.TRA.txt


title: colonTreg_6-2_noGvHD

description:
re-isolated
224-4183_C_ra


title: colonTreg_6-1_noGvHD

description:
re-isolated
221-4181_C_ra


title: h66B_TCR

tag: Sex

value: Pembrolizumab

tag: disease state


title: spleenTreg_7-2_noGvHD_Tcra_R2

description:
re-isolated
mTRA_239_Tx198-12-4208_S_ra_A.RunAB.clonotypes.TRA.txt

tag: treg donor

tag: recipient