title: Control LSK 3

data_processing:
FastQC (v0.11.8) to check FASTQ quality.
Trim Galore (v0.6.4) for adapter trimming
Bowtie2 (v2.3.5) to align reads
Picard (v2.21.1) to remove duplicate reads.
Samtools (v1.9) to remove reads multimapped (MAPQ > 30), unmapped, not primary aligned, failing platform, and mitchondrial reads.
Reads shifted + 4bp and - 5bp for the plus and negative strand respectively.
MACS2 to call peaks
Genome_build: mm10
Supplementary_files_format_and_content: Gene count matrix

source_name: WT LSK HSPCs

extract_protocol:
LSK HSPCs were isolated by fluorescence-activated cell sorting and subjected to Omni-ATAC-seq protocl
ATAC-seq libraries of DNA fregments were prepared accordingly to the OMNI-ATAC-seq protocol.

tag: strain background

tag: genotype/variation

value: LSK HSPC


title: SV439_CO_KO_CT5

status: Public on Apr 01 2024

type: SRA

city: Freising

first: Yunhui

last: Niu

address: Gregor-Mendel-Str. 2

country: Germany

institute: Technische Universität München

library_source: transcriptomic

data_processing:
The quality of Next Generation Sequencing data was assessed with FastQC v0.11.5
Adapter content and low quality reads were removed using Trimmomatic v0.39 (Anthony et al., 2014)
Trimmed FASTQ files were then mapped against the mouse mm10 genome with the STAR v2.7.5c aligner
Format conversions were performed using samtools v1.3.1 (Li et al., 2009). The featureCounts program v1.4.6 (Liao et al., 2014) was used to count reads located within an exon, do not overlap multiple features, with a threshold of MAPQ > = 4 and are not chimeric.
DESeq2 version 1.22.0 (RRID:SCR_015687 (Love et al., 2014) was used to normalize the read count matrix and perform differential expression analysis.
Assembly: mm10
Supplementary files format and content: txt file includes feature counts for all samples in one table

molecule: total RNA

organism: Mus musculus

source_name: Colon

extract_protocol:
RNA was harvested using NucleoSpin® RNA kit (MACHEREY-NAGEL).
RNA libraries for RNA-seq were prepared using TruSeq Stranded mRNA Library Prep Kit following manufacturer's protocols.

tag: tissue

value: Colon

tag: genotype

tag: time


title: Control LSK 2

city: NY

first: Tomer

last: Itkin

state: NY

address: 1300 York Avenue, A-869

department: Medicine

country: USA

institute: Weill Cornell Medicine

library_source: genomic

molecule: genomic DNA

growth_protocol: LSK cells were sorted and co-cultured with E4orf1 vascular niche cells in StemSpan serum free media supplemented with SCF, Flt3, and TPO and with knockout serum replacemnt media

treatment_protocol: Fli-1 alleles deletion was induced using 4-OHT

value: WT

value: BM

tag: cell type


title: hASC_D15_A_rep1

city: Oslo

first: Philippe

last: Collas

address: PO Box 1112 Blindern

department: Institute of Basic Medical Sciences

country: Norway

institute: University of Oslo

description:
22-21426_S3
day15_bulk_v_float.counts
adipogenesis_rpkm_tmm.tab
day15_bulk.v.float.tab
nucleolus_adipogenesis_DE.tab

data_processing:
RNA-Seqpairedreads were aligned to the hg38 genome with Hisat2(v2.1.0)using theensemblannotation(v95).
Reads ingeneswere countedusingfeaturecounts, with multimapping readsfractionally assigned using --fractionand-M.
Low abundance genes were filtered out of further analysis using edgeR's function "filterByExpr".
Normalisation between library sizes to get FPKM values was done using the trimmed mean of M-values method.
Normalisationfor differential expression was bylimma-voom(Law et al. 2014)and tests calculatedbylmFitandeBayes.
Calculations of changing expression over time were done pairwiseProlifvday0,day0 vday3 andday3 vday15;first within each donor and then combined across donors.
Assembly: hg38 ensembl95
Supplementary files format and content: Bigwig RNA-Seq coverage file generated with deeptools bamCoverage (binsize 50bp)
Supplementary files format and content: Raw read counts per gene, one column per sample
Supplementary files format and content: RPKM for each gene passing the filtering threshold in each sample.  Includes Prolif, day0, 1, 3 and 9 timepoints from previous study (GSE176020; Potolitsyna et al., 2022).
Supplementary files format and content: Differential expression analysis tables including logFC and adj.P.Val.  Analysis 1) bulk day15 versus adipocytes isolated from day15.  Analysis 2) Adipogenesis timecourse analysis with Prolif, day0 and day3 timepoints from previous study (GSE176020; Potolitsyna et al,. 2022).

molecule: polyA RNA

organism: Homo sapiens

source_name: Human primary adipose stem cells

growth_protocol: Human normal adipose tissue-derived mesenchymal stem cells were isolated from subcutaneous liposuction material. Cells were expanded in DMEM/F12 containing 10% fetal calf serum, and 20 ng/ml basic fibroblast growth factor (FGF2; Sigma-Aldrich F5392). Cells at passage 5-8 were used in three independent differentiations into the adipocyte lineage.

extract_protocol:
For RNA-seq, total mRNA was isolated from cells at aforementioned stages of differentiation using the Qiagen RNeasy kit and processed for Illumina library preparation and sequencing.
Illumina sequencing libraries were generated as per Illumina protocols.

treatment_protocol: For adipogenic differentiation, confluent cells were cultivated for 48h in DMEM/F12/10% fetal bovine serum without growth factors. Adipogenesis was then induced by addition of 10 µg/ml insulin, 200 µM indomethacin, 0.5 µM 1-methyl-3 isobutylxanthine and 1 µM dexamethasone. Medium was renewed every 3 days until day 9. Cells were then maintained in DMEM/F12/10% fetal bovine serum and 10 µg/ml insulin until day 15. RNA from the total cell population ("bulk") was harvested by direct lysis in the well. For mature adipocyte isolation, D15 samples were trypsinized, resuspended in HBSS, 0,5% BSA, and centrifuged 200g for 5 min. Floating mature adipocytes were harvested in Trizol and processed for RNA extraction.

value: Human primary adipose stem cells

tag: time point

tag: adipose depot

value: Abdominal subcutaneous

tag: culture

value: Cultured in vitro

tag: cell fraction

value: bulk


title: Control LSK 1


title: hASC_D15_A_floating_rep1

description:
16-21420_S16
day15_bulk_v_float.counts
adipogenesis_rpkm_tmm.tab
day15_bulk.v.float.tab
nucleolus_adipogenesis_DE.tab

value: adipocytes


title: KO LSK 3 [ChIP]

data_processing:
FastQC (v0.11.8) to check FASTQ quality.
Trim Galore (v0.6.4) for adapter trimming
Bowtie2 (v2.3.5) to align reads
Picard (v2.21.1) to remove duplicate reads.
Samtools (v1.9) to remove reads multimapped (MAPQ > 30), unmapped, not primary aligned, failing platform, and mitchondrial reads.
MACS2 to call peaks
Genome_build: mm10
Supplementary_files_format_and_content: narrowPeak

source_name: LSK HSPC

extract_protocol:
LSK HSPCs were isolated by fluorescence-activated cell sorting and subjected to Nuclei extraction using NP40 buffer and digested into nucleosomes using Mnase.
ChIP-seq libraries of fragmented DNA were prepared using the KAPA Hyper Kit (KAPA) according to the manufacturer’s instructions.

tag: organ

value: Fli-1 knockout

value: LSK HSPCs

tag: antibody


title: hASC_D15_A_floating_rep2

description:
17-21421_S24
day15_bulk_v_float.counts
adipogenesis_rpkm_tmm.tab
day15_bulk.v.float.tab
nucleolus_adipogenesis_DE.tab


title: VUMC0261

type: RNA

city: Nashville

first: Cong

last: Wang

state: TN

address: 2525 West End Ave

institute: Vanderbilt University

hyb_protocol: NanoString nCounter miRNA Expression Assay manufacturer's protocol

data_processing: miRNAs were normalized in the following steps: 1) background correction to remove non-specific binding. The background level was calculated as an aggregate summary of seven negative control probes with no target transcript. Mean of the calculated background level for each sample was then subtracted from the adjusted probe value to remove background noise. 2) Normalization of miRNA counts using mean counts of three spike-in synthetic control miRNAs (ath-miR159a, cel-miR-248, and osa-miR414) to control for variances on the starting material and the miRNA purification efficiency. 3) Sample content normalization using geometric mean counts of the top 50 miRNAs expressed excluding hsa-miR-320e, hsa-miR-16-5p and hsa-miR-451a because these miRNAs may be related to red blood cell contamination or hemolysis. After these steps, the expression level of each miRNA was calculated.

label: NanoString

source_name: plasma

extract_protocol: Qiagen’s miRNeasy Plasma Kit manufacturer's protocol

tag: study site (plco=1 swmhs=2 sccs=3 mec=4)

tag: case control status (control=0 case=1)

tag: sets

tag: sex (female=0 male=1)

tag: age at diagnosis

tag: race (white=1 black=2 other=3)


title: KO LSK 3

source_name: Fli-1 KO LSK HSPCs

tag: strain background

tag: genotype/variation


title: VUMC0262


title: SV480_CO_KO_CT1

tag: tissue

tag: genotype


title: SV400_CO_KO_CT9

tag: time


title: VUMC0265


title: hASC_D15_A_floating_rep3

description:
18-21422_S32
day15_bulk_v_float.counts
adipogenesis_rpkm_tmm.tab
day15_bulk.v.float.tab
nucleolus_adipogenesis_DE.tab

tag: cell type

tag: time point


title: VUMC0260


title: hASC_D15_G_floating_rep2

description:
14-21418_S2
day15_bulk_v_float.counts
adipogenesis_rpkm_tmm.tab
day15_bulk.v.float.tab
nucleolus_adipogenesis_DE.tab

tag: adipose depot

value: Gluteal

tag: culture


title: hASC_D15_G_floating_rep1

description:
13-21417_S53
day15_bulk_v_float.counts
adipogenesis_rpkm_tmm.tab
day15_bulk.v.float.tab
nucleolus_adipogenesis_DE.tab

tag: cell fraction


title: MCF-7 cells, siUSP36-3

city: xinxing

first: huijie

last: yang

address: Hongqi District, Xinxiang City

country: China

institute: Xinxiang Medical University

data_processing:
Using BGISEQ-500 sequencing platform
The sequencing data was filtered with SOAPnuke (v1.5.2) by (1) Removing reads containing sequencing adapter; (2) Removing reads whose low-quality base ratio (base quality less than or equal to 5) is more than 20%; (3) Removing reads whose unknown base ('N' base) ratio is more than 5%, afterwards clean reads were obtained and stored in FASTQ format.
Reads Per Kilobase of exon per Megabase of library size (RPKM) were calculated using a protocol from Chepelev et al., Nucleic Acids Research, 2009. In short, exons from all isoforms of a gene were merged to create one meta-transcript. The number of reads falling in the exons of this meta-transcript were counted and normalized by the size of the meta-transcript and by the size of the library.

source_name: Breast cancer

growth_protocol: MCF-7 cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS) and antibiotics in humidified atmosphere with 5% CO2 at 37℃

extract_protocol:
Lysis of cells with Trizol regent and harvesting of RNA.
RNA libraries for RNA-seq were prepared using BGISEQ-500 protocols.

treatment_protocol: MCF-7 cells were transfected with siControl and siUSP36 for 48 h.

value: Breast cancer

tag: cell line

value: MCF-7 cells

value: ER positive breast cancer cell

tag: treatment


title: SV438_CO_WT_CT17


title: VUMC0258

tag: study site (plco=1 swmhs=2 sccs=3 mec=4)

tag: case control status (control=0 case=1)


title: MCF-7 cells, siUSP36-2


title: VUMC0257

tag: sets

tag: sex (female=0 male=1)


title: VUMC0259

tag: age at diagnosis

tag: race (white=1 black=2 other=3)


title: MCF-7 cells, siUSP36-1


title: ID27_Target_P3

city: Noda

first: Hiroyasu

last: Aoki

state: Chiba

address: 2669 Yamazaki

department: Research Institute for Biomedical Sciences

country: Japan

institute: Tokyo University of Science

library_source: transcriptomic single cell

data_processing:
Data processing of scRNAseq was performed as described in Shichino et al. (Shichino et al., 2021).
Adapter trimming of sequencing data was performed by using cutadapt 3.4. Filtered reads were chunked to 16 parts for parallel processing by using Seqkit 0.15.0. Filtered cell barcode reads were annotated by Python script provided by BD with minor modifications to be compatible with Python3.8. Associated cDNA reads were mapped to reference RNA (build GRCh38 release-104) using bowtie2-2.4.2 by the following parameters: -p 2 -D 20 -R 3 -N 0 -L 8 -I S,1,0.75 –norc –seed 656565 –reorder. Then, cell barcode information of each read was added to the bowtie2-mapped BAM files, and read counts of each gene in each cell barcode were counted by using mawk. Resulted count data was converted to genes x cells matrix file, and the knee-plot inflection threshold was detected using DropletUtils package in R version 4.1.2. In addition, we further estimate background beads by using emptyDrops formula in DropletUtils package. Cells of total read count over inflection point were considered valid cells.
For sample tag data, adapter trimming of sequencing data was performed using cutadapt 3.4 (cutadapt). Filtered reads were chunked to 64 parts for parallel processing using Seqkit 0.15.0 (Seqkit). Filtered cell barcode reads were annotated using the Python script provided by BD with a minor modification for compatible to Python3.8. Associated sample tag reads were mapped to known barcode fasta using bowtie2-2.4.2 (bowtie) with the following parameters: -p 2 -D 50 -R 20 -N 0 -L 14 -i S,1,0.75 -norc -seed 656565 -reorder -trim-to 3:40 -score-min L,-9,0 -mp 3,3 -np 3 -rdg 3,3.
The cell barcode information for each read was added to the bowtie2-mapped BAM files, and read counts of each Tag in each cell barcode were counted using mawk. The resulting count data were converted to Tags x cells matrix file using the data.table package in R 3.6.3, and the top 1M cell barcodes were extracted. For assignment of each tags to each cell barcodes, read counts of each tag in each valid cell barcode, which defined by the cDNA matrix, were extracted from tag/cell barcode expression matrix. Unassigned cell barcodes were labeled as ?not-detected? cells. The sum of the total read counts of each tag was normalized to the minimum sum count of each tags, and log2 fold-change between first most tag counts and second most tag counts within each cell barcode. Each cell barcode was ranked by the fold-change ascending order, and top N cells were identified as doublets (N was calculated as theoretically detectable doublets calculated using the Poisson?s distribution based on the number of loaded cells, total Rhapsody well number, and number of tags used).
For AbSeq data, adapter trimming of sequencing data was performed using cutadapt 3.4. Filtered reads were chunked into 64 parts for parallel processing using Seqkit 0.15.0. Filtered cell barcode reads were annotated by Python script provided by BD with minor modifications to be compatible with Python3.8. Associated AbSeq reads were mapped to known barcode fasta by using bowtie2-2.4.2 by the following parameters: -p 2 -D 20 -R 3 -N 0 -L 8 -I S,1,0.75 -norc -seed 656565 -reorder-trim-to 3:39 -score-min L,-9,0 -mp 3,3 -np 3 -rdg 3,3. Then, cell barcode information of each read was added to the bowtie2-mapped BAM files, and read counts of each AbSeq tag in each cell barcode were counted using mawk. Resulted count data were converted to tags x cells matrix file by using the data.table package in R version 4.1.2, and top 1M cell barcodes were extracted.
Raw data of single-cell TCR sequencing were processed by pipeline produced by ImmunoGeneTeqs, Inc. Briefly, sequencing reads were separated by cell barcode, then adapter trimming and quality filtering of sequencing data were performed using cutadapt 3.4. PCR and sequencing errors were corrected by lighter v1.1.2 with the following parameters: -newQual 25 -maxcor 4 -K 20. Error-corrected sequence reads were processed by MiXCR55 to generate the list of TCRalpha or TCRbeta sequences for each cell barcode, using reference human TCR V/D/J sequences registered in the international IMGT information system. Quality filtering of cell barcodes was performed with the following criteria: (1) more than 32 TCR reads were detected. (2) the proportion of the most frequent TCR sequence in cell barcodes was over 0.6. (3) the cell barcode was annotated to the T cell cluster in scRNAseq/AbSeq analysis. Parameters used in MiXCR is summarized as Supplementary Table S4.
Finally, the most frequent sequence of TCRalpha and TCRbeta was adopted and paired in each cell barcode. Identified TCRalpha or TCRbeta sequence was imported into the meta.data matrix in Seurat object of scRNAseq/AbSeq data based on the cellular barcode identities. T-cell clones were determined as cells with the same TCRbeta sequence defined by V segment, J segment, and CDR3 nucleotide sequence. The frequency of each clone was calculated as the proportion of cells belonging to the clone in all T cells whose TCRbeta sequence was assigned.
Assembly: GRCh38 release-104
Assembly: IMGT reference sequences of human TRA and TRB
Supplementary files format and content: (scRNAseq, Abseq) tab-delimited text files include raw tag-count values for each sample
Supplementary files format and content: (scTCRseq) correspondence table between cell barcodes and TCR sequences
Raw data of single-cell TCR sequencing were processed by pipeline produced by ImmunoGeneTeqs, Inc., as described previously (GSE198211). Briefly, sequencing reads were separated by cell barcode, then adapter trimming and quality filtering of sequencing data were performed using cutadapt 3.4. PCR and sequencing errors were corrected by lighter v1.1.2 with the following parameters: -newQual 25 -maxcor 4 -K 20. Error-corrected sequence reads were processed by MiXCR to generate the list of TCRα or TCRβ sequences for each cell barcode, using reference mouse TCR V/D/J sequences registered in the international IMGT information system. Quality filtering of cell barcodes was performed with the following criteria: (1) more than 32 TCR reads were detected. (2) the proportion of the most frequent TCR sequence in cell barcodes was over 0.6. (3) the cell barcode was annotated to the T cell cluster in scRNAseq/AbSeq analysis. Finally, the most frequent sequence of TCRα and TCRβ was adopted and paired in each cell barcode. Identified TCRα or TCRβ sequence was imported into the meta.data matrix in Seurat object of scRNAseq/AbSeq data based on the cellular barcode identities. T-cell clones were determined as cells with the same TCRβ sequence defined by V segment, J segment, and CDR3 nucleotide sequence. The frequency of each clone was calculated as the proportion of cells belonging to the clone in all T cells whose TCRβ sequence was assigned. The 1 - Pielou index was used to evaluate the clonality of clusters.
Assembly: GRCh38 release-104
Assembly: IMGT reference sequences of human TRA and TRB
Supplementary files format and content: (scRNAseq, Abseq) tab-delimited text files include raw tag-count values for each sample
Supplementary files format and content: (scTCRseq) correspondence table between cell barcodes and TCR sequences

source_name: Peripheral blood T cells

extract_protocol:
PBMCs were isolated from whole blood with BD vacutainer CPT tubes (BD), resuspended in CELLBANKER 1 (Takara), and stored in -80°C.
PBMC thawed as described above were resuspended with Cell Staining Buffer (BioLegend) supplemented with Human TruStain FcX. After 2-3 minutes, 2x AbSeq master mix of Immune Discovery Panel (BD Biosciences) prepared as the provider?s protocol was added to the cell suspension, and stained for 15 minutes on ice. Then, the antibody mix 3 (Supplementary Table S1) with Sample Tag oligonucleotide-conjugated antibodies (Single-Cell Multiplexing Kit, BD Biosciences) was added to the cell suspension and left on ice for 15 minutes for staining. Stained cells were washed with Cell Staining Buffer.
Non-naive CD4+ and CD8+ cells (ID2 and ID27) or only non-naive CD8+ cells (ID10, ID19, ID22, ID31, and ID35) were sorted using FACS Aria II or Aria III and FACSDiva software (v8.0.1). Nonviable cells were excluded from the analysis based on forward and side scatter profiles and PI staining. Data were analyzed using FlowJo software (version 10.8.1).
Live cells were stained with Calcein AM (Nacalai) and counted using Flow-Count fluorospheres and a Cytoflex flow cytometer. About 25,000 - 30,000 labeled cells were loaded on a BD Rhapsody cartridge (BD) and processed using BD Rhapsody? Cartridge Reagent Kit (BD) following the manufacturer?s instructions. Single-cell mRNA, AbSeq barcodes, and Sample Tag barcodes were captured by BD Rhapsody beads coated with poly(T) oligonucleotide with a unique cell barcode and molecular barcode on each bead. Single-cell cDNA synthesis was performed using BD Rhapsody cDNA Kit (BD) following the manufacturer?s protocol. Targeted mRNA, TCRseq, and Abseq Index libraries were prepared using Immune Response Panel HS (BD) according to the manufacturer?s instructions with the following modifications. (1) We used the original set of primers for TCR amplification, as summarized in Table S2. (2) We performed additional PCRs to introduce a unique dual index for sequencing (3rd PCR for mRNA and TCR), as described in Table S3. (3) We used ProNex Size-Selection DNA purification System (Promega) for the purification of PCR products, as summarized in Table S8. Amplified libraries were quantified using a KAPA SYBR Fast qPCR Kit (KAPA Biosystems), and size distribution was analyzed by Microchip Electrophoresis System MultiNA (Shimadzu). Targeted RNAseq, TCRseq, and AbSeq libraries were sequenced on an Illumina Novaseq 6000 S4 flowcell (67 bp read 1 and 140 bp read 2) using NovaSeq 6000 S4 Reagent Kit v1.5 to a depth of approximately 20,000 reads, 7,000 reads, and 10,000 reads per cell, respectively.

treatment_protocol: Thirty-eight healthcare workers (over 20 years old) at Nara Medical University in Japan received two doses of Pfizer-BNT162b2 vaccine between 10 March and 30 March 2021.

value: Three shots of Pfizer-BNT162b2 vaccine

value: CD14- CD19- CD3+ [CD45RA- or CD27-] T cells

tag: age

tag: Sex

value: male

tag: condition

value: 1 week after receiving the second dose


title: ID27_TCR_P3


title: SV399_CO_KO_CT17

tag: tissue

value: Colon

tag: genotype

tag: time


title: ID27_Abseq_P3

tag: cell type


title: SV479_CO_KO_CT1


title: hASC_D15_G_floating_rep3

description:
15-21419_S9
day15_bulk_v_float.counts
adipogenesis_rpkm_tmm.tab
day15_bulk.v.float.tab
nucleolus_adipogenesis_DE.tab

tag: time point

tag: adipose depot

tag: culture


title: KO LSK 2

tag: strain background

tag: genotype/variation

value: BM

value: LSK HSPC


title: VUMC0256

tag: study site (plco=1 swmhs=2 sccs=3 mec=4)

tag: case control status (control=0 case=1)


title: VUMC0255

tag: sets

tag: sex (female=0 male=1)


title: SV455_CO_WT_CT1


title: SV468_CO_WT_CT1


title: MCF-7 cells, siControl-2

tag: cell line

tag: treatment

value: siControl


title: MCF-7 cells, siControl-3


title: KO LSK 1


title: VUMC0253

tag: age at diagnosis

tag: race (white=1 black=2 other=3)


title: ID2_TCR_P3

tag: age

tag: Sex

tag: condition


title: SV446_CO_WT_CT5

tag: tissue

tag: genotype

tag: time


title: ID2_Target_P3

tag: cell type


title: MCF-7 cells, siControl-1


title: VUMC0254


title: SV445_CO_KO_CT21


title: SV437_CO_KO_CT17


title: SV417_CO_WT_CT17


title: ID2_Abseq_P3


title: UM101-TNF replicate 1

city: Baltimore

first: Jeffrey

middle: David

last: Hasday

state: MD

address: 20 Penn St

institute: University of Maryland School of Medicine

data_processing:
Sequence quality evaluated
Reads are trimmed
Map reads alligned to genome (Homo sapiens GRCh38) using Tophat
Expression quantification (generate hit counts for genes/exons
Compare gene hit counts between pairs of treatments
Assembly: Analyze gene ontology
Supplementary files format and content: tab limited text file contains raw counts for each sample

source_name: pulmonary artery

growth_protocol: Cells were maintained in Ednothelial Cell Growth Medium MV2 at 37°C, used at passage 3-10, and studied at postconfluence

extract_protocol:
Total RNA was isolated from hPAEC using Qiagen Rneasy and 2 µg was used per reaction
RNASeq library prepared by GENEwiz: (1) mRNA enrichment, mRNA fragmentation, and random priming; (2) 1st and 2nd strand cDNA synthesis; (3) end repain, 5' phosphorylation, and dA tailing; (4) Adaptor ligation, and PCR enrichment.

treatment_protocol: Cells were pretreated with nothing (control) or 0.1% (final concentration DMSO alone of 10µM SB203580 or 25µM SF7044 or UM101 for 60 min, then with 10 ng/ml human TNFa for 3h at 37°C.

value: pulmonary artery

value: primary culture

value: endothelial cells

value: WT


title: UM101-TNF replicate 3


title: VUMC0252


title: UM101-TNF replicate 2


title: VUMC0250


title: NaraCOVID_TP4_CD8_039_TCR

data_processing:
Data processing of Bulk TCRseq was performed as described in Aoki et al. (Aoki et al., 2021).
Adapter trimming and quality filtering of sequencing data were performed using Cutadapt-3.2 and PRINSEQ-0.20.4. Sequencing data were processed by MiXCR-3.0.5. In MiXCR, filtered reads were aligned to reference mouse TCR V/D/J sequences registered in the international ImMunoGeneTics (IMGT) information system. The Variable (V) and Joining (J) segments of TCRs were represented in IMGT gene nomenclature. T-cell clones were determined as TCR reads with the same TCR V segment, J segment, and CDR3 nucleotide sequence.
Inter-sample contamination within samples using same UDIs were excluded by "Decontaminate" function of vdjtools with the parameter "-r 10".
For bulk TCRseq data, we regarded the CD4+-CD8+ overlapping clones in the same participant as contamination during T-cell isolation from PBMC because CD4+ and CD8+ T-cell repertoire are mutually exclusive (Aoki 2021). Thus, these overlapping clones were categorized into either CD4+ or CD8+ according to their dominance in CD4+ and CD8+ repertoire using VDJtools-1.2.1. The sequencing reads of bulk TCR-seq samples were normalized to 100,000 reads by the ?DownSample? command of VDJtools-1.2.1. To preserve the minor clonotypes, we performed the normalization ten times and pooled these down-sampled repertoire data for each sample, by ?JoinSamples? command of VDJtools-1.2.1.
Assembly: IMGT reference sequences of human TRA and TRB
Supplementary files format and content: Text files in VDJtools format include read count and frequency, complementarity determining region 3 (CDR3) nucleotide and amino acid sequences, Variable(V)/ Diversity(D)/ Joining(J) segment names, and V, D and J segment boundaries within CDR3 nucleotide sequence of individual clones

extract_protocol:
PBMCs were isolated from whole blood with BD vacutainer CPT tubes (BD), resuspended in CELLBANKER 1 (Takara), and stored in -80°C
CD4+ and CD8+ T cells were magnetically isolated from PBMC. PBMC were thawed by warming frozen cryovials in a 37°C water bath. Cells were stained for 20 minutes at 4°C with 1st antibody mix (Antibody Mix 1 in Supplementary Table S1). After washing with MACS buffer [0.5% BSA (Nacalai), 2 mmol/L EDTA (NIPPON GENE) in PBS (Nacalai)], Cells were stained for 20 minutes at 4°C with 2nd antibody mix; BD IMag anti-PE Magnetic Particles-DM (Clone E31-1459, BD) and anti-APC-Biotin (clone APC003, BioLegend). Then, the cell suspension was diluted with MACS buffer and set on a magnet plate (DynaMag96SideSkirted, Veritas). The positive fraction was collected as CD4+ T cells. The negative fraction was stained with BD IMag Streptavidin Particles Plus-DM (BD) for 20 minutes at 4°C. Then, the cell suspension was diluted with MACS buffer and set on a magnet plate. The positive fraction was collected as CD8+ T cells and B cells. The counts of CD4+ T cells, CD8+ T cells, and B cells were determined using Flow-Count fluorospheres (Beckman) and a Cytoflex flow cytometer (Beckman) and FlowJo software (version 10.8.1; BD Biosciences).
Bulk TCR sequencing library was prepared according to the previous report (Aoki et al., 2023).  In brief, mRNA in T-cell lysates were captured by incubating with 10 ?L of Dynabeads M270 streptavidin (Thermo Fisher Scientific) bound to 2.5 pmol of BioEcoP-dT25-adapter primers for 30 minutes at room temperature under gentle rotation. mRNA-trapped beads were suspended in 10 µL of RT mix and incubated for 60 min at 42ºC to perform reverse transcription (RT) and template-switching. The composition of the RT mix was as follows; 1× First Strand buffer (Thermo Fisher Scientific), 1 mM dNTP (Roche), 2.5 mM DTT (Thermo Fisher Scientific), 1 M betaine (Sigma-Aldrich), 9 mM MgCl2 (NIPPON GENE), 1 U/µL RNaseIn Plus RNase Inhibitor (Promega), 10 U/µL Superscript II (Thermo Fisher Scientific), and 1 µM of i5-TSO. To amplify the TCR cDNA containing complementarity determining region 3 (CDR3), nested PCR of the TCR locus was performed using KAPA Hifi Hotstart ReadyMix (KAPA Biosystems), followed by the purification of PCR product using an AM Pure XP kit (Beckman Coulter) at a 0.7:1 beads-to-sample ratio. The third PCR was performed to amplify TCR libraries and add adaptor sequences for the next-generation sequencer. The third-PCR products were purified as second PCR. Final TCR libraries, whose lengths were about 600 base pairs, were quantified using a KAPA SYBR Fast qPCR Kit (KAPA Biosystems), and size distribution was analyzed by Microchip Electrophoresis System MultiNA (Shimadzu). TCR libraries were sequenced using an Illumina Novaseq 6000 S4 flowcell (67 bp read 1 and 140 bp read 2) (Illumina) using NovaSeq 6000 S4 Reagent Kit v1.5 (200 cycles, Illumina). Only read2 contained the sequence regarding the definition of T-cell clones. Oligonucleotides and reaction conditions for library preparation were summarized in Table S2 and S3, respectively.

treatment_protocol: Thirty-eight healthcare workers (≥20 years old) at Nara Medical University in Japan received two doses of Pfizer-BNT162b2 vaccine between 10 March and 30 March 2021.

value: CD8+ T cells and CD19+ B cells

tag: date

value: 1 month after receiving the second dose


title: NaraCOVID_TP4_CD8_041_TCR


title: SV398_CO_WT_CT17


title: NaraCOVID_TP4_CD8_040_TCR


title: VUMC0251


title: SV416_CO_KO_CT17


title: SF7044-TNF replicate 3


title: SV478_CO_KO_CT5


title: SV454_CO_WT_CT1


title: VUMC0248


title: VUMC0249


title: NaraCOVID_TP4_CD8_038_TCR


title: SV444_CO_KO_CT21


title: SF7044-TNF replicate 2


title: NaraCOVID_TP4_CD8_037_TCR

value: female


title: NaraCOVID_TP4_CD8_036_TCR


title: VUMC0247


title: SV434_CO_WT_CT21


title: SB203580-TNF replicate 2


title: SB203580-TNF replicate 3


title: NaraCOVID_TP4_CD8_033_TCR


title: NaraCOVID_TP4_CD8_035_TCR


title: SV397_CO_WT_CT13


title: SV477_CO_KO_CT5


title: NaraCOVID_TP4_CD8_031_TCR


title: SV453_CO_WT_CT17


title: VUMC0242


title: NaraCOVID_TP4_CD8_030_TCR


title: NaraCOVID_TP4_CD8_032_TCR


title: SF7044-TNF replicate 1


title: SV415_CO_WT_CT9


title: VUMC0240


title: VUMC0244


title: DMSO-TNF replicatee 2

value: DMSO/TNFa


title: VUMC0243


title: VUMC0246


title: SV443_CO_WT_CT5


title: SB203580-TNF replicate 1


title: VUMC0241


title: NaraCOVID_TP4_CD8_029_TCR


title: DMSO-TNF replicatee 1


title: NaraCOVID_TP4_CD8_026_TCR


title: NaraCOVID_TP4_CD8_027_TCR


title: SV433_CO_WT_CT21


title: VUMC0237


title: Control (untreated) replicate 3

value: untreated


title: VUMC0245


title: SV396_CO_WT_CT13


title: MyriadR_1965

city: Ann Arbor

first: Simpa

middle: S

last: Salami

state: MI

address: 1500 E Medical Center Dr

department: Urology

institute: University of Michigan

data_processing:
libraries were normalized using TMM method prior to downstream processing
Assembly: GRCh38.p1
Supplementary files format and content: tab delimited text file with raw counts for each sample
Supplementary files format and content: tab delimited text file with logCPM for each sample

source_name: Clear Cell RCC

extract_protocol:
1ug - 6μg of total RNA was used in a reverse transcription reaction, and the obtained cDNA was taken to a second-strand DNA synthesis as described
Briefly, following second-strand synthesis, we used the Sciclone G3 NGS workstation (Perkin Elmer) with the Kappa HT library preparation kit to produce the libraries

value: Clear Cell RCC


title: NaraCOVID_TP4_CD8_024_TCR

value: Three shots of Pfizer-BNT162b2 vaccine


title: SV412_CO_WT_CT9


title: VUMC0238


title: Control (untreated) replicate 1


title: MyriadR_2234_2


title: MyriadR_1484


title: SV476_CO_WT_CT21


title: SV450_CO_KO_CT13


title: VUMC0236


title: NaraCOVID_TP4_CD8_022_TCR

value: male


title: SV442_CO_WT_CT5


title: Control (untreated) replicate 2


title: VUMC0235


title: NaraCOVID_TP4_CD8_021_TCR


title: NaraCOVID_TP4_CD8_023_TCR


title: NaraCOVID_TP4_CD8_025_TCR


title: VUMC0232


title: NaraCOVID_TP4_CD8_020_TCR


title: SV424_CO_WT_CT13


title: VUMC0233


title: SV410_CO_KO_CT9


title: VUMC0231


title: NaraCOVID_TP4_CD8_019_TCR


title: MYCN WT, RUNX1T1 Heterozygote, Coelic ganglia, replicate 1

city: Randwick

first: Murray

last: Norris

state: NSW

address: Lowy Cancer Research Centre

country: Australia

institute: Children's Cancer Institute

data_processing:
Illumina paired-end sequencing data was alligned using STAR to mouse genome build mm10
BAM files were sorted and indexed using SAMtools
Gene expression and isoform expression counts were calculated using RSEM
Assembly: mm10
Supplementary files format and content: tab-delimited text files containing raw and TPM counts for all samples

source_name: Coelic ganglia

extract_protocol:
Total RNA was isolated using the RNeasy Micro kit
Whole transcriptome sequencing was performed at the Ramaciotti Centre for Genomics with SMARTer Stranded Total RNA-seq v2  preparation kit. Illumina NovaSeq 6000 S1 pair-end 100bp reads to obtain average depth of 100 million reads per sample.

value: Coelic ganglia

value: Neuroblastoma

value: MYCN wild-type, Runx1t1 Heterozygote


title: MyriadR_47


title: MyriadR_842


title: MyriadR_1580


title: MyriadR_495


title: SV471_CO_KO_CT21

value: Colon


title: NaraCOVID_TP4_CD8_018_TCR


title: VUMC0230


title: VUMC0229


title: SV449_CO_KO_CT13


title: VUMC0234


title: NaraCOVID_TP4_CD8_017_TCR


title: VUMC0227


title: MyriadR_106


title: SV441_CO_WT_CT21


title: NaraCOVID_TP4_CD8_016_TCR


title: VUMC0226


title: NaraCOVID_TP4_CD8_015_TCR


title: MyriadR_943


title: SV421_CO_WT_CT13


title: VUMC0228


title: NaraCOVID_TP4_CD8_014_TCR


title: MyriadR_894


title: SV395_CO_KO_CT13


title: SV385_CO_KO_CT1


title: SV470_CO_KO_CT21


title: NaraCOVID_TP4_CD8_012_TCR


title: NaraCOVID_TP4_CD8_013_TCR


title: VUMC0224


title: MyriadR_260


title: VUMC0225


title: SV440_CO_KO_CT5


title: NaraCOVID_TP4_CD8_011_TCR


title: VUMC0222


title: MyriadR_124


title: NaraCOVID_TP4_CD8_008_TCR


title: VUMC0221


title: MyriadR_837


title: MyriadR_1873


title: NaraCOVID_TP4_CD8_007_TCR


title: MyriadR_2019


title: MyriadR_35


title: SV384_CO_KO_CT1


title: VUMC0223


title: NaraCOVID_TP4_CD8_006_TCR


title: SV448_CO_KO_CT13


title: NaraCOVID_TP4_CD8_005_TCR


title: VUMC0217


title: NaraCOVID_TP4_CD8_010_TCR


title: SV420_CO_WT_CT9


title: OTI-WT1_replicate 1

city: Yangsan

first: YUNA

last: JO

address: 49, Busandaehak-ro

country: South Korea

institute: Pusan National University

data_processing:
Before proceeding with the analysis of the raw sequence, a quality check is performed using the FASTQC program v.0.11.7 to ensure that there are no problems with the data.
Trimming for illumina paired-end was performed using the Trimmomatic 0.38 program.
Mapping to a known reference genome using the HISAT2 program version 2.1.0
After read mapping, transcript assembly was performed using the StringTie version 2.1.3b
Assembly: Mus musculus mm10
Supplementary files format and content: 1. raw data --> fastq (fastq.gz) file, read sequence and quality information  2. mapping --> bam, bam.bai file, mapping information of mapped reads  3. trasncript assembly --> txt or excel file, expression profiling result after trasncript assembly based on mm10 reference gtf annotation

source_name: tumor

extract_protocol:
To analyze the function of CD8+ T cells in the TME, we sorted tumor-infiltrating CD8+ T cells from tumor-bearing mice.
TruSeq Stranded Total RNA Reference Guide(1000000040499 v00)

value: tumor

value: primary cells

value: Tumor-infiltrating CD8 T cells


title: SV402_CO_KO_CT9


title: MyriadR_2278


title: VUMC0220


title: VUMC0216


title: SV401_CO_WT_CT9


title: VUMC0219


title: MyriadR_766


title: KOOTI-WT6_replicate 2


title: MyriadR_2159


title: OTI-WT2_replicate 2


title: VUMC0218


title: NaraCOVID_TP4_CD8_004_TCR


title: KOOTI-WT5_replicate 1


title: MyriadR_1415


title: MyriadR_2110


title: NaraCOVID_TP4_CD8_003_TCR


title: NaraCOVID_TP4_CD8_002_TCR


title: KOOTI-WT_po_replicate 3


title: NaraCOVID_TP4_CD4_041_TCR

value: CD4+ T cells


title: VUMC0215


title: MyriadR_80


title: VUMC0212


title: VUMC0211


title: VUMC0214


title: brain,CerM,Bio rep 1

city: Shanghai

first: Guanhao

address: Siping Road

institute: Tongji University

data_processing:
A cDNA library constructed by technology from the pooled RNA from < MWTL1,MWTL2,MWTL3,MCORTL1,MCORTL2,MCORTL3,WTMI1,WTMI2,WTMI3,CORTMI1,CORTMI2,CORTMI3> samples of homo sapiens was sequenced run with Illumina Novaseq TM 6000 sequence platform.
The raw paire-end reads were trimmed and quality controlled by SeqPrep and Sickle with default parameters.
Then clean reads were separately aligned to reference genome with orientation mode using HISAT2 software.
The mapped reads of each sample were assembled by StringTie in a reference-based approach.
The expression level of each transcript was calculated according to the transcripts per million reads (TPM) method. Transcript abundances were quantified using RSEM software tool.
Assembly: Mus_musculus GRCm39
Supplementary files format and content: tab-delimited text files include FPKM values for each Sample

source_name: hippocampus

extract_protocol:
Total RNA was extracted from the tissue using TRIzol® Reagent (Plant RNA Purification Reagent for plant tissue) according the manufacturer’s instructions (Invitrogen) and genomic DNA was removed using DNase I (TaKara).
RNA-seq transcriptome library was prepared with 1μg of total RNA using TruSeqTM RNA sample preparation Kit from Illumina (San Diego, CA).

value: hippocampus

value: CerM


title: VUMC0213


title: MyriadR_2103


title: MyriadR_969


title: brain,CerM,Bio rep 3


title: NaraCOVID_TP4_CD4_038_TCR

value: 1 month after receiving the second dose


title: brain,veh,Bio rep 3

value: vehicle


title: MyriadR_2011


title: brain,CerM,Bio rep 2


title: brain,veh,Bio rep 1


title: NaraCOVID_TP4_CD4_037_TCR

value: female


title: NaraCOVID_TP4_CD4_040_TCR


title: MyriadR_1772


title: VUMC0208


title: MyriadR_1362


title: MyriadR_1311


title: NaraCOVID_TP4_CD4_039_TCR


title: NaraCOVID_TP4_CD8_001_TCR

value: CD8+ T cells and CD19+ B cells


title: VUMC0209


title: small intestine, MSC+Mel_rep3

city: Dalian

first: Jinsong

last: Yan

state: Liaoning

address: 467 Zhongshan Road, Dalian 116027, Liaoning, P. R. China

department: Department of Hematology

institute: the Second Hospital of Dalian Medical University

data_processing:
For the gene expression, we mapped the sequencing data to reference genome (mm10) using STAR, and defined transcript coordinates according to the gene annotation format file (GTF file) from GENCODE (GRCm38).
The Gene abundances are given as Reads Per Kilobase per Million mapped reads (RPKM) with “cuffnorm” command using the Cufflinks package.
Assembly: mm10
Supplementary files format and content: Excel file includes RPKM values for each Sample

source_name: small intestine

extract_protocol:
Total RNA of small intestine cells from mouse were extracted using the QIAamp AllPrep RNA Mini Kit (Cat# 80204, QIAGEN), according to the manufacturer's specifications.
Libraries were constructed using the TruSeq RNA Sample Preparation Kits (Illumina), and the quality of the library was assessed using Bioanalyzer 2100 (Agilent Technologies).

value: small intestine

value: mesenchymal stem cell + Melphalan


title: MyriadR_1376

value: Clear Cell RCC


title: DMSO-TNF replicatee 3

value: pulmonary artery

value: primary culture

value: endothelial cells

value: WT

value: DMSO/TNFa


title: brain,veh,Bio rep 2


title: NaraCOVID_TP4_CD4_033_TCR

value: Three shots of Pfizer-BNT162b2 vaccine


title: VUMC0207


title: NaraCOVID_TP4_CD4_032_TCR

value: male


title: VUMC0203


title: NaraCOVID_TP4_CD4_036_TCR

value: CD4+ T cells


title: VUMC0206


title: MyriadR_1126


title: MyriadR_1016


title: NaraCOVID_TP4_CD4_035_TCR


title: small intestine, Mel+MSC_rep1

value: Melphalan+ mesenchymal stem cell


title: NaraCOVID_TP4_CD4_031_TCR


title: MyriadR_81


title: VUMC0205


title: VUMC0204


title: NaraCOVID_TP4_CD4_030_TCR


title: NaraCOVID_TP4_CD4_029_TCR


title: VUMC0199


title: small intestine, MSC+Mel_rep1


title: MyriadR_1027


title: small intestine, Mel+MSC_rep2


title: VUMC0202


title: small intestine, MSC+Mel_rep2


title: MyriadR_1220


title: MyriadR_2198


title: small intestine, Melphalan_rep1

value: Melphalan treatment


title: MyriadR_1606


title: small intestine, Mel+MSC_rep3


title: VUMC0200


title: small intestine, Melphalan_rep2


title: NaraCOVID_TP4_CD4_027_TCR

value: 1 month after receiving the second dose


title: VUMC0198


title: VUMC0196


title: NaraCOVID_TP4_CD4_026_TCR


title: small intestine, Melphalan_rep3


title: NaraCOVID_TP4_CD4_025_TCR


title: small intestine, Control_rep3

value: wild type control


title: small intestine, Control_rep2


title: VUMC0195


title: small intestine, Control_rep1


title: NaraCOVID_TP4_CD4_024_TCR

value: female


title: MyriadR_1234


title: MyriadR_516


title: NaraCOVID_TP4_CD4_022_TCR


title: MyriadR_866


title: MyriadR_1649


title: MyriadR_525


title: E16.5_3b

city: Jerusalem

first: Batia

last: Azria

state: Israel

address: Jerusalem

country: Israel

institute: Hebrew university of Jerusalem

data_processing:
Quality control applied with trim_galore V0.6.0 deploying cutadapt V1.18 and fastqc v0.11.7. Paired-end bisulfite treated FASTQ files were aligned by Bismark V0.13.1 deploying Bowtie2 aligner to mm10 assembly. Methylation calling performed with bismark_methylation_extractor followed by Bismark cov2cytp and a custom AWK script transforming files to methylKit input format. Differential methylated regions were obtain with methylKit R-package V1.10.0 and earlier consistent versions. Genome_build: mm10.
Assembly: mm10
Supplementary files format and content: all processed data are in methkit format

source_name: Liver

growth_protocol: All animal experiments were performed in accordance with the guidelines of the Hebrew University Institutional Committee for the Use of Animals for Research. Female C57BL mice aged 10-12 weeks, were maintained in a Specific Pathogen-Free (SPF) animal facility (Hebrew University-Hadassah Medical School Jerusalem, Israel), housed under regular 12h light-dark cycle.  Tet2/Tet3 DKO mice were generated by injecting 1,000 genome-equivalent copies of AAV8 carrying the gene for Cre under the hepatocyte-specific Tbg promoter (Penn vector) into the jugular vein of three-month-old Tet2F/Tet3F mice.

extract_protocol: Partial hepatectomy was carried out on 12 weeks old mice by removing 2/3 of the liver, including resection of the left lower, upper lobes and the right upper lobe.  Mice were anaesthetized and subjected to liver perfusion. Whole liver was then digested by collagenase (Roche) and hepatocytes removed and isolated by means of a Percoll gradient. Cells were then sorted by FACS according to size. Hepatoblasts were prepared from E11.5 and E16.5 mouse embryos by collecting fetal liver samples from littermate siblings which were then minced and digested for 30 min at 37°C with 0.025% collagenase IV (Sigma-Aldrich, C4-22-1G). After gentle pipetting, the cell suspension was filtered through a 40-mm cell strainer and washed twice with RPMI medium containing 10% FBS. The cells were then stained for 40 minutes at 4°C with moderate agitation with Liv2 (mouse) mAb (MBL, D118-3) for E11.5 hepatoblast, and Dlk1-APC-conjugated mouse Pref-1/DLK1/FA1 antibody (R&D Systems, FAB8634A-100) for E16.5 hepatoblast. Positive hepatoblast cells were then isolated by Flow cytometry. DNA was isolated from purified cells, incubated in lysis buffer (25 mM Tris-HCl (pH  8), 2 mM EDTA, 0.2% SDS, 200 mM NaCl) supplemented with 300 μg/ml proteinase K (Roche) followed by phenol:chloroform extraction and ethanol precipitation. The methylation profile was checked using the RRBS assay [Boyle, 2012]. Briefly, the DNA was digested with the MspI enzyme to yield CpG enriched fragments. Adapter sequences were ligated at both ends, and DNA fragments were then treated with sodium-bisulfite, allowing for easy distinction between methylated and unmethylated bases. The DNA was then amplified by PCR to produce the final libraries.

value: Liver

value: hepatoblast from E16.5 embryo

value: Dlk1+ hepatoblast of E16.5 liver embryo


title: MyriadR_819

value: Clear Cell RCC


title: VUMC0193


title: VUMC0192


title: NaraCOVID_TP4_CD4_021_TCR


title: NaraCOVID_TP4_CD4_023_TCR


title: VUMC0191


title: MyriadR_706


title: NaraCOVID_TP4_CD4_020_TCR


title: E16.5_2

treatment_protocol: Partial hepatectomy was carried out on 12 weeks old mice by removing 2/3 of the liver, including resection of the left lower, upper lobes and the right upper lobe.


title: VUMC0194


title: VUMC0189


title: NaraCOVID_TP4_CD4_019_TCR


title: E16.5_1


title: NaraCOVID_TP4_CD4_018_TCR


title: MyriadR_1745


title: PI-5

city: Guangzhou

first: Caiguang

last: Liu

address: No. 58 Zhongshan Road 2, Guangzhou, Guangdong Province, P.R. China.

institute: The First Affiliated Hospital, Sun Yat-sen University

description: Treatment

data_processing:
The transcriptome sequencing and analysis were conducted by OE biotech Co., Ltd. (Shanghai, China). Raw data (raw reads) were processed using Trimmomatic. The reads containing poly-N and the low quality reads were removed to obtain the clean reads. Then the clean reads were mapped to reference genome using hisat2.
FPKM value of each gene was calculated using cufflinks, and the read counts of each gene were obtained by htseq-count. DEGs were identified using the DESeq (2012) R package functions estimateSizeFactors and nbinomTest. P value < 0.05 and foldChange >2 or foldChange < 0.5 was set as the threshold for significantly differential expression. Hierarchical cluster analysis of DEGs was performed to explore genes expression pattern. GO enrichment and KEGG pathway enrichment analysis of DEGs were respectively performed using R based on the hypergeometric distribution.
FPKM and read counts value of each transcript (protein_coding) was calculated using bowtie2 and eXpress. DEGs were identified using the DESeq (2012) functions estimateSizeFactors and nbinomTest. P value < 0.05 and foldChange >2 or foldChange < 0.5 was set as the threshold for significantly differential expression. Hierarchical cluster analysis of DEGs was performed to explore transcripts expression pattern. GO enrichment and KEGG pathway enrichment analysis of DEGs were respectively performed using R based on the hypergeometric distribution.
The reads were reassembled using StringTie. Then gene structure extension and novel transcripts identification were performed by comparing the reference genome and the known annotated genes using cuffcompare software.
Assembly: mm10
Supplementary files format and content: FPKM_allsamples.txt: Tab-delimited text file includes FPKM values for each Sample.

extract_protocol:
Total RNA was extracted using the mirVana miRNA Isolation Kit (Ambion) following the manufacturer’s protocol. RNA integrity was evaluated using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). The samples with RNA Integrity Number (RIN) ≥ 7 were subjected to the subsequent analysis.
The libraries were constructed using TruSeq Stranded mRNA LTSample Prep Kit (Illumina, San Diego, CA, USA) according to the manufacturer’s instructions. Then these libraries were sequenced on the Illumina sequencing platform and 125bp/150bp paired-end reads were generated.

value: Colon

value: Pro-inflammatory diet


title: MyriadR_182


title: NaraCOVID_TP4_CD4_017_TCR


title: PI-4

description: Treatment


title: VUMC0188


title: MyriadR_662


title: PI-3

description: Treatment


title: VUMC0186


title: MyriadR_1267


title: NaraCOVID_TP4_CD4_016_TCR


title: E16.5_3


title: VUMC0184


title: MyriadR_1269


title: NaraCOVID_TP4_CD4_014_TCR


title: MyriadR_96


title: VUMC0183


title: CN-5

description: Control

value: Normal diet


title: VUMC0187


title: VUMC0182


title: VUMC0181


title: NaraCOVID_TP4_CD4_011_TCR


title: MyriadR_1860


title: VUMC0185


title: VUMC0178


title: PI-2

description: Treatment

value: WT


title: MyriadR_2165


title: NaraCOVID_TP4_CD4_010_TCR


title: VUMC0177


title: NaraCOVID_TP4_CD4_008_TCR


title: CN-2

description: Control


title: MyriadR_595


title: MyriadR_356


title: MyriadR_902


title: PI-1

description: Treatment


title: CN-1

description: Control


title: NaraCOVID_TP4_CD4_007_TCR


title: CN-4

description: Control


title: VUMC0176


title: MyriadR_151


title: NaraCOVID_TP4_CD4_006_TCR


title: MyriadR_1799


title: 50% WT GFP+ BMDMs and 50% N4bp1 KO BMDMs + TLR7 stimulation

city: South San Francisco

first: Rohit

last: Reja

state: CA

address: 1 DNA Way

institute: Genentech

data_processing:
10X immune profiling single-cell RNA sequencing data were processed with our in-house pipeline. Briefly, the expression library were first demultiplexed followed by alignment to the custom mouse reference genome (GRCm38 + GFP sequence) to measure the expression of GFP using Cell Ranger (version 6.0.1). A gene by cell barcode matrix was generated and the downstream analysis was performed using Seurat.
Seurat (version 4.0.4) was used for all downstream analysis. Cells with detected genes between 500 and 5000, and with less than 5% mitochondrial mapped reads were kept for downstream analysis. After filtering, we detected  36,174 cells across the whole dataset. After normalizing the  data, we performed linear dimensionality reduction using PCA. We ranked the principal components (PCs) by the amount of variance explained by each and found that after the first 20 PCs the explained variance plateaus. To visualize the data in two-dimensional space, Uniform Manifold Approximation and Projection (UMAP) was performed using the first 20 PCs. We then used the UMAP dimensions to determine the nearest neighbor of each cell and perform clustering by setting the resolution parameter to 0.15.
Assembly: mm10

source_name: Bone Marrow

extract_protocol:
Bone marrow cells from femurs and tibia of wild-type C57BL/6N mice were cultured in Roswell Park Memorial Institute (RPMI) medium  supplemented with 10% heat-inactivated fetal bovine serum, 2 mM glutamine, 50 Î¼M 2-mercaptoethanol (Gibco), 100 U/ml penicillin, and 100 Î¼g/ml streptomycin (Gibco) and 50 ng/ml  of recombinant M-CSF (R&D Systems) on non-tissue culture treated 15 cm plates. On day 5 of culture, cells were harvested and 5 million cells were re-plated onto non-tissue culture  treated 10 cm plates in fresh media (same as that described above). After 2 additional days of culture, BMDMs from individual mice  were harvested by scraping.
scRNAseq library constructions were carried out using the 10X Chromium Controller and the Chromium Next GEM Single Cell 3’ Reagent Kits  (V3.1) in accordance with the manufacture’s protocol. Prior to loading into the chromium controller, cells were washed once with PBS and resuspended in PBS at 700-1200 cells/ul for a target cell recovery of 7000 cells.

treatment_protocol: Treatment with TLR7 for 8h

tag: background

value: Bone Marrow

value: N4bp1 knockout


title: CN-3

description: Control


title: VUMC0175

tag: study site (plco=1 swmhs=2 sccs=3 mec=4)


title: NaraCOVID_TP4_CD4_005_TCR

value: Three shots of Pfizer-BNT162b2 vaccine

value: male


title: MyriadR_1753


title: VUMC0173

tag: case control status (control=0 case=1)


title: MyriadR_576


title: VUMC0174

tag: sets


title: NaraCOVID_TP4_CD4_004_TCR

value: CD4+ T cells


title: VUMC0172

tag: sex (female=0 male=1)


title: NaraCOVID_TP4_CD4_003_TCR


title: VUMC0171


title: Foxf1 multiome job snATACseq

city: Cincinnati

first: Guolun

state: OH

address: 3333 Burnet Avenue

institute: Cincinnati Children's Hospital Medical Center

description: 10x Genomics

library_source: genomic single cell

data_processing:
The demultiplexing, barcoded processing, gene counting and aggregation were made using the Cell Ranger ARC software v2.0.0 (https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/what-is-cell-ranger)
Assembly: mm10
Supplementary files format and content: gex_matrix.h5
Supplementary files format and content: fragments and bw files

source_name: lung mesoderm

extract_protocol:
Freshly collected Foxf1+ cells from Foxf1GFP mice lung at E18.5 were flushed with ice-cold PBS to remove luminal content. The lungs were finely minced into small pieces on ice in RPMI1640 containing 0.2 mg/ml Liberase TM and 100U/ml Deoxyribonuclease I. After digestion, cell suspensions were passed through a 70 µm cell strainer. Red blood cells were lysed in Ammonium-Chloride-Potassium (ACK) lysis buffer. The isolated cells were washed and re-suspended in autoMACS running buffer and stained with a Viability Dye, washed and resuspended in autoMACS running buffer to stain CD45 and CD31. CD45- cells were further purified according to the presence or absence or GFP signal. Single nuclei were prepared according to Nuclei Isolation from Complex Tissues for Single Cell Multiome ATAC + Gene Expression Sequencing (CG0000375) and were resuspended in nuclei buffer (2000207) for library construction.
Library was constructed and analyzed according to the manufacture’s instructions (Single Cell Multiome ATAC + Gene Expression v1 protocol, 10x Genomics). Briefly, the single nuclei were prepared and resuspended for Tn5 transposition in the master mix and loaded together with partitioning oil and gel beads into the chromium next GEM chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA contained in every single GEM was retrotranscripted to cDNA. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the appropriated sized fragments were selected with SPRIselect reagent for subsequent library construction.
Single Cell Multiome ATAC + Gene Exp

value: lung mesoderm

value: mesoderm derived cells

tag: sorted cell type

value: Foxf1+ cells


title: MyriadR_2068


title: MyriadR_904


title: 50% WT GFP+ BMDMs and 50% WT BMDMs + TLR7 stimulation

value: wild type


title: MyriadR_790


title: NaraCOVID_TP4_CD4_001_TCR

tag: treatment

value: 1 month after receiving the second dose


title: MyriadR_691


title: VUMC0169

tag: age at diagnosis


title: MyriadR_1178


title: 50% WT GFP+ BMDMs and 50% N4bp1 KO BMDMs unstimulated


title: VUMC0167

tag: race (white=1 black=2 other=3)


title: 50% WT GFP+ BMDMs and 50% WT BMDMs unstimulated


title: Tail of striatum biological replicate 2

city: La Jolla

first: Kay

last: Tye

address: 10010 N Torrey Pines Rd

department: Systems Neurobiology Laboratory

institute: Salk Institute

description:
TS-2
10X Single Cell 3' v3.1 Single Index

data_processing:
Fastq demultiplexing with CellRanger (v5.0.0)
Read alignment and single cell data parsing with CellRanger (v5.0.0)
Reads aligned to CellRanger 2020-A reference genome (mm10) and annotation (GENCODE vM23/Ensembl 98)
Empty droplets were called using the emptyDrops model from DropletUtils (v1.10.2), where droplets with less than 100 counts were used to profile ambient RNA and over 1000 were assumed non-empty
Minimal quality filtering for each barcode was performed by setting a floor of 1000 features per barcode for downstream inclusion to ensure the dataset is entirely composed of high-quality nuclei. Next, to remove highly likely multiplet barcodes, barcodes were filtered out if their count depth was more than 5 median absolute deviations above the median count depth. Barcodes were then removed if their proportion of ribosomal or mitochondrial reads was more than 5 interquartile ranges above the 75th percentile.
Heterotypic doublets were identified by creating simulated artificial doublets in scDblFinder (v1.4.1),
Assembly: mm10
Supplementary files format and content: Barcodes: Tab-separated values files containing cell barcodes and matrix files
Supplementary files format and content: Features: Tab separated values files containing the gene identities in the matrix
Supplementary files format and content: Matrix: raw barcode by feature matrix files giving counts for each combination

source_name: brain

extract_protocol:
All sacrifices were performed during the dark period of the light cycle. Animals were anesthetized via combined intraperitoneal injection of 150 mg/kg ketamine and 15 mg/kg xylazine. Once unconscious, mice animals were transcardially perfused with ice-cold, carbogen-bubbled (95% O2, 5% CO2), nuclease-free, 0.22 µm sterile-filtered artificial cerebrospinal fluid (ACSF) with a composition of 93 mM N-methyl-D-glucamine, 2.5 mM KCl, 1.2 mM NaH2PO4, 30 mM NaHCO3, 20 mM HEPES, 25 mM glucose, 5 mM sodium ascorbate, 2 mM thiourea, 3 mM sodium pyruvate, 13.2 mM trehalose, 12 mM N-acetyl-cysteine, 0.5 mM CaCl2, 10 mM MgSO4, and 93 mM HCl, at pH 7.3-7.4.(Tasic et al., 2018; Ting et al., 2014) Following transcardial perfusion, brains were immediately extracted and submerged into ice-cold carbogen-bubbled ACSF, with less than 5 minutes between the beginning of perfusion and final submersion after extraction. Brains were serially sectioned in ice-cold, carbogen-bubbled ACSF on a VT1000S vibratome (Leica) with polytetrafluoroethane-coated razor blades (Ted Pella) at 0.15 mm/sec and 100 Hz, dividing the whole cerebrum into 400 µm coronal slices. Target regions were microdissected from these slices under a stereomicroscope using a sterile blunt-end needle (22 gauge for CeA, ASt, and tail of striatum, 16 gauge for dorsal striatum). All regions were targeted using Paxinos and Franklin’s the Mouse Brain in Stereotaxic Coordinates as reference (Paxinos and Franklin, 2019). Extracted tissue samples were recovered in ice-cold, nuclease-free, 0.22 µm sterile-filtered cryoprotective nuclear storage buffer, composed of 0.32 M sucrose, 5 mM CaCl2, 3 mM magnesium acetate, 10 mM Trizma hydrochloride buffer (pH 8.0), 1 mM dithiothreitol, 0.02 U/µl SUPERase•In RNAse Inhibitor (Invitrogen), and 1X cOmplete Protease Inhibitor Cocktail with EDTA (Roche). Tissue was then snap frozen using a metal CoolRack M90 (Biocision) pre-chilled to -80˚C and stored at -80˚C until nuclear isolation. Cryopreserved tissue pieces were slow-thawed by incubation at 4˚C for 1 hour prior to isolation. Tissue pieces were then pooled and resuspended in nuclear isolation medium composed of 0.25 M sucrose, 25 mM KCl, 5 mM MgCl2, 10 mM Trizma hydrochloride buffer (pH 7.4), 1 mM dithiothreitol, 0.04 U/µl RNasin Plus RNAse Inhibitor (Promega), 1X cOmplete Protease Inhibitor Cocktail with EDTA (Roche), and 0.1% Triton-X. The pooled tissue pieces in nuclear isolation medium were transferred to a 2 mL Dounce tissue grinder. Tissue was homogenized by 5 strokes from the loose pestle and 15 followed by the tight pestle, and the resulting homogenate was filtered through a 40 µm Flowmi cell strainer (Bel-Art) into a 1.5 ml Lo-Bind tube (Eppendorf). The homogenate was then centrifuged with a swinging bucket rotor at 4˚C and 1000 x g for 8 minutes. Nuclei were then washed with nuclear flow buffer composed of DPBS with 1% bovine serum albumin, 1 mM dithiothreitol, and 0.04 U/µl RNasin Plus RNAse Inhibitor (Promega) and centrifuged at 4˚C and 500 x g for 5 minutes, which was subsequently repeated. Nuclei were finally resuspended in nuclear flow buffer containing 3 µm DRAQ7 (Cell Signaling Technology) and again filtered through a 40 µm Flowmi cell strainer into a 5 ml round-bottom polystyrene tube. Each isolation took under 45 minutes to perform, from homogenization to final suspension.
Library construction was performed acording to manufacturers' instructions (10X Single Cell 3' v3.1 Single Index). GCs were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip. RNA from nuclei in each droplet was reverse-transcribed to cDNA, which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with DynaBeads, PCR-amplified and appropriately-sized fragments were selected with SPRIselect reagents for subsequent library construction. During subsequent steps, Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.

value: brain

tag: tissue region

value: tail of striatum (TS)

tag: age

value: 8 weeks

tag: strain


title: Tail of striatum biological replicate 3

description:
TS-3
10X Single Cell 3' v3.1 Single Index

tag: tissue

tag: Sex


title: Tail of striatum biological replicate 1

description:
TS-1
10X Single Cell 3' v3.1 Single Index

tag: genotype


title: MyriadR_2033

value: Clear Cell RCC


title: VUMC0165


title: NaraCOVID_TP3_CD8_041_TCR

tag: cell type

value: CD8+ T cells and CD19+ B cells

value: 1 week after receiving the second dose


title: NaraCOVID_TP3_CD8_039_TCR

tag: date


title: VUMC0166


title: NaraCOVID_TP4_CD4_002_TCR


title: Foxf1 multiome job snRNAseq

description: 10x Genomics


title: VUMC0170


title: Dorsal striatum biological replicate 4

description:
DS-4
10X Single Cell 3' v3.1 Single Index

value: dorsal striatum (DS)


title: NaraCOVID_TP3_CD8_038_TCR


title: VUMC0164


title: NaraCOVID_TP3_CD8_040_TCR


title: MyriadR_9


title: Dorsal striatum biological replicate 3

description:
DS-3
10X Single Cell 3' v3.1 Single Index


title: MyriadR_478


title: NaraCOVID_TP4_CD4_015_TCR


title: MyriadR_1824


title: NaraCOVID_TP3_CD8_037_TCR

value: female


title: MyriadR_2234_1


title: Dorsal striatum biological replicate 2

description:
DS-2
10X Single Cell 3' v3.1 Single Index


title: MyriadR_1846


title: NaraCOVID_TP4_CD4_012_TCR


title: VUMC0163


title: VUMC0161


title: NaraCOVID_TP3_CD8_036_TCR


title: MyriadR_886


title: Dorsal striatum biological replicate 1

description:
DS-1
10X Single Cell 3' v3.1 Single Index


title: NaraCOVID_TP3_CD8_033_TCR


title: Central amygdala biological replicate 2

description:
CeA-2
10X Single Cell 3' v3.1 Single Index

value: central amygdala (CeA)


title: MyriadR_850


title: VUMC0160


title: VUMC0159


title: Central amygdala biological replicate 3

description:
CeA-3
10X Single Cell 3' v3.1 Single Index


title: NaraCOVID_TP3_CD8_032_TCR

value: Three shots of Pfizer-BNT162b2 vaccine


title: NaraCOVID_TP3_CD8_035_TCR


title: Central amygdala biological replicate 1

description:
CeA-1
10X Single Cell 3' v3.1 Single Index

value: male


title: VUMC0158


title: VUMC0157


title: MyriadR_244


title: Amygdalostriatal transition area biological replicate 2

description:
ASt-2
10X Single Cell 3' v3.1 Single Index

value: amygdalostriatal transition area (ASt)


title: MyriadR_180


title: VUMC0156


title: NaraCOVID_TP3_CD8_030_TCR


title: NaraCOVID_TP3_CD8_029_TCR


title: MyriadR_1968


title: Amygdalostriatal transition area biological replicate 1

description:
ASt-1
10X Single Cell 3' v3.1 Single Index


title: NaraCOVID_TP3_CD8_031_TCR


title: Notch1 OE LSK HSPCs

data_processing:
Cell Ranger 3.1.0 count pipeline was used to generate UMI count matrices
Assembly: mm10-2020-A
Supplementary files format and content: h5 filtered feature counts

source_name: LSK HSPCs isolated from co-cultures

extract_protocol: LSK HSPCs were harvested isolated and sorted from flushed and crushed murine tibias and femurs. LSK HSPCs (WT, Fli-1ROSAΔ , Fli-1ROSAΔN1-ICiOE, and N1-ICiOE) were introduced into co-culture with vascular niche ECs. 4-OHT was introduced into cultures to induce genetic manipulation. Cultures were harvested and LSK HSPCs were resorted and processed for 10X Chromium scRNA-seq analysis.

value: LSK HSPCs

tag: time point

value: 48 hours post 4-OH induction

tag: condition

value: In vitro cultures

value: Notch1 OE LSK HSPCs


title: Fli-1 KO and Notch1 OE LSK HSPCs

value: Fli-1 KO and Notch1 OE LSK HSPCs


title: MyriadR_2007


title: NaraCOVID_TP3_CD8_027_TCR


title: MyriadR_1161


title: VUMC0154

tag: study site (plco=1 swmhs=2 sccs=3 mec=4)

tag: case control status (control=0 case=1)


title: Fli-1 KO LSK HSPCs

value: Fli-1 KO LSK HSPCs


title: VUMC0155

tag: sets

tag: sex (female=0 male=1)


title: NaraCOVID_TP3_CD8_025_TCR

tag: treatment


title: MyriadR_1927


title: WT LSK HSPCs

description: WT

value: WT LSK HSPCs


title: NaraCOVID_TP3_CD8_024_TCR


title: VUMC0151

tag: age at diagnosis


title: B16-F10 HOIP-KO rep 4

city: Kyoto

first: Kazuhiro

last: Iwai

address: Yoshida Konoecho, Sakyo-ku

institute: Kyoto university

data_processing:
In order to remove technical sequences, including adapters, polymerase chain reaction (PCR) primers, or fragments thereof, and quality of bases lower than 20, pass filter data of fastq format were processed by Cutadapt (V1.9.1) to be high quality clean data.
Firstly, reference genome sequences (GRCm38) and gene model annotation files . Secondly, Hisat2 (v2.0.1) was used to index reference genome sequence. Finally, clean data were aligned to reference genome via software Hisat2 (v2.0.1).
In the beginning transcripts in fasta format are converted from known gff annotation file and indexed properly. Then, with the file as a reference gene file, HTSeq (v0.6.1) estimated gene and isoform expression levels from the pair-end clean data.
Supplementary files format and content: mm10
Supplementary files format and content: Excel files include FPKM values for each Sample

source_name: Subcutaneous tumor

growth_protocol: B16-F10 cells, which maintained in DMEM supplemented with 10% fetal bovine serum (FBS) and antibiotics in humidified atmosphere with 5% CO2 at 37C, were mixed with matrigel, and then subcutaneously injected  in NSG mice.

extract_protocol:
Clude RNA was harvested from tumor tissue using ISOGEN RNA isolation solution (Nippon gene). Then sequencing-grade RNA was purified using Rneasy mini plus kit (Qiagen). After polyA was enriched, 500 ng of mRNA was used for the construction of sequencing libraries.
RNA libraries for RNA-seq were prepared using NEBNext Ultra II Directional RNA Library Prep Kit for Illumina following manufacturer's protocols.

treatment_protocol: Tumors were established In vivo for 2 weeks.

value: Subcutaneous tumor

tag: cell line

value: Melanoma

value: HOIP-KO


title: MyriadR_2061

value: Clear Cell RCC


title: VUMC0150

data_processing: miRNAs were normalized in the following steps: 1) background correction to remove non-specific binding. The background level was calculated as an aggregate summary of seven negative control probes with no target transcript. Mean of the calculated background level for each sample was then subtracted from the adjusted probe value to remove background noise. 2) Normalization of miRNA counts using mean counts of three spike-in synthetic control miRNAs (ath-miR159a, cel-miR-248, and osa-miR414) to control for variances on the starting material and the miRNA purification efficiency. 3) Sample content normalization using geometric mean counts of the top 50 miRNAs expressed excluding hsa-miR-320e, hsa-miR-16-5p and hsa-miR-451a because these miRNAs may be related to red blood cell contamination or hemolysis. After these steps, the expression level of each miRNA was calculated.

extract_protocol: Qiagen’s miRNeasy Plasma Kit manufacturer's protocol

tag: race (white=1 black=2 other=3)


title: VUMC0153


title: MyriadR_1844


title: B16-F10 HOIP-KO rep 3


title: B16-F10 HOIP-KO rep 2


title: NaraCOVID_TP3_CD8_022_TCR

value: CD8+ T cells and CD19+ B cells

tag: age

value: 1 week after receiving the second dose


title: MyriadR_1685


title: MyriadR_2065


title: NaraCOVID_TP3_CD8_026_TCR


title: B16-F10 HOIP-KO rep 1


title: VUMC0147


title: NaraCOVID_TP3_CD8_023_TCR


title: MyriadR_1091


title: VUMC0148


title: B16-F10 control rep 4

value: control


title: NaraCOVID_TP3_CD8_020_TCR

value: Three shots of Pfizer-BNT162b2 vaccine

value: female


title: NaraCOVID_TP3_CD8_019_TCR


title: B16-F10 control rep 3


title: NaraCOVID_TP3_CD8_018_TCR

value: male


title: VUMC0145


title: VUMC0142


title: B16-F10 control rep 2


title: VUMC0149


title: MyriadR_992


title: MyriadR_40


title: NaraCOVID_TP3_CD8_017_TCR


title: VUMC0144


title: B16-F10 control rep 1


title: NaraCOVID_TP3_CD8_021_TCR


title: MyriadR_1551


title: VUMC0141


title: NaraCOVID_TP3_CD8_016_TCR


title: VUMC0140


title: NaraCOVID_TP3_CD8_015_TCR


title: primary murine hepatocytes, C29, rep3

city: Gent

first: Karolien

last: De Bosscher

address: Technologiepark-Zwijnaarde 75

country: Belgium

institute: VIB-UGent Center for Medical Biotechnology

data_processing:
Data analysis was performed using a dedicated Snakemake pipeline. Trim-Galore (version 0.6.6-0) was used to trim low-quality ends from reads (with phred score < 30), in addition to adapter removal.
Trimmed reads were pre-mapped to PhiX genome using STAR (version 2.7.6a) with a genome index setting --genomeSAindexNbases 5, disabled splicing and a maximum of 2 mismatches allowed. The PhiX-unmapped reads were then aligned to the mouse genome GRCm38 using the splice junctions of the Ensembl 101 annotation, allowing only uniquely mapped reads and a maximum of 4 mismatches.
The position-sorted output BAM files were converted to count data using HTSeq (version 0.12.4) in the ‘union’ mode.
Assembly: GRCm38
Supplementary files format and content: tab-delimited text files include read counts for each Sample

source_name: liver

growth_protocol: Mice were maintained in a pathogen-free animal facility under standard 12h light/12 dark cycle, with access to chow and water ad libitum. For the hepatic RNA expression studies, C57Bl/6J (wild-type) male mice, 11 weeks of age, were randomly divided in 4 groups of 6 mice.

extract_protocol:
RNA from the murine livers was isolated using TRIzol Reagent, according to the manufacturer’s protocol (Invitrogen).
RNA sequencing libraries were generated in biological triplicate (3 mice were used per condition) using the TruSeq stranded mRNA protocol. Libraries were subjected to single-end 100bp sequencing on Illumina NovaSeq6000, yielding 12-20 million reads per sample.

treatment_protocol: After overnight starvation, the mice were intraperitoneally injected with solvent control, GW7647 (4mg/kg) and/or Compound 29 (10mg/kg) dissolved in Ringer solution. The mice were sacrificed 4h after injection. Liver tissue was isolated, snap frozen and stored at -80°C.

value: liver

value: primary murie hepatocytes


title: MyriadR_431


title: MyriadR_1127


title: MyriadR_1002


title: NaraCOVID_TP3_CD8_014_TCR


title: NaraCOVID_TP3_CD8_013_TCR


title: MyriadR_1074


title: primary murine hepatocytes, C29, rep2


title: primary murine hepatocytes, DMSO, rep3

value: DMSO


title: NaraCOVID_TP3_CD8_012_TCR


title: VUMC0139

source_name: plasma


title: primary murine hepatocytes, C29, rep1


title: MyriadR_743


title: VUMC0136


title: MyriadR_570


title: VUMC0137


title: NaraCOVID_TP3_CD8_011_TCR


title: NaraCOVID_TP3_CD8_010_TCR


title: primary murine hepatocytes, DMSO, rep1


title: MH7A cells-TNFα treatment and LED-15min/3times_10

city: Harbin

first: Wuqi

last: Song

state: Heilongjiang

address: Baojian Road

department: Department of Microbiology

institute: Wu Lien-Teh Institute

description: Sample ID: 10

data_processing:
Illumina bcl2fastq 2.0 software used for basecalling.
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence, then mapped to mm10 whole genome using HISAT2
Transcript abundance values were obtained from gene-level count matrices were generated by StringTie
Gene counts were normalized using geometric mean (GEO) in DESeq2 package and log2 transformed. Removal of unwanted variation (RUV) was used to remove batch effects and genes with no counts in over 50% of the samples were removed.
Differential gene expression by limma voom was performed on normalized gene counts, using batch effects and the first two PCAs of aligning and sequencing as covariates.
Genome_build: Genome_build: mm10
Supplementary_files_format_and_content: Supplementary_files_format_and_content: tab-delimited text file includes gene abundance for each sample

extract_protocol:
Total RNA was extracted from each tissue sample using the Trizol method.
After incubation of RNA with a biotin-modified probe, the probe was captured by a streptavidin magnetic bead to eliminate rRNA. The obtained samples were purified by AMPure XP magnetic beads, and then buffer composed of elution, fragmented, and random primers was added and the samples were incubated at 94°C. Thermal fracture led to fragments distributed between 100 and 300 bp. Using first-strand synthesis buffer and Invitrogen SuperScript IV reverse transcriptase, this fragmented RNA was used as a template to synthesize the first-strand cDNA. Actinomycin D was added to the first-strand synthesis buffer to prevent the reverse transcription of DNA templates and to ensure the reverse transcription specificity of RNA templates. After that, the double-stranded synthesis premix system was added to the synthesized first-strand cDNA, and the product was purified by Agencourt AMpure XP magnetic beads. From this reaction we obtained purified double-stranded cDNA and then added a 3' end plus "A" buffer reaction system to this system. Finally, ligation buffer and a double-stranded sequencing linker was added to the library; we used T4 DNA ligase to attach the Illumina sequencing linker to the DNA library. The library was purified using the Agencourt SPRI select Nucleic Acid Fragment Screening kit.

value: 630nm LED-15min/3 times and 10ng/ml TNF-alpha


title: VUMC0135

city: Nashville

first: Cong

address: 2525 West End Ave

institute: Vanderbilt University


title: primary murine hepatocytes, GW+C29, rep3


title: NaraCOVID_TP3_CD8_008_TCR


title: NaraCOVID_TP4_CD4_013_TCR

value: CD4+ T cells

value: 1 month after receiving the second dose


title: primary murine hepatocytes, GW+C29, rep2


title: VUMC0138


title: MH7A cells-TNFα treatment and LED-15min/3times_6

description: Sample ID: 6


title: VUMC0133


title: NaraCOVID_TP3_CD8_007_TCR


title: MH7A cells-TNFα treatment and LED-15min/3times_4

description: Sample ID: 4


title: VUMC0131


title: primary murine hepatocytes, GW+C29, rep1


title: VUMC0132


title: primary murine hepatocytes, GW, rep3

value: GW


title: NaraCOVID_TP3_CD8_006_TCR


title: MH7A cells-TNFα treatment_14

description: Sample ID: 14

value: 10ng/ml TNF-alpha


title: VUMC0130


title: NaraCOVID_TP3_CD8_005_TCR


title: VUMC0129


title: primary murine hepatocytes, DMSO, rep2


title: MH7A cells-TNFα treatment_2

description: Sample ID: 2


title: MH7A cells-TNFα treatment_8

description: Sample ID: 8


title: primary murine hepatocytes, GW, rep2


title: primary murine hepatocytes, GW, rep1


title: NaraCOVID_TP3_CD8_004_TCR


title: Monocytes THP-1: Control vs Colpvp

city: Mexico City

first: Krötzsch

last: Edgar

address: Calz México-Xochimilco 289, Coapa, Col. Arenal de Guadalupe, Tlalpan

country: Mexico

institute: Instituto Nacional de Rehabilitación LGII

description:
Biological sample 1 of 2, untreated, macrophages
Biological sample 2 of 2, colpvp, macrophages

hyb_protocol: Hybridization was performed using equal amounts of labeled cDNA with the hybridization solution UniHyb (TeleChem International INC). The arrays were incubated  ( 42°C, 14 hrs), and washed  with 1X SCC/0.05 % SDS at RT.

scan_protocol: GenePix 4100A and GenePix software were used for the acquisition and quantification of array images using 10µm resolution

data_processing: ArrayPro Analyzer (Media Cibernetics) was used to calculate the Alexa555 and Alexa647 density mean value and background mean value for each spot. genArise (http://www.ifc.unam.mx/genarise/) was used for  the microarray data performing background correction, lowess normalization, intensity filter, replicates analysis and selecting differentially expressed genes.

source_name: Total RNA extracted from untreated monocytes

label_protocol: 10 µg of total RNA for cDNA synthesis, incorporating dUTP-Alexa555 or dUTP-Alexa647 using the First-Strand cDNA labeling kit (Invitrogen). Incorporation of fluorophore was analyzed by using the absorbance at 555 or 650 nm.

growth_protocol: The human monocyte cell line (THP-1) was cultured with RPMI-1640 medium, supplemented with 10% FBS, 1mM sodium pyruvate, 0.1mM non-essential aminoacids, 0.1mM of glutamine, 100 U / ml penicillin and 100 μg/ml streptomycin.

extract_protocol: Total RNA extracted using Trizol following manufacturer's instructions

treatment_protocol: Controls were untreated cell cultures, Colpvp were incubated for 3 h with FBS-free medium and then with 3% collagen-PVP prepared in the corresponding medium without FBS and incubated for 24 h at 37 °C and 5% CO2

value: Macrophage

source_name: Total RNA extracted from Colpvp treated monocytes


title: VUMC0127


title: NaraCOVID_TP3_CD8_002_TCR

city: Noda

first: Hiroyasu

address: 2669 Yamazaki

institute: Tokyo University of Science

data_processing:
Data processing of Bulk TCRseq was performed as described in Aoki et al. (Aoki et al., 2021).
Adapter trimming and quality filtering of sequencing data were performed using Cutadapt-3.2 and PRINSEQ-0.20.4. Sequencing data were processed by MiXCR-3.0.5. In MiXCR, filtered reads were aligned to reference mouse TCR V/D/J sequences registered in the international ImMunoGeneTics (IMGT) information system. The Variable (V) and Joining (J) segments of TCRs were represented in IMGT gene nomenclature. T-cell clones were determined as TCR reads with the same TCR V segment, J segment, and CDR3 nucleotide sequence.
Inter-sample contamination within samples using same UDIs were excluded by "Decontaminate" function of vdjtools with the parameter "-r 10".
For bulk TCRseq data, we regarded the CD4+-CD8+ overlapping clones in the same participant as contamination during T-cell isolation from PBMC because CD4+ and CD8+ T-cell repertoire are mutually exclusive (Aoki 2021). Thus, these overlapping clones were categorized into either CD4+ or CD8+ according to their dominance in CD4+ and CD8+ repertoire using VDJtools-1.2.1. The sequencing reads of bulk TCR-seq samples were normalized to 100,000 reads by the ?DownSample? command of VDJtools-1.2.1. To preserve the minor clonotypes, we performed the normalization ten times and pooled these down-sampled repertoire data for each sample, by ?JoinSamples? command of VDJtools-1.2.1.
Assembly: IMGT reference sequences of human TRA and TRB
Supplementary files format and content: Text files in VDJtools format include read count and frequency, complementarity determining region 3 (CDR3) nucleotide and amino acid sequences, Variable(V)/ Diversity(D)/ Joining(J) segment names, and V, D and J segment boundaries within CDR3 nucleotide sequence of individual clones

source_name: Peripheral blood T cells

extract_protocol:
PBMCs were isolated from whole blood with BD vacutainer CPT tubes (BD), resuspended in CELLBANKER 1 (Takara), and stored in -80°C
CD4+ and CD8+ T cells were magnetically isolated from PBMC. PBMC were thawed by warming frozen cryovials in a 37°C water bath. Cells were stained for 20 minutes at 4°C with 1st antibody mix (Antibody Mix 1 in Supplementary Table S1). After washing with MACS buffer [0.5% BSA (Nacalai), 2 mmol/L EDTA (NIPPON GENE) in PBS (Nacalai)], Cells were stained for 20 minutes at 4°C with 2nd antibody mix; BD IMag anti-PE Magnetic Particles-DM (Clone E31-1459, BD) and anti-APC-Biotin (clone APC003, BioLegend). Then, the cell suspension was diluted with MACS buffer and set on a magnet plate (DynaMag96SideSkirted, Veritas). The positive fraction was collected as CD4+ T cells. The negative fraction was stained with BD IMag Streptavidin Particles Plus-DM (BD) for 20 minutes at 4°C. Then, the cell suspension was diluted with MACS buffer and set on a magnet plate. The positive fraction was collected as CD8+ T cells and B cells. The counts of CD4+ T cells, CD8+ T cells, and B cells were determined using Flow-Count fluorospheres (Beckman) and a Cytoflex flow cytometer (Beckman) and FlowJo software (version 10.8.1; BD Biosciences).
Bulk TCR sequencing library was prepared according to the previous report (Aoki et al., 2023).  In brief, mRNA in T-cell lysates were captured by incubating with 10 ?L of Dynabeads M270 streptavidin (Thermo Fisher Scientific) bound to 2.5 pmol of BioEcoP-dT25-adapter primers for 30 minutes at room temperature under gentle rotation. mRNA-trapped beads were suspended in 10 µL of RT mix and incubated for 60 min at 42ºC to perform reverse transcription (RT) and template-switching. The composition of the RT mix was as follows; 1× First Strand buffer (Thermo Fisher Scientific), 1 mM dNTP (Roche), 2.5 mM DTT (Thermo Fisher Scientific), 1 M betaine (Sigma-Aldrich), 9 mM MgCl2 (NIPPON GENE), 1 U/µL RNaseIn Plus RNase Inhibitor (Promega), 10 U/µL Superscript II (Thermo Fisher Scientific), and 1 µM of i5-TSO. To amplify the TCR cDNA containing complementarity determining region 3 (CDR3), nested PCR of the TCR locus was performed using KAPA Hifi Hotstart ReadyMix (KAPA Biosystems), followed by the purification of PCR product using an AM Pure XP kit (Beckman Coulter) at a 0.7:1 beads-to-sample ratio. The third PCR was performed to amplify TCR libraries and add adaptor sequences for the next-generation sequencer. The third-PCR products were purified as second PCR. Final TCR libraries, whose lengths were about 600 base pairs, were quantified using a KAPA SYBR Fast qPCR Kit (KAPA Biosystems), and size distribution was analyzed by Microchip Electrophoresis System MultiNA (Shimadzu). TCR libraries were sequenced using an Illumina Novaseq 6000 S4 flowcell (67 bp read 1 and 140 bp read 2) (Illumina) using NovaSeq 6000 S4 Reagent Kit v1.5 (200 cycles, Illumina). Only read2 contained the sequence regarding the definition of T-cell clones. Oligonucleotides and reaction conditions for library preparation were summarized in Table S2 and S3, respectively.


title: VUMC0128


title: Alveolospheres with Bleomycin treatment (HashTag)

city: Kashiwa

first: BIN

last: WU

address: #304, 6-6-2, Kashiwanoha

department: Research

institute: ImmunoGeneTeqs

data_processing:
Pair-end Fastq files (R1: cell barcode reads, R2: RNA reads) were processed as follows. Adapter trimming of sequencing data was performed using cutadapt 3.4. Filtered reads were chunked into 16 parts for parallel processing by using Seqkit 0.14.0. Filtered cell barcode reads were annotated by Python script provided by BD Biosciences with minor modification for compatibility to Python 3.8. Reference RNA sequences were built by concatenating cDNA and ncRNA fasta files of the Ensembl database (build GRCm38 release-101). Associated cDNA reads were mapped to reference RNA using bowtie2-2.4.2 by the following parameters: -p 2 --very-sensitive-local -N 1 -norc -seed 656565 -reorder. Then, cell barcode information of each read was added to the bowtie2-mapped BAM files by the python script and pysam 0.15.4 (https://github.com/pysam-developers/pysam), and read counts of each gene in each cell barcode were counted using mawk. Resultant count data was converted to a single-cell gene-expression matrix file by using R-3.6.3. The inflection point of the knee-plot (total read count versus the rank of the read count) was detected using DropletUtils package in R-3.6.3. Cells of which total read count was over inflection point were considered as valid cells.
For assignment of each tags to each cell barcodes, read counts of each tag in each valid cell barcode, which defined by the cDNA matrix, were extracted from tag/cell barcode expression matrix. Unassigned cell barcodes were labeled as not-detected cells. Then, sum of the total read counts of each tags were normalized to the minimum sum count of each tags, and log2 fold-change between first most tag counts and second most tag counts within each cell barcode. Each cell barcode were ranked by the fold-change ascending order, and top 3.01% (sample1), 3.73% (sample2), and 4.43% (sample3) cells were identified as doublets, which were theoretically detectable doublets calculated by the Poisson\u2019s distribution based on the number of loaded cells, total Rhapsody well number, and number of tags used. Finally, remained cell barcodes were assigned to the first most counted tags. Each tag expression in each cell barcode were log2(x+1)-transformed, z-scaled by each cell barcode, and visualized by using pheatmap package in R 3.6.3.
Assembly: GRCm38
Supplementary files format and content: tab-delimited text files include raw tag-count values for each sample

source_name: Alveolospheres

growth_protocol: Alveolopheres were generated by co-culturing murine lung fibroblasts and epithelial cells. Briefly, single-cell suspension was obtained from murine lungs by enzymatic digestion by collagenase and DNase I as described previously (Tsukui et al., 2015), and lung fibroblasts and epithelial cells were isolated by cell sorting. Resulted cells were suspended in MTEC Plus medium (described below) to yield 4 x 10^6 cells/ml of fibroblasts and 2 x 10^5 cells/ml of lung epithelial cells per well. 1x10^5 lung fibroblast and 5 x 10^3 lung epithelial cells/25µL were combined and suspended with an equal volume of Matrigel (BD Biosciences) at 4°C, and 90µL of the suspension was seeded onto each insert (BD Biosciences or Greiner) placed on a 24-well culture dish. Then, cells were cultured at 37°C for 30 min, 500 µL of MTEC Plus medium were added, and further cultured at 37°C with 5% CO2 in a humidified atmosphere. The MTEC Plus medium used for culture consisted of medium DMEM/F-12 (Sigma-Aldrich), mouse epidermal cell growth factor (25 ng/mL; R&D Systems), pituitary extract (30 µg/mL; Sigma-Aldrich), and insulin-transferrin-sodium selenite (1×; Thermo Fisher Scientific), penicillin/streptomycin (1×; Sigma-Aldrich), cholera toxin (100 ng/mL; Sigma-Aldrich), and fetal bovine serum (5%). Retinoic acid (Sigma-Aldrich) was added at the time of medium addition, and Y-27632 Rho-associated kinase inhibitor (10 μM; Fujifilm Wako Pure Chemicals) only immediately after seeding. The culture medium was changed every two days for 14 days.

extract_protocol:
Lungs were minced into 0.5 mm2 with a razor blade and digested with Collagenase solution [RPMI-1640 (Nacalai Tesque) supplemented with 10% FBS (MP Bio Japan), 10 mM HEPES pH7.2-7.4 (Cytiva (Global Life Sciences Technologies Japan)), 0.2% Collagenase (Fujifilm Wako Pure Chemicals), 2 kU/mL DNase I (Merck)] , and 0.96 mg/mL Dispase (Roche) at 37 ºC for 50 minutes. Cell suspensions were washed and resuspended with 25% Percoll PLUS (Cytiva). After under layered 65% Percoll PLUS, cell suspensions were centrifuged at 20ºC for 1,000 ×g for 20 minutes, and the middle layer was collected. Resultant cell suspensions were washed and resuspended with 1000 μL of preparation medium [RPMI-1640 supplemented with 5% FBS and 10 mM HEPES]. Each single-cell suspension cell concentration was counted using Flow-count fluorospheres (Beckman Coulter) and a Cytoflex flow cytometer (Beckman Coulter). Cells were stained with 2.5 μg/ml of Totalseq anti-mouse Hashtag-A antibodies (BioLegend) at 4ºC for 30 minutes and washed thrice with Cell Staining Buffer (BioLegend) and pooled.
Pooled single-cell suspensions were subjected to a BD Rhapsody system with BD Rhapsody Targeted & Abseq Reagent kit (BD Biosciences) following the manufacture’s instructions. 20000 cells were subjected to the BD Rhapsody workflow. After reverse transcription, Exonuclease I treatment of the resultant BD Rhapsody beads was performed at 37ºC for 60 minutes with 1,200 rpm on a Thermomixer C with Thermotop. Resultant beads were immediately chilled on ice; the supernatant was removed and washed with 1 mL of WTA wash buffer [10 mM Tris-HCl (pH 8.0), 50 mM NaCl, 1 mM EDTA, and 0.05% Tween-20], 200 μL of BD Rhapsody lysis buffer (for inactivation of enzyme), once with 1 ml of WTA wash buffer, twice with 500 μL of WTA wash buffer, resuspended with 200 μL of Beads resuspension buffer. During the washing step, beads-containing DNA LoBind tubes (Eppendorf Japan) were replaced twice. Exonuclease I-treated BD Rhapsody beads were subjected to TAS- Seq workflow for cDNA and/or hashtag library amplification (Shichino S., bioRxiv, 2021). 150 ng of amplified cDNA libraries was subjected to fragmentation/end-repair/A-tailing using NEBNext Ultra II FS DNA Library Prep Kit for Illumina. Ligated products were purified by AmPure XP beads and 9 cycles of Barcoding PCR were performed. Resultant products were purified twice by double size selection with 0.5× → 0.3× (final 0.8×) AmPure XP beads and eluted with 30 μL of 10 mM Tris-HCl pH8.0. Size distribution of amplified products was analyzed by MultiNA system with appropriate dilutions. Resultant libraries and barcoded hashtag libraries were quantified using the KAPA Library Quantification Kit. Pooled libraries were sequenced by Illumina Novaseq 6000 sequencer.
BD Rhapsody WTA

treatment_protocol: Alveolopheres were stimulated with 0.1 ug/ml bleomycin for 48 hours from day 8 post co-culture.

tag: strain

value: C57BL/6J or C57BL/6-Tg

value: WT or CAG-EGFP

value: lung

value: Bleomycin

value: Day 10 and Day 14


title: Fibroblasts hTERT-BJ1: Control vs Colpvp

description:
Biological sample 1 of 2, untreated, fibroblast
Biological sample 2 of 2, colpvp, fibroblast

source_name: Total RNA extracted from untreated fibroblasts

growth_protocol: hTERT-BJ1 were cultured with Dulbecco's modified Eagle's Medium,supplemented with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin.

value: Fibroblast

source_name: Total RNA extracted from Colpvp treated fibroblasts


title: NaraCOVID_TP3_CD8_003_TCR

value: Three shots of Pfizer-BNT162b2 vaccine

value: CD8+ T cells and CD19+ B cells

value: 1 week after receiving the second dose


title: Alveolospheres with Bleomycin treatment (cDNA)

tag: tissue


title: VUMC0126

last: Wang


title: NaraCOVID_TP3_CD8_001_TCR

tag: Sex

value: male


title: P. nyererei, testes, rep4

city: Cambridge

first: Miguel

middle: Vasconcelos

last: Almeida

address: Tennis Court Road

department: Department of Biochemistry

country: United Kingdom

institute: University of Cambridge

description:
Pny_tes_rep4
rawCounts_mRNA_GenesAndTEs_Pnye.txt
NormCounts_mRNA_TEs_Pnye.txt

data_processing:
Illumina adapters and reads with low-quality calls were filtered out with Trimmomatic v0.39 (Bolger et al., 2014) using options SLIDINGWINDOW:4:28 MINLEN:36.
We mapped trimmed reads to the genome, using STAR v2.5.4b (Dobin et al., 2013) with options --outTmpDir --readFilesCommand zcat ‑‑genomeDir ‑‑readFilesIn {R1} {R2} --outSAMtype BAM SortedByCoordinate ‑‑outFilterType BySJout --outFilterMultimapNmax 150 ‑‑winAnchorMultimapNmax 150 --alignSJoverhangMin 8 --alignSJDBoverhangMin 3 ‑‑outFilterMismatchNmax 999 --outFilterMismatchNoverReadLmax 0.04 --alignIntronMin 20 --alignIntronMax 10000000 --alignMatesGapMax 100000000 --outFileNamePrefix. Reads from Lake Malawi cichlid species (A. calliptera, M. zebra, T. sp. "mauve") were all mapped to the A. calliptera genome fAstCal1.2. A. burtoni, P. nyererei, and O. niloticus reads were mapped to their genomes: AstBur1.0, PunNye1.0, and  O_niloticus_UMD_NMBU respectively. All these genomes are available at Ensembl.
The resulting BAM files were used as inputs for TEtranscripts v2.2.1 (Jin et al., 2015) with options -t -c --GTF --TE --stranded reverse ‑‑project --SortByPos.
For A. calliptera samples, gene expression (without TEs) was quantified from trimmed reads using salmon v1.5.1 (Patro et al., 2017), with options --seqBias --gcBias - validateMappings -l A -1 ‑2 -o -i.
DESeq2 (Love et al., 2014) and custom scripts were used to calculate log-normalized and TPM counts, generate plots and conduct statistical tests on an R framework (R Core Team, 2021).
Assembly: fAstCal1.2; AstBur1.0; PunNye1.0; O_niloticus_UMD_NMBU
Supplementary files format and content: rawCounts_mRNA_GenesAndTEs_Acal.txt: Tab-delimited file includes raw counts for genes and TEs in each A. calliptera sample. Output of TEtranscripts.
Supplementary files format and content: NormCounts_mRNA_TEs_Acal.txt: Tab-delimited file includes DESeq2 normalized counts for TEs in each A. calliptera sample.
Supplementary files format and content: rawCounts_mRNA_GenesAndTEs_Malawi_curatedTEAnnotation.txt: Tab-delimited file includes raw counts for genes and TEs in each Lake Malawi cichlid sample. Output of TEtranscripts, using a curated TE annotation.
Supplementary files format and content: NormCounts_mRNA_TEs_Malawi_curatedTEAnnotation.txt: Tab-delimited file includes DESeq2 normalized counts for TEs in each Lake Malawi cichlid sample. Using a curated TE annotation.
Supplementary files format and content: rawCounts_mRNA_Acal_brain_gonads.txt: Tab-delimited file includes raw counts in each A. calliptera sample. From Salmon output.
Supplementary files format and content: lengthScaledTPM_mRNA_Acal_brain_gonads.txt: Tab-delimited file includes length scaled TPM counts in each A. calliptera sample. From Salmon output.
Supplementary files format and content: rawCounts_mRNA_GenesAndTEs_Abur.txt: Tab-delimited file includes raw counts for genes and TEs in each A. burtoni sample. Output of TEtranscripts.
Supplementary files format and content: NormCounts_mRNA_TEs_Abur.txt: Tab-delimited file includes DESeq2 normalized counts for TEs in each A. burtoni sample.
Supplementary files format and content: rawCounts_mRNA_GenesAndTEs_Pnye.txt: Tab-delimited file includes raw counts for genes and TEs in each P. nyererei sample. Output of TEtranscripts.
Supplementary files format and content: NormCounts_mRNA_TEs_Pnye.txt: Tab-delimited file includes DESeq2 normalized counts for TEs in each P. nyererei sample.
Supplementary files format and content: rawCounts_mRNA_GenesAndTEs_Onil.txt: Tab-delimited file includes raw counts for genes and TEs in each O. niloticus sample. Output of TEtranscripts.
Supplementary files format and content: NormCounts_mRNA_TEs_Onil.txt: Tab-delimited file includes DESeq2 normalized counts for TEs in each O. niloticus sample.

organism: Pundamilia nyererei

source_name: Testes

extract_protocol:
Tissue pieces were transferred to BeadBug tubes prefilled with 0.5 mm Zirconium beads (Merck, #Z763772) and 500-600 µl of TRIzol (Life Technologies, #15596026) was added to the tubes and mixed vigorously. Afterwards, we conducted the homogenisation using a BeadBug microtube homogeniser (Sigma, #Z764140) at approximately 4oC (in cold room). Each sample was homogenised with five BeadBug runs at maximum speed (4000 rpm) for 60 seconds each. Supernatant was then removed into a clean 1.5 mL tube. Centrifuged the lysates again, this time at maximum speed (approximately 21,000 G) for 5 minutes at 4oC. Transferred supernatant into a clean tube without disturbing the pellet and tissue debris. Mixed supernatant thoroughly 1:1 with 100% ethanol, pipetted the mix into a column provided in the Direct-zol RNA Miniprep Plus kit (Zymo Research, #R2072) and followed manufacturer’s instructions, using the recommended in-column DNase I treatment.
Quality control, library preparation (directional, with poly-A enrichment), and mRNA sequencing (Illumina, PE150) was performed by Novogene. A. calliptera brain libraries and P.nyererei gonad libraries were prepared differently with the following protocol. We used 50-250 ng of total RNA for library production with the NEBNext® Poly(A) mRNA Magnetic Isolation Module (NEB, #E7490), in conjunction with the NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina® (NEB, #E7760) and the NEBNext® Multiplex Oligos for Illumina® (96 Unique Dual Index Primer Pairs, NEB #E6440). Samples were then pooled in equimolar amounts and sequenced on a NovaSeq 6000 system (PE150 on one lane of an S1 Flowcell).

value: Testes

tag: genotype

value: wild-type