title: CB-ATO DP early sample 2

description:
immature T cell population generated in ATOs from T cell differentiation of human cord blood hematopoietic stem cells
GCAMhsExp.GEO.txt

growth_protocol:
Cord blood ATOs were generated as previously described (Seet et al, 2017, Protocol exchange). MS5-hDLL1  cells were harvested by trypsinization and resuspended in serum free ATO culture medium (“RB27”) composed of RPMI 1640 (Corning, Manassas, VA), 4% B27 supplement (ThermoFisher Scientific, Grand Island, NY), 30 µM L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate (Sigma-Aldrich, St. Louis, MO) reconstituted in PBS, 1% penicillin/streptomycin (Gemini Bio-Products, West Sacramento, CA), 1% Glutamax (ThermoFisher Scientific, Grand Island, NY), 5 ng/ml rhFLT3L and 5 ng/ml rhIL-7 (Peprotech, Rocky Hill, NJ). RB27 was made fresh weekly. 1.5x105 MS5-hDLL1 cells were combined with 5x103 purified CD34+CD3- cells per ATO in 1.5 ml Eppendorf tubes and centrifuged at 300 g for 5 min at 4˚C in a swinging bucket centrifuge. Supernatants were carefully removed, and the cell pellet was resuspended by brief vortexing. ATOs were plated on a 0.4 µm Millicell transwell insert (EMD Millipore, Billerica, MA; Cat. PICM0RG50) placed in a 6-well plate containing 1 ml RB27 per well. Medium was changed completely every 3-4 days by aspiration from around the cell insert followed by replacement with 1 ml with fresh RB27/cytokines. ATO cells were harvested by adding FACS buffer (PBS/0.5% bovine serum album/2mM EDTA) to each well and briefly disaggregating the ATO by pipetting with a 1 ml “P1000” pipet, followed by passage through a 50 µm nylon strainer. Flow cytometry cell sorting of CB-ATO derived double positive cell population used the following surface phenotypes: DP; CD4+CD8+CD3-.
The hESC line H1 (WiCell, Madison, WI) was maintained on Matrigel coated plates (growth factor reduced, no phenol red; BD Biosciences, San Jose, CA), and cultured in mTESR medium (Stem Cell Technologies, Vancouver, BC).  PSC-ATOs were generated using purified hEMPs and followed a two-step procedure including a hematopoietic induction phase and a T-cell differentiation phase.  MS5-hDLL4 cells were harvested by trypsinization and resuspended in hematopoietic induction medium composed of EGM2 (Lonza Ref CC-4176) supplemented with ROCK inhibitor Y-27632 dihydrochloride (10uM) (Tocris Bioscience, Cat. 1254) and TGF-βRI inhibitor SB-431542 (“SB blocker”) (10 uM) (Tocris Bioscience, Cat. 1614). 5x105 MS5-hDLL4 cells were combined with 0.5-1x104 purified hEMP per PSC-ATO in 1.5 ml Eppendorf tubes and centrifuged at 300 g for 5 min at 4˚C in a swinging bucket centrifuge. Multiple (up to 12) PSC-ATO cell volumes were prepared per tube. Supernatants were carefully removed and the cell pellet was resuspended by brief vortexing, and resuspended in hematopoietic induction medium at a volume of 6 µl per PSC-ATO. 6 µl of cells were plated as PSC-ATOs on a 0.4 µm Millicell transwell insert (EMD Millipore, Billerica, MA; Cat. PICM0RG50) (up to 2 PSC-ATOs per insert were plated) and placed in 6-well plates containing 1 ml hematopoietic induction medium per well. Medium was changed completely every 2-3 days for 1 weekM with medium composed of EGM2 with SB blocker (10uM). At day 7, medium was change to EGM2 + SB blocker (10uM) with the hematopoietic cytokines TPO 5ng/ml (Peprotech, Cat. 300-18), FLT3L 5ng/ml (Peprotech, Cat. 300-19), and SCF 50ng/ml (Peprotech, Cat. 300-07). This medium was changed every 2-3 days for an additional 7 days. At day 14, T cell differentiation was induced by changing the medium to “RB27” (described above) supplemented with 10 ng/ml SCF, 5 ng/ml FLT3L, and 5 ng/ml IL-7 (Peprotech, Cat. 200-07). Medium was changed completely every 3-4 days. PSC-ATO cells were harvested by adding MACS buffer (PBS/0.5% bovine serum album/2mM EDTA) to each well and briefly disaggregating the ATO by pipetting with a 1 ml “P1000” pipet, followed by passage through a 50 µm nylon strainer. Flow cytometry cell sorting of ESC-ATO derived  double positive cell population used the following surface phenotypes: DP; CD4+CD8+CD3-. Cord blood ATOs were generated as previously described (Seet et al, 2017, Protocol exchange). MS5-hDLL1  cells were harvested by trypsinization and resuspended in serum free ATO culture medium (“RB27”) composed of RPMI 1640 (Corning, Manassas, VA), 4% B27 supplement (ThermoFisher Scientific, Grand Island, NY), 30 µM L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate (Sigma-Aldrich, St. Louis, MO) reconstituted in PBS, 1% penicillin/streptomycin (Gemini Bio-Products, West Sacramento, CA), 1% Glutamax (ThermoFisher Scientific, Grand Island, NY), 5 ng/ml rhFLT3L and 5 ng/ml rhIL-7 (Peprotech, Rocky Hill, NJ). RB27 was made fresh weekly. 1.5x105 MS5-hDLL1 cells were combined with 5x103 purified CD34+CD3- cells per ATO in 1.5 ml Eppendorf tubes and centrifuged at 300 g for 5 min at 4˚C in a swinging bucket centrifuge. Supernatants were carefully removed, and the cell pellet was resuspended by brief vortexing. ATOs were plated on a 0.4 µm Millicell transwell insert (EMD Millipore, Billerica, MA; Cat. PICM0RG50) placed in a 6-well plate containing 1 ml RB27 per well. Medium was changed completely every 3-4 days by aspiration from around the cell insert followed by replacement with 1 ml with fresh RB27/cytokines. ATO cells were harvested by adding FACS buffer (PBS/0.5% bovine serum album/2mM EDTA) to each well and briefly disaggregating the ATO by pipetting with a 1 ml “P1000” pipet, followed by passage through a 50 µm nylon strainer. Flow cytometry cell sorting of CB-ATO derived double positive cell population used the following surface phenotypes: DP; CD4+CD8+CD3-. Postnatal human thymi were obtained under IRB exemption as discarded waste from patients undergoing cardiac surgery at Children’s Hospital Los Angeles (CHLA). Thymic fragments were finely dissected in RPMI and disrupted by pipetting to release thymocytes into suspension, followed by passage through a 70 µm nylon strainer. Cells were analyzed fresh on the same or following day. Flow cytometry cell sorting of thymic SP4 T cell population used the following surface phenotypes: CD4 single positive (CD4SP; TCRαβ+CD3+CD8+CD4-CD45RA+).

treatment_protocol: The freshly sorted CB-ATO derived DP population was pelleted for RNA extraction

value: CD4+ CD8+ CD3-


title: C1307202G_MDCF2_Treatment

status: Public on Aug 20 2019

type: protein

city: St. Louis

first: Jeanette

last: Gehrig

state: Missouri

address: 4515 McKinley

country: USA

institute: Washington University School of Medicine

hyb_protocol: Following the manufacturer’s protocol and utilizing SOMAmer reagents immobilized on streptavidin beads, proteins from plasma samples were tagged with NHS-biotin reagent, captured as a SOMAmer reagent/protein complex, cleaved, denatured, eluted and hybridized to a custom Agilent DNA microarray. Additional details can be found at http://www.tandfonline.com/doi/abs/10.1586/erm.10.89 and Gold L et al. N Biotechnol. 2012 Jun 15;29(5):543-9.

scan_protocol: Microarrays were scanned with an Agilent SureScan scanner at 5 um resolution, and the Cy3 fluorescence readout was quantified. Additional information can be found in Gold L et al. N Biotechnol. 2012 Jun 15;29(5):543-9.

data_processing: Raw signal values were processed using Somalogic's SOMAscan standardization procedures, including hybridization normalization, plate scaling, median scaling, and final somamer calibration as described in http://www.tandfonline.com/doi/abs/10.1586/erm.10.89.

molecule: protein

organism: Homo sapiens

source_name: non-fasted plasma

extract_protocol: Non-fasting venous blood was collected into EDTA-coated tubes, spun, separated into plasma, aliquoted and snap-frozen at -80C until sample processing.

treatment_protocol: Non-fasting plasma collected in EDTA-coated tubes

tag: group

tag: gender

value: Female

tag: age

tag: wlz

tag: waz

tag: laz


title: C1321201G_MDCF2_Baseline

label_protocol: Following the manufacturer’s protocol and utilizing SOMAmer reagents immobilized on streptavidin beads, proteins from plasma samples were tagged with NHS-biotin reagent, captured as a SOMAmer reagent/protein complex, cleaved, denatured, eluted and hybridized to a custom Agilent DNA microarray. Additional details can be found at http://www.tandfonline.com/doi/abs/10.1586/erm.10.89 and Gold L et al. N Biotechnol. 2012 Jun 15;29(5):543-9.

value: Male


title: UBC.CreER_VHL2B.lox-lox_rep2

type: SRA

city: NASHVILLE

first: Aguirre

middle: A

last: de Cubas

state: TN

address: 2220 Pierce Ave

department: Hematology and Oncology

institute: Vanderbilt University Medical Center

library_source: transcriptomic

data_processing:
Sequencing reads were aligned against the Mouse GENCODE genome, Version M14 (January 2017 freeze, GRCm38, Ensembl 89) using the Spliced Transcripts Alignment to a Reference (STAR) software (PMID: 26187010 and 23104886).
Reads were preprocessed and index using SAMtools (PMID: 19505943).
Mapped reads were assigned to gene features and quantified using featureCounts (PMID: 24227677).
Genome_build: Mouse GENCODE genome, Version M14 (January 2017 freeze, GRCm38, Ensembl 89)
Supplementary_files_format_and_content: Raw Counts

molecule: polyA RNA

organism: Mus musculus

source_name: UBC.CreER_VHL2B.lox-lox, retina

growth_protocol: Mice expressing Cre-recombinase ubiquitously [Tg(UBC-cre/ERT2), denoted in the current study as UBCCreER/+ with CreER negative animals denoted as UBC+/+] were bred with Vhlfl/fl mice.  Vhl2B/+ mice were also crossed with Vhlfl/fl mice to yield Vhlfl/2B offspring.  Breeding UBCCreER/+; Vhlfl/fl males with Vhlfl/2B females produced experimental litters with genotypes: (1 and 2) non-mutant littermate control -- UBC+/+; Vhlfl/fl, (littermate control with 1 WT Vhl allele and 1 2B mutant Vhl allele -- UBC+/+; Vhlfl/2B , (3) conditional Vhl null -- UBCCreER/+; Vhlfl/fl, (4) conditional Vhl type 2B mutant -- UBCCreER/+; Vhlfl/2B. Where possible, animals were distributed across sex to avoid the introduction of sex biasing.

extract_protocol:
Following induction of the genetic mutations as described above, retinas were collected from P7 mice and immediately digested in TRIzol to extract mRNA transcripts (n=2 for each genotype). RNA was further isolated with 1-Bromo-3-chloropropane (Sigma) and ethanol and purified with an RNeasy Plus Kit (Qiagen).  Using an Agilent Bioanlayzer, the Virginia Tech Biocomplexity Institute ensured the isolated mRNA was of a sufficiently high quality for RNA sequencing.
Eight samples (4 genotypes x 2 replicates per genotype) were sent to GeneWiz (South Plainfield, NJ) for additional quality assessment, library generation, and sequencing on an Illumina HiSeq with a 2x150 configuration.

treatment_protocol: Pups were intraperitoneally (i.p.) injected each day with 1 mg/ml of tamoxifen (Fisher) from postnatal day 1 (P1) to 3. Retinal harvest occurred on P7. Cre-recombination activity following tamoxifen exposure was verified using a Cre reporter mouse line.

tag: experimental group

tag: tissue

value: retina

tag: genotype


title: THY CD4SP sample 3

city: Los Angeles

first: Gay

middle: Miriam

last: Crooks

state: California

address: 610 Charles E. Young Drive, East

department: Pathology & Laboratory Med

institute: University of California Los Angeles

description:
Mature T cell population isolated from fresh human thymus
GCAMhsExp.GEO.txt

data_processing:
The STAR ultrafast universal RNA-seq aligner v2.5.2b (Dobin et al., 2013) was used to generate the genome index and perform paired-end alignments. Reads were aligned to a genome index that includes both the genome sequence (GRCh38 primary assembly) and the exon/intron structure of known gene models (Gencode v26 basic genome annotation).
Alignment files were used to generate strand-specific, gene-level count summaries with STAR's built-in gene counter. Only protein-coding, long-noncoding, anti-sense and T-cell receptor genes in the Gencode v26 annotation were considered (98% of total counts on average). Independent filtering was applied as follows: genes with less than one count per sample on average, count outliers or low mappability were filtered out for downstream analysis
Counts were normalized per-sample in units of FPKMs after correcting for gene mappable length and sample total counts.
Genome_build: GRCh38 primary assembly
Supplementary_files_format_and_content: Tab delimited. Table of per-gene and per-sample expression estimates in units of FPKMs

molecule: total RNA

source_name: human thymus

growth_protocol:
Postnatal human thymi were obtained under IRB exemption as discarded waste from patients undergoing cardiac surgery at Children’s Hospital Los Angeles (CHLA). Thymic fragments were finely dissected in RPMI and disrupted by pipetting to release thymocytes into suspension, followed by passage through a 70 µm nylon strainer. Cells were analyzed fresh on the same or following day. Flow cytometry cell sorting of thymic SP4 T cell population used the following surface phenotypes: CD4 single positive (CD4SP; TCRαβ+CD3+CD8+CD4-CD45RA+).
The hESC line H1 (WiCell, Madison, WI) was maintained on Matrigel coated plates (growth factor reduced, no phenol red; BD Biosciences, San Jose, CA), and cultured in mTESR medium (Stem Cell Technologies, Vancouver, BC).  PSC-ATOs were generated using purified hEMPs and followed a two-step procedure including a hematopoietic induction phase and a T-cell differentiation phase.  MS5-hDLL4 cells were harvested by trypsinization and resuspended in hematopoietic induction medium composed of EGM2 (Lonza Ref CC-4176) supplemented with ROCK inhibitor Y-27632 dihydrochloride (10uM) (Tocris Bioscience, Cat. 1254) and TGF-βRI inhibitor SB-431542 (“SB blocker”) (10 uM) (Tocris Bioscience, Cat. 1614). 5x105 MS5-hDLL4 cells were combined with 0.5-1x104 purified hEMP per PSC-ATO in 1.5 ml Eppendorf tubes and centrifuged at 300 g for 5 min at 4˚C in a swinging bucket centrifuge. Multiple (up to 12) PSC-ATO cell volumes were prepared per tube. Supernatants were carefully removed and the cell pellet was resuspended by brief vortexing, and resuspended in hematopoietic induction medium at a volume of 6 µl per PSC-ATO. 6 µl of cells were plated as PSC-ATOs on a 0.4 µm Millicell transwell insert (EMD Millipore, Billerica, MA; Cat. PICM0RG50) (up to 2 PSC-ATOs per insert were plated) and placed in 6-well plates containing 1 ml hematopoietic induction medium per well. Medium was changed completely every 2-3 days for 1 weekM with medium composed of EGM2 with SB blocker (10uM). At day 7, medium was change to EGM2 + SB blocker (10uM) with the hematopoietic cytokines TPO 5ng/ml (Peprotech, Cat. 300-18), FLT3L 5ng/ml (Peprotech, Cat. 300-19), and SCF 50ng/ml (Peprotech, Cat. 300-07). This medium was changed every 2-3 days for an additional 7 days. At day 14, T cell differentiation was induced by changing the medium to “RB27” (described above) supplemented with 10 ng/ml SCF, 5 ng/ml FLT3L, and 5 ng/ml IL-7 (Peprotech, Cat. 200-07). Medium was changed completely every 3-4 days. PSC-ATO cells were harvested by adding MACS buffer (PBS/0.5% bovine serum album/2mM EDTA) to each well and briefly disaggregating the ATO by pipetting with a 1 ml “P1000” pipet, followed by passage through a 50 µm nylon strainer. Flow cytometry cell sorting of ESC-ATO derived  double positive cell population used the following surface phenotypes: DP; CD4+CD8+CD3-. Cord blood ATOs were generated as previously described (Seet et al, 2017, Protocol exchange). MS5-hDLL1  cells were harvested by trypsinization and resuspended in serum free ATO culture medium (“RB27”) composed of RPMI 1640 (Corning, Manassas, VA), 4% B27 supplement (ThermoFisher Scientific, Grand Island, NY), 30 µM L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate (Sigma-Aldrich, St. Louis, MO) reconstituted in PBS, 1% penicillin/streptomycin (Gemini Bio-Products, West Sacramento, CA), 1% Glutamax (ThermoFisher Scientific, Grand Island, NY), 5 ng/ml rhFLT3L and 5 ng/ml rhIL-7 (Peprotech, Rocky Hill, NJ). RB27 was made fresh weekly. 1.5x105 MS5-hDLL1 cells were combined with 5x103 purified CD34+CD3- cells per ATO in 1.5 ml Eppendorf tubes and centrifuged at 300 g for 5 min at 4˚C in a swinging bucket centrifuge. Supernatants were carefully removed, and the cell pellet was resuspended by brief vortexing. ATOs were plated on a 0.4 µm Millicell transwell insert (EMD Millipore, Billerica, MA; Cat. PICM0RG50) placed in a 6-well plate containing 1 ml RB27 per well. Medium was changed completely every 3-4 days by aspiration from around the cell insert followed by replacement with 1 ml with fresh RB27/cytokines. ATO cells were harvested by adding FACS buffer (PBS/0.5% bovine serum album/2mM EDTA) to each well and briefly disaggregating the ATO by pipetting with a 1 ml “P1000” pipet, followed by passage through a 50 µm nylon strainer. Flow cytometry cell sorting of CB-ATO derived double positive cell population used the following surface phenotypes: DP; CD4+CD8+CD3-. Postnatal human thymi were obtained under IRB exemption as discarded waste from patients undergoing cardiac surgery at Children’s Hospital Los Angeles (CHLA). Thymic fragments were finely dissected in RPMI and disrupted by pipetting to release thymocytes into suspension, followed by passage through a 70 µm nylon strainer. Cells were analyzed fresh on the same or following day. Flow cytometry cell sorting of thymic SP4 T cell population used the following surface phenotypes: CD4 single positive (CD4SP; TCRαβ+CD3+CD8+CD4-CD45RA+).

extract_protocol:
All samples were extracted using miRNeasy Micro Kit (Qiagen).
1.5ng of total RNA was input to generate sequencing libraries with SMARTer Stranded Total RNA-Seq (Pico) Kit (Clonetech, Cat. 635005).

treatment_protocol: The freshly sorted thymic SP4 population was pelleted for RNA extraction

tag: phenotype

value: CD4+ CD8- CD3+ TCRab+ CD45RA+


title: Cint_24

city: Cincinnati

first: Zhenhua

last: Luo

state: Ohio

address: 3333 Burnet Avenue

institute: Cincinnati Children's Hospital Medical Center

data_processing:
The RNA-seq reads were aligned to the human genome (GRCh37/hg19) using TopHat (version 2.1.0)
Transcript quantifications were performed by Cufflinks v2.2.1
Transcript abundances were normalized in reads per kilobase of exon per million mapped reads (RPKM) for single end RNA-seq and fragments per kilobase of exon per million mapped reads (FPKM) for paired end RNA-seq
Genome_build: hg19
Supplementary_files_format_and_content: tab-delimited text files include RPKM or FPKM values for each sample

source_name: Liver

extract_protocol:
Total RNA was isolated from liver biopsies using the miRNeasy Kit (QIAGEN). RNA purity was assessed using the Agilent 2100 Bioanalyzer. Total RNA was converted to cDNA using High-Capacity cDNA Reverse Transcrition Kit (ABI).
The cDNA was then used for Illumina sequencing library preparation by TruSeq Stranded mRNA Library Prep Kit. DNA fragments were further end repaired to generate blunt ends with 5′ phosphatase and 3′ hydroxyls and adapters were ligated for single end or paired end sequencing on Illumina HiSeq 2500.

tag: disease state

value: Biliary Atresia

value: Liver


title: C1307201G_MDCF2_Baseline


title: CB-ATO DP early sample 1

source_name: human cord blood


title: C1321202G_MDCF2_Treatment


title: UBC.CreER_VHL2B.lox-lox_rep1


title: Bp.c-B60


title: C1304201G_MDCF2_Baseline


title: C1302202G_MDCF2_Treatment


title: B98


title: no stroke healthy mouse, rep 3

city: University Park

first: Gong

last: Chen

state: PA

address: Old Main

institute: Penn State University

data_processing:
quality check using FastQC v0.11.3
reads were aligned to GRCm38 using HISAT2 version 2.0.4
counts summarized using featureCounts v1.5.1 (count) or StringTie v1.3.4 (RPKM)
differential analysis by DESeq2 v1.16.1
Supplementary_files_format_and_content: a txt file containing read count from all samples generated by featureCounts

source_name: no stroke healthy mouse, rep 3

extract_protocol:
total RNA were extracted according to protocols accompanying with Macherey-Nagel NucleoSpin RNA kit, then sent to sequencing center for polyA enrichment
libraries were constructed using KAPA RNA kit at UCLA Technology Center for Genomics & Bioinformatics

tag: injury

value: no injury

tag: agent

value: no virus


title: THY CD4SP sample 1


title: THY CD4SP sample 2


title: no stroke healthy mouse, rep 2

source_name: no stroke healthy mouse, rep 2


title: C1302201G_MDCF2_Baseline


title: BS.209_B1_SAM

value: SAM


title: UBC.CreER_VHL.lox-lox_rep1

source_name: UBC.CreER_VHL.lox-lox, retina

value: VHL-/-


title: B96


title: THY CD8SP sample 3

growth_protocol:
Postnatal human thymi were obtained under IRB exemption as discarded waste from patients undergoing cardiac surgery at Children’s Hospital Los Angeles (CHLA). Thymic fragments were finely dissected in RPMI and disrupted by pipetting to release thymocytes into suspension, followed by passage through a 70 µm nylon strainer. Cells were analyzed fresh on the same or following day. Flow cytometry cell sorting of thymic SP8 T cell population used the following surface phenotypes: CD8 single positive (CD8SP; TCRαβ+CD3+CD8α+CD4-CD45RA+)
The hESC line H1 (WiCell, Madison, WI) was maintained on Matrigel coated plates (growth factor reduced, no phenol red; BD Biosciences, San Jose, CA), and cultured in mTESR medium (Stem Cell Technologies, Vancouver, BC).  PSC-ATOs were generated using purified hEMPs and followed a two-step procedure including a hematopoietic induction phase and a T-cell differentiation phase.  MS5-hDLL4 cells were harvested by trypsinization and resuspended in hematopoietic induction medium composed of EGM2 (Lonza Ref CC-4176) supplemented with ROCK inhibitor Y-27632 dihydrochloride (10uM) (Tocris Bioscience, Cat. 1254) and TGF-βRI inhibitor SB-431542 (“SB blocker”) (10 uM) (Tocris Bioscience, Cat. 1614). 5x105 MS5-hDLL4 cells were combined with 0.5-1x104 purified hEMP per PSC-ATO in 1.5 ml Eppendorf tubes and centrifuged at 300 g for 5 min at 4˚C in a swinging bucket centrifuge. Multiple (up to 12) PSC-ATO cell volumes were prepared per tube. Supernatants were carefully removed and the cell pellet was resuspended by brief vortexing, and resuspended in hematopoietic induction medium at a volume of 6 µl per PSC-ATO. 6 µl of cells were plated as PSC-ATOs on a 0.4 µm Millicell transwell insert (EMD Millipore, Billerica, MA; Cat. PICM0RG50) (up to 2 PSC-ATOs per insert were plated) and placed in 6-well plates containing 1 ml hematopoietic induction medium per well. Medium was changed completely every 2-3 days for 1 weekM with medium composed of EGM2 with SB blocker (10uM). At day 7, medium was change to EGM2 + SB blocker (10uM) with the hematopoietic cytokines TPO 5ng/ml (Peprotech, Cat. 300-18), FLT3L 5ng/ml (Peprotech, Cat. 300-19), and SCF 50ng/ml (Peprotech, Cat. 300-07). This medium was changed every 2-3 days for an additional 7 days. At day 14, T cell differentiation was induced by changing the medium to “RB27” (described above) supplemented with 10 ng/ml SCF, 5 ng/ml FLT3L, and 5 ng/ml IL-7 (Peprotech, Cat. 200-07). Medium was changed completely every 3-4 days. PSC-ATO cells were harvested by adding MACS buffer (PBS/0.5% bovine serum album/2mM EDTA) to each well and briefly disaggregating the ATO by pipetting with a 1 ml “P1000” pipet, followed by passage through a 50 µm nylon strainer. Flow cytometry cell sorting of ESC-ATO derived  double positive cell population used the following surface phenotypes: DP; CD4+CD8+CD3-. Cord blood ATOs were generated as previously described (Seet et al, 2017, Protocol exchange). MS5-hDLL1  cells were harvested by trypsinization and resuspended in serum free ATO culture medium (“RB27”) composed of RPMI 1640 (Corning, Manassas, VA), 4% B27 supplement (ThermoFisher Scientific, Grand Island, NY), 30 µM L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate (Sigma-Aldrich, St. Louis, MO) reconstituted in PBS, 1% penicillin/streptomycin (Gemini Bio-Products, West Sacramento, CA), 1% Glutamax (ThermoFisher Scientific, Grand Island, NY), 5 ng/ml rhFLT3L and 5 ng/ml rhIL-7 (Peprotech, Rocky Hill, NJ). RB27 was made fresh weekly. 1.5x105 MS5-hDLL1 cells were combined with 5x103 purified CD34+CD3- cells per ATO in 1.5 ml Eppendorf tubes and centrifuged at 300 g for 5 min at 4˚C in a swinging bucket centrifuge. Supernatants were carefully removed, and the cell pellet was resuspended by brief vortexing. ATOs were plated on a 0.4 µm Millicell transwell insert (EMD Millipore, Billerica, MA; Cat. PICM0RG50) placed in a 6-well plate containing 1 ml RB27 per well. Medium was changed completely every 3-4 days by aspiration from around the cell insert followed by replacement with 1 ml with fresh RB27/cytokines. ATO cells were harvested by adding FACS buffer (PBS/0.5% bovine serum album/2mM EDTA) to each well and briefly disaggregating the ATO by pipetting with a 1 ml “P1000” pipet, followed by passage through a 50 µm nylon strainer. Flow cytometry cell sorting of CB-ATO derived double positive cell population used the following surface phenotypes: DP; CD4+CD8+CD3-. Postnatal human thymi were obtained under IRB exemption as discarded waste from patients undergoing cardiac surgery at Children’s Hospital Los Angeles (CHLA). Thymic fragments were finely dissected in RPMI and disrupted by pipetting to release thymocytes into suspension, followed by passage through a 70 µm nylon strainer. Cells were analyzed fresh on the same or following day. Flow cytometry cell sorting of thymic SP4 T cell population used the following surface phenotypes: CD4 single positive (CD4SP; TCRαβ+CD3+CD8+CD4-CD45RA+).

treatment_protocol: The freshly sorted thymic SP8 population was pelleted for RNA extraction

value: CD4- CD8+ CD3+ TCRab+ CD45RA+


title: THY CD8SP sample 2


title: B84-B200


title: B82


title: WT.CreER_VHL2B-lox_rep2

source_name: WT.CreER_VHL2B-lox, retina


title: C1304202G_MDCF2_Treatment


title: BS.207_B1_SAM


title: B99


title: WT.CreER_VHL2B-lox_rep1


title: WT.CreER_VHL.lox-lox_rep2

source_name: WT.CreER_VHL.lox-lox, retina

value: VHL+/+


title: UBC.CreER_VHL.lox-lox_rep2


title: B79


title: WT.CreER_VHL.lox-lox_rep1


title: BS.178_B1_SAM


title: THY CD8SP sample 1


title: B80


title: BS.184_B1_SAM


title: B77


title: THY DP early sample 3

description:
immature T cell population isolated from fresh human thymus
GCAMhsExp.GEO.txt

growth_protocol:
Postnatal human thymi were obtained under IRB exemption as discarded waste from patients undergoing cardiac surgery at Children’s Hospital Los Angeles (CHLA). Thymic fragments were finely dissected in RPMI and disrupted by pipetting to release thymocytes into suspension, followed by passage through a 70 µm nylon strainer. Cells were analyzed fresh on the same or following day. Flow cytometry cell sorting of thymic  double positive cell population used the following surface phenotypes: DP; CD4+CD8+CD3-.
The hESC line H1 (WiCell, Madison, WI) was maintained on Matrigel coated plates (growth factor reduced, no phenol red; BD Biosciences, San Jose, CA), and cultured in mTESR medium (Stem Cell Technologies, Vancouver, BC).  PSC-ATOs were generated using purified hEMPs and followed a two-step procedure including a hematopoietic induction phase and a T-cell differentiation phase.  MS5-hDLL4 cells were harvested by trypsinization and resuspended in hematopoietic induction medium composed of EGM2 (Lonza Ref CC-4176) supplemented with ROCK inhibitor Y-27632 dihydrochloride (10uM) (Tocris Bioscience, Cat. 1254) and TGF-βRI inhibitor SB-431542 (“SB blocker”) (10 uM) (Tocris Bioscience, Cat. 1614). 5x105 MS5-hDLL4 cells were combined with 0.5-1x104 purified hEMP per PSC-ATO in 1.5 ml Eppendorf tubes and centrifuged at 300 g for 5 min at 4˚C in a swinging bucket centrifuge. Multiple (up to 12) PSC-ATO cell volumes were prepared per tube. Supernatants were carefully removed and the cell pellet was resuspended by brief vortexing, and resuspended in hematopoietic induction medium at a volume of 6 µl per PSC-ATO. 6 µl of cells were plated as PSC-ATOs on a 0.4 µm Millicell transwell insert (EMD Millipore, Billerica, MA; Cat. PICM0RG50) (up to 2 PSC-ATOs per insert were plated) and placed in 6-well plates containing 1 ml hematopoietic induction medium per well. Medium was changed completely every 2-3 days for 1 weekM with medium composed of EGM2 with SB blocker (10uM). At day 7, medium was change to EGM2 + SB blocker (10uM) with the hematopoietic cytokines TPO 5ng/ml (Peprotech, Cat. 300-18), FLT3L 5ng/ml (Peprotech, Cat. 300-19), and SCF 50ng/ml (Peprotech, Cat. 300-07). This medium was changed every 2-3 days for an additional 7 days. At day 14, T cell differentiation was induced by changing the medium to “RB27” (described above) supplemented with 10 ng/ml SCF, 5 ng/ml FLT3L, and 5 ng/ml IL-7 (Peprotech, Cat. 200-07). Medium was changed completely every 3-4 days. PSC-ATO cells were harvested by adding MACS buffer (PBS/0.5% bovine serum album/2mM EDTA) to each well and briefly disaggregating the ATO by pipetting with a 1 ml “P1000” pipet, followed by passage through a 50 µm nylon strainer. Flow cytometry cell sorting of ESC-ATO derived  double positive cell population used the following surface phenotypes: DP; CD4+CD8+CD3-. Cord blood ATOs were generated as previously described (Seet et al, 2017, Protocol exchange). MS5-hDLL1  cells were harvested by trypsinization and resuspended in serum free ATO culture medium (“RB27”) composed of RPMI 1640 (Corning, Manassas, VA), 4% B27 supplement (ThermoFisher Scientific, Grand Island, NY), 30 µM L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate (Sigma-Aldrich, St. Louis, MO) reconstituted in PBS, 1% penicillin/streptomycin (Gemini Bio-Products, West Sacramento, CA), 1% Glutamax (ThermoFisher Scientific, Grand Island, NY), 5 ng/ml rhFLT3L and 5 ng/ml rhIL-7 (Peprotech, Rocky Hill, NJ). RB27 was made fresh weekly. 1.5x105 MS5-hDLL1 cells were combined with 5x103 purified CD34+CD3- cells per ATO in 1.5 ml Eppendorf tubes and centrifuged at 300 g for 5 min at 4˚C in a swinging bucket centrifuge. Supernatants were carefully removed, and the cell pellet was resuspended by brief vortexing. ATOs were plated on a 0.4 µm Millicell transwell insert (EMD Millipore, Billerica, MA; Cat. PICM0RG50) placed in a 6-well plate containing 1 ml RB27 per well. Medium was changed completely every 3-4 days by aspiration from around the cell insert followed by replacement with 1 ml with fresh RB27/cytokines. ATO cells were harvested by adding FACS buffer (PBS/0.5% bovine serum album/2mM EDTA) to each well and briefly disaggregating the ATO by pipetting with a 1 ml “P1000” pipet, followed by passage through a 50 µm nylon strainer. Flow cytometry cell sorting of CB-ATO derived double positive cell population used the following surface phenotypes: DP; CD4+CD8+CD3-. Postnatal human thymi were obtained under IRB exemption as discarded waste from patients undergoing cardiac surgery at Children’s Hospital Los Angeles (CHLA). Thymic fragments were finely dissected in RPMI and disrupted by pipetting to release thymocytes into suspension, followed by passage through a 70 µm nylon strainer. Cells were analyzed fresh on the same or following day. Flow cytometry cell sorting of thymic SP4 T cell population used the following surface phenotypes: CD4 single positive (CD4SP; TCRαβ+CD3+CD8+CD4-CD45RA+).

treatment_protocol: The freshly sorted thymic DP population was pelleted for RNA extraction


title: THY DP early sample 2


title: BS.177_B1_SAM


title: THY DP early sample 1


title: BS.175_B1_SAM


title: ESC (iPSC-ATO CD8SP sample 2

description:
Mature T cell population generated in ATOs from hematopoietic differentiation of human induced pluripotent stem cells
GCAMhsExp.GEO.txt

source_name: human embryonic stem cells-derived (iPSC  line)

growth_protocol:
PSC (iPSC)-ATOs were generated using purified hEMPs and followed a two-step procedure including a hematopoietic induction phase and a T-cell differentiation phase.  MS5-hDLL4 cells were harvested by trypsinization and resuspended in hematopoietic induction medium composed of EGM2 (Lonza Ref CC-4176) supplemented with ROCK inhibitor Y-27632 dihydrochloride (10uM) (Tocris Bioscience, Cat. 1254) and TGF-βRI inhibitor SB-431542 (“SB blocker”) (10 uM) (Tocris Bioscience, Cat. 1614). 5x105 MS5-hDLL4 cells were combined with 0.5-1x104 purified hEMP per PSC-ATO in 1.5 ml Eppendorf tubes and centrifuged at 300 g for 5 min at 4˚C in a swinging bucket centrifuge. Multiple (up to 12) PSC-ATO cell volumes were prepared per tube. Supernatants were carefully removed and the cell pellet was resuspended by brief vortexing, and resuspended in hematopoietic induction medium at a volume of 6 µl per PSC-ATO. 6 µl of cells were plated as PSC-ATOs on a 0.4 µm Millicell transwell insert (EMD Millipore, Billerica, MA; Cat. PICM0RG50) (up to 2 PSC-ATOs per insert were plated) and placed in 6-well plates containing 1 ml hematopoietic induction medium per well. Medium was changed completely every 2-3 days for 1 weekM with medium composed of EGM2 with SB blocker (10uM). At day 7, medium was change to EGM2 + SB blocker (10uM) with the hematopoietic cytokines TPO 5ng/ml (Peprotech, Cat. 300-18), FLT3L 5ng/ml (Peprotech, Cat. 300-19), and SCF 50ng/ml (Peprotech, Cat. 300-07). This medium was changed every 2-3 days for an additional 7 days. At day 14, T cell differentiation was induced by changing the medium to “RB27” (described above) supplemented with 10 ng/ml SCF, 5 ng/ml FLT3L, and 5 ng/ml IL-7 (Peprotech, Cat. 200-07). Medium was changed completely every 3-4 days. PSC-ATO cells were harvested by adding MACS buffer (PBS/0.5% bovine serum album/2mM EDTA) to each well and briefly disaggregating the ATO by pipetting with a 1 ml “P1000” pipet, followed by passage through a 50 µm nylon strainer. Flow cytometry cell sorting of PSC-ATO derived SP8 T cell population used the following surface phenotypes: CD8 single positive (CD8SP; TCRαβ+CD3+CD8α+CD4-CD45RA+)
The hESC line H1 (WiCell, Madison, WI) was maintained on Matrigel coated plates (growth factor reduced, no phenol red; BD Biosciences, San Jose, CA), and cultured in mTESR medium (Stem Cell Technologies, Vancouver, BC).  PSC-ATOs were generated using purified hEMPs and followed a two-step procedure including a hematopoietic induction phase and a T-cell differentiation phase.  MS5-hDLL4 cells were harvested by trypsinization and resuspended in hematopoietic induction medium composed of EGM2 (Lonza Ref CC-4176) supplemented with ROCK inhibitor Y-27632 dihydrochloride (10uM) (Tocris Bioscience, Cat. 1254) and TGF-βRI inhibitor SB-431542 (“SB blocker”) (10 uM) (Tocris Bioscience, Cat. 1614). 5x105 MS5-hDLL4 cells were combined with 0.5-1x104 purified hEMP per PSC-ATO in 1.5 ml Eppendorf tubes and centrifuged at 300 g for 5 min at 4˚C in a swinging bucket centrifuge. Multiple (up to 12) PSC-ATO cell volumes were prepared per tube. Supernatants were carefully removed and the cell pellet was resuspended by brief vortexing, and resuspended in hematopoietic induction medium at a volume of 6 µl per PSC-ATO. 6 µl of cells were plated as PSC-ATOs on a 0.4 µm Millicell transwell insert (EMD Millipore, Billerica, MA; Cat. PICM0RG50) (up to 2 PSC-ATOs per insert were plated) and placed in 6-well plates containing 1 ml hematopoietic induction medium per well. Medium was changed completely every 2-3 days for 1 weekM with medium composed of EGM2 with SB blocker (10uM). At day 7, medium was change to EGM2 + SB blocker (10uM) with the hematopoietic cytokines TPO 5ng/ml (Peprotech, Cat. 300-18), FLT3L 5ng/ml (Peprotech, Cat. 300-19), and SCF 50ng/ml (Peprotech, Cat. 300-07). This medium was changed every 2-3 days for an additional 7 days. At day 14, T cell differentiation was induced by changing the medium to “RB27” (described above) supplemented with 10 ng/ml SCF, 5 ng/ml FLT3L, and 5 ng/ml IL-7 (Peprotech, Cat. 200-07). Medium was changed completely every 3-4 days. PSC-ATO cells were harvested by adding MACS buffer (PBS/0.5% bovine serum album/2mM EDTA) to each well and briefly disaggregating the ATO by pipetting with a 1 ml “P1000” pipet, followed by passage through a 50 µm nylon strainer. Flow cytometry cell sorting of ESC-ATO derived  double positive cell population used the following surface phenotypes: DP; CD4+CD8+CD3-. Cord blood ATOs were generated as previously described (Seet et al, 2017, Protocol exchange). MS5-hDLL1  cells were harvested by trypsinization and resuspended in serum free ATO culture medium (“RB27”) composed of RPMI 1640 (Corning, Manassas, VA), 4% B27 supplement (ThermoFisher Scientific, Grand Island, NY), 30 µM L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate (Sigma-Aldrich, St. Louis, MO) reconstituted in PBS, 1% penicillin/streptomycin (Gemini Bio-Products, West Sacramento, CA), 1% Glutamax (ThermoFisher Scientific, Grand Island, NY), 5 ng/ml rhFLT3L and 5 ng/ml rhIL-7 (Peprotech, Rocky Hill, NJ). RB27 was made fresh weekly. 1.5x105 MS5-hDLL1 cells were combined with 5x103 purified CD34+CD3- cells per ATO in 1.5 ml Eppendorf tubes and centrifuged at 300 g for 5 min at 4˚C in a swinging bucket centrifuge. Supernatants were carefully removed, and the cell pellet was resuspended by brief vortexing. ATOs were plated on a 0.4 µm Millicell transwell insert (EMD Millipore, Billerica, MA; Cat. PICM0RG50) placed in a 6-well plate containing 1 ml RB27 per well. Medium was changed completely every 3-4 days by aspiration from around the cell insert followed by replacement with 1 ml with fresh RB27/cytokines. ATO cells were harvested by adding FACS buffer (PBS/0.5% bovine serum album/2mM EDTA) to each well and briefly disaggregating the ATO by pipetting with a 1 ml “P1000” pipet, followed by passage through a 50 µm nylon strainer. Flow cytometry cell sorting of CB-ATO derived double positive cell population used the following surface phenotypes: DP; CD4+CD8+CD3-. Postnatal human thymi were obtained under IRB exemption as discarded waste from patients undergoing cardiac surgery at Children’s Hospital Los Angeles (CHLA). Thymic fragments were finely dissected in RPMI and disrupted by pipetting to release thymocytes into suspension, followed by passage through a 70 µm nylon strainer. Cells were analyzed fresh on the same or following day. Flow cytometry cell sorting of thymic SP4 T cell population used the following surface phenotypes: CD4 single positive (CD4SP; TCRαβ+CD3+CD8+CD4-CD45RA+).

treatment_protocol: The freshly sorted PSC (iPSC)-ATO derived SP8 population was pelleted for RNA extraction


title: ESC (iPSC)-ATO CD8SP sample 1

source_name: human embryonic stem cells-derived (iPSC line)


title: BS.170_B3_Post-SAM_MAM2


title: BS.170_B2_Post-SAM_MAM1


title: B75


title: B72


title: ESC (TCR)-ATO CD8SP sample 3

description:
Mature T cell population generated in ATOs from hematopoietic differentiation of human embryonic stem cells
GCAMhsExp.GEO.txt

source_name: human embryonic stem cells-derived (H1 line)

growth_protocol:
PSC (TCR)-ATOs were generated using purified hEMPs and followed a two-step procedure including a hematopoietic induction phase and a T-cell differentiation phase.  MS5-hDLL4 cells were harvested by trypsinization and resuspended in hematopoietic induction medium composed of EGM2 (Lonza Ref CC-4176) supplemented with ROCK inhibitor Y-27632 dihydrochloride (10uM) (Tocris Bioscience, Cat. 1254) and TGF-βRI inhibitor SB-431542 (“SB blocker”) (10 uM) (Tocris Bioscience, Cat. 1614). 5x105 MS5-hDLL4 cells were combined with 0.5-1x104 purified hEMP per PSC-ATO in 1.5 ml Eppendorf tubes and centrifuged at 300 g for 5 min at 4˚C in a swinging bucket centrifuge. Multiple (up to 12) PSC-ATO cell volumes were prepared per tube. Supernatants were carefully removed and the cell pellet was resuspended by brief vortexing, and resuspended in hematopoietic induction medium at a volume of 6 µl per PSC-ATO. 6 µl of cells were plated as PSC-ATOs on a 0.4 µm Millicell transwell insert (EMD Millipore, Billerica, MA; Cat. PICM0RG50) (up to 2 PSC-ATOs per insert were plated) and placed in 6-well plates containing 1 ml hematopoietic induction medium per well. Medium was changed completely every 2-3 days for 1 weekM with medium composed of EGM2 with SB blocker (10uM). At day 7, medium was change to EGM2 + SB blocker (10uM) with the hematopoietic cytokines TPO 5ng/ml (Peprotech, Cat. 300-18), FLT3L 5ng/ml (Peprotech, Cat. 300-19), and SCF 50ng/ml (Peprotech, Cat. 300-07). This medium was changed every 2-3 days for an additional 7 days. At day 14, T cell differentiation was induced by changing the medium to “RB27” (described above) supplemented with 10 ng/ml SCF, 5 ng/ml FLT3L, and 5 ng/ml IL-7 (Peprotech, Cat. 200-07). Medium was changed completely every 3-4 days. PSC-ATO cells were harvested by adding MACS buffer (PBS/0.5% bovine serum album/2mM EDTA) to each well and briefly disaggregating the ATO by pipetting with a 1 ml “P1000” pipet, followed by passage through a 50 µm nylon strainer. Flow cytometry cell sorting of PSC-ATO derived SP8 T cell population used the following surface phenotypes: CD8 single positive (CD8SP; TCRαβ+CD3+CD8α+CD4-CD45RA+)
The hESC line H1 (WiCell, Madison, WI) was maintained on Matrigel coated plates (growth factor reduced, no phenol red; BD Biosciences, San Jose, CA), and cultured in mTESR medium (Stem Cell Technologies, Vancouver, BC).  PSC-ATOs were generated using purified hEMPs and followed a two-step procedure including a hematopoietic induction phase and a T-cell differentiation phase.  MS5-hDLL4 cells were harvested by trypsinization and resuspended in hematopoietic induction medium composed of EGM2 (Lonza Ref CC-4176) supplemented with ROCK inhibitor Y-27632 dihydrochloride (10uM) (Tocris Bioscience, Cat. 1254) and TGF-βRI inhibitor SB-431542 (“SB blocker”) (10 uM) (Tocris Bioscience, Cat. 1614). 5x105 MS5-hDLL4 cells were combined with 0.5-1x104 purified hEMP per PSC-ATO in 1.5 ml Eppendorf tubes and centrifuged at 300 g for 5 min at 4˚C in a swinging bucket centrifuge. Multiple (up to 12) PSC-ATO cell volumes were prepared per tube. Supernatants were carefully removed and the cell pellet was resuspended by brief vortexing, and resuspended in hematopoietic induction medium at a volume of 6 µl per PSC-ATO. 6 µl of cells were plated as PSC-ATOs on a 0.4 µm Millicell transwell insert (EMD Millipore, Billerica, MA; Cat. PICM0RG50) (up to 2 PSC-ATOs per insert were plated) and placed in 6-well plates containing 1 ml hematopoietic induction medium per well. Medium was changed completely every 2-3 days for 1 weekM with medium composed of EGM2 with SB blocker (10uM). At day 7, medium was change to EGM2 + SB blocker (10uM) with the hematopoietic cytokines TPO 5ng/ml (Peprotech, Cat. 300-18), FLT3L 5ng/ml (Peprotech, Cat. 300-19), and SCF 50ng/ml (Peprotech, Cat. 300-07). This medium was changed every 2-3 days for an additional 7 days. At day 14, T cell differentiation was induced by changing the medium to “RB27” (described above) supplemented with 10 ng/ml SCF, 5 ng/ml FLT3L, and 5 ng/ml IL-7 (Peprotech, Cat. 200-07). Medium was changed completely every 3-4 days. PSC-ATO cells were harvested by adding MACS buffer (PBS/0.5% bovine serum album/2mM EDTA) to each well and briefly disaggregating the ATO by pipetting with a 1 ml “P1000” pipet, followed by passage through a 50 µm nylon strainer. Flow cytometry cell sorting of ESC-ATO derived  double positive cell population used the following surface phenotypes: DP; CD4+CD8+CD3-. Cord blood ATOs were generated as previously described (Seet et al, 2017, Protocol exchange). MS5-hDLL1  cells were harvested by trypsinization and resuspended in serum free ATO culture medium (“RB27”) composed of RPMI 1640 (Corning, Manassas, VA), 4% B27 supplement (ThermoFisher Scientific, Grand Island, NY), 30 µM L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate (Sigma-Aldrich, St. Louis, MO) reconstituted in PBS, 1% penicillin/streptomycin (Gemini Bio-Products, West Sacramento, CA), 1% Glutamax (ThermoFisher Scientific, Grand Island, NY), 5 ng/ml rhFLT3L and 5 ng/ml rhIL-7 (Peprotech, Rocky Hill, NJ). RB27 was made fresh weekly. 1.5x105 MS5-hDLL1 cells were combined with 5x103 purified CD34+CD3- cells per ATO in 1.5 ml Eppendorf tubes and centrifuged at 300 g for 5 min at 4˚C in a swinging bucket centrifuge. Supernatants were carefully removed, and the cell pellet was resuspended by brief vortexing. ATOs were plated on a 0.4 µm Millicell transwell insert (EMD Millipore, Billerica, MA; Cat. PICM0RG50) placed in a 6-well plate containing 1 ml RB27 per well. Medium was changed completely every 3-4 days by aspiration from around the cell insert followed by replacement with 1 ml with fresh RB27/cytokines. ATO cells were harvested by adding FACS buffer (PBS/0.5% bovine serum album/2mM EDTA) to each well and briefly disaggregating the ATO by pipetting with a 1 ml “P1000” pipet, followed by passage through a 50 µm nylon strainer. Flow cytometry cell sorting of CB-ATO derived double positive cell population used the following surface phenotypes: DP; CD4+CD8+CD3-. Postnatal human thymi were obtained under IRB exemption as discarded waste from patients undergoing cardiac surgery at Children’s Hospital Los Angeles (CHLA). Thymic fragments were finely dissected in RPMI and disrupted by pipetting to release thymocytes into suspension, followed by passage through a 70 µm nylon strainer. Cells were analyzed fresh on the same or following day. Flow cytometry cell sorting of thymic SP4 T cell population used the following surface phenotypes: CD4 single positive (CD4SP; TCRαβ+CD3+CD8+CD4-CD45RA+).

treatment_protocol: The freshly sorted PSC (TCR)-ATO derived SP8 population was pelleted for RNA extraction


title: BS.170_B1_SAM


title: B70


title: ESC (TCR)-ATO CD8SP sample 1


title: B69


title: BS.167_B1_SAM


title: BS.159_B1_SAM


title: ESC-ATO CD4SP sample 3

growth_protocol:
PSC-ATOs were generated using purified hEMPs and followed a two-step procedure including a hematopoietic induction phase and a T-cell differentiation phase.  MS5-hDLL4 cells were harvested by trypsinization and resuspended in hematopoietic induction medium composed of EGM2 (Lonza Ref CC-4176) supplemented with ROCK inhibitor Y-27632 dihydrochloride (10uM) (Tocris Bioscience, Cat. 1254) and TGF-βRI inhibitor SB-431542 (“SB blocker”) (10 uM) (Tocris Bioscience, Cat. 1614). 5x105 MS5-hDLL4 cells were combined with 0.5-1x104 purified hEMP per PSC-ATO in 1.5 ml Eppendorf tubes and centrifuged at 300 g for 5 min at 4˚C in a swinging bucket centrifuge. Multiple (up to 12) PSC-ATO cell volumes were prepared per tube. Supernatants were carefully removed and the cell pellet was resuspended by brief vortexing, and resuspended in hematopoietic induction medium at a volume of 6 µl per PSC-ATO. 6 µl of cells were plated as PSC-ATOs on a 0.4 µm Millicell transwell insert (EMD Millipore, Billerica, MA; Cat. PICM0RG50) (up to 2 PSC-ATOs per insert were plated) and placed in 6-well plates containing 1 ml hematopoietic induction medium per well. Medium was changed completely every 2-3 days for 1 weekM with medium composed of EGM2 with SB blocker (10uM). At day 7, medium was change to EGM2 + SB blocker (10uM) with the hematopoietic cytokines TPO 5ng/ml (Peprotech, Cat. 300-18), FLT3L 5ng/ml (Peprotech, Cat. 300-19), and SCF 50ng/ml (Peprotech, Cat. 300-07). This medium was changed every 2-3 days for an additional 7 days. At day 14, T cell differentiation was induced by changing the medium to “RB27” (described above) supplemented with 10 ng/ml SCF, 5 ng/ml FLT3L, and 5 ng/ml IL-7 (Peprotech, Cat. 200-07). Medium was changed completely every 3-4 days. PSC-ATO cells were harvested by adding MACS buffer (PBS/0.5% bovine serum album/2mM EDTA) to each well and briefly disaggregating the ATO by pipetting with a 1 ml “P1000” pipet, followed by passage through a 50 µm nylon strainer. Flow cytometry cell sorting of thymic SP4 T cell population used the following surface phenotypes: CD4 single positive (CD4SP; TCRαβ+CD3+CD8+CD4-CD45RA+).
The hESC line H1 (WiCell, Madison, WI) was maintained on Matrigel coated plates (growth factor reduced, no phenol red; BD Biosciences, San Jose, CA), and cultured in mTESR medium (Stem Cell Technologies, Vancouver, BC).  PSC-ATOs were generated using purified hEMPs and followed a two-step procedure including a hematopoietic induction phase and a T-cell differentiation phase.  MS5-hDLL4 cells were harvested by trypsinization and resuspended in hematopoietic induction medium composed of EGM2 (Lonza Ref CC-4176) supplemented with ROCK inhibitor Y-27632 dihydrochloride (10uM) (Tocris Bioscience, Cat. 1254) and TGF-βRI inhibitor SB-431542 (“SB blocker”) (10 uM) (Tocris Bioscience, Cat. 1614). 5x105 MS5-hDLL4 cells were combined with 0.5-1x104 purified hEMP per PSC-ATO in 1.5 ml Eppendorf tubes and centrifuged at 300 g for 5 min at 4˚C in a swinging bucket centrifuge. Multiple (up to 12) PSC-ATO cell volumes were prepared per tube. Supernatants were carefully removed and the cell pellet was resuspended by brief vortexing, and resuspended in hematopoietic induction medium at a volume of 6 µl per PSC-ATO. 6 µl of cells were plated as PSC-ATOs on a 0.4 µm Millicell transwell insert (EMD Millipore, Billerica, MA; Cat. PICM0RG50) (up to 2 PSC-ATOs per insert were plated) and placed in 6-well plates containing 1 ml hematopoietic induction medium per well. Medium was changed completely every 2-3 days for 1 weekM with medium composed of EGM2 with SB blocker (10uM). At day 7, medium was change to EGM2 + SB blocker (10uM) with the hematopoietic cytokines TPO 5ng/ml (Peprotech, Cat. 300-18), FLT3L 5ng/ml (Peprotech, Cat. 300-19), and SCF 50ng/ml (Peprotech, Cat. 300-07). This medium was changed every 2-3 days for an additional 7 days. At day 14, T cell differentiation was induced by changing the medium to “RB27” (described above) supplemented with 10 ng/ml SCF, 5 ng/ml FLT3L, and 5 ng/ml IL-7 (Peprotech, Cat. 200-07). Medium was changed completely every 3-4 days. PSC-ATO cells were harvested by adding MACS buffer (PBS/0.5% bovine serum album/2mM EDTA) to each well and briefly disaggregating the ATO by pipetting with a 1 ml “P1000” pipet, followed by passage through a 50 µm nylon strainer. Flow cytometry cell sorting of ESC-ATO derived  double positive cell population used the following surface phenotypes: DP; CD4+CD8+CD3-. Cord blood ATOs were generated as previously described (Seet et al, 2017, Protocol exchange). MS5-hDLL1  cells were harvested by trypsinization and resuspended in serum free ATO culture medium (“RB27”) composed of RPMI 1640 (Corning, Manassas, VA), 4% B27 supplement (ThermoFisher Scientific, Grand Island, NY), 30 µM L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate (Sigma-Aldrich, St. Louis, MO) reconstituted in PBS, 1% penicillin/streptomycin (Gemini Bio-Products, West Sacramento, CA), 1% Glutamax (ThermoFisher Scientific, Grand Island, NY), 5 ng/ml rhFLT3L and 5 ng/ml rhIL-7 (Peprotech, Rocky Hill, NJ). RB27 was made fresh weekly. 1.5x105 MS5-hDLL1 cells were combined with 5x103 purified CD34+CD3- cells per ATO in 1.5 ml Eppendorf tubes and centrifuged at 300 g for 5 min at 4˚C in a swinging bucket centrifuge. Supernatants were carefully removed, and the cell pellet was resuspended by brief vortexing. ATOs were plated on a 0.4 µm Millicell transwell insert (EMD Millipore, Billerica, MA; Cat. PICM0RG50) placed in a 6-well plate containing 1 ml RB27 per well. Medium was changed completely every 3-4 days by aspiration from around the cell insert followed by replacement with 1 ml with fresh RB27/cytokines. ATO cells were harvested by adding FACS buffer (PBS/0.5% bovine serum album/2mM EDTA) to each well and briefly disaggregating the ATO by pipetting with a 1 ml “P1000” pipet, followed by passage through a 50 µm nylon strainer. Flow cytometry cell sorting of CB-ATO derived double positive cell population used the following surface phenotypes: DP; CD4+CD8+CD3-. Postnatal human thymi were obtained under IRB exemption as discarded waste from patients undergoing cardiac surgery at Children’s Hospital Los Angeles (CHLA). Thymic fragments were finely dissected in RPMI and disrupted by pipetting to release thymocytes into suspension, followed by passage through a 70 µm nylon strainer. Cells were analyzed fresh on the same or following day. Flow cytometry cell sorting of thymic SP4 T cell population used the following surface phenotypes: CD4 single positive (CD4SP; TCRαβ+CD3+CD8+CD4-CD45RA+).

treatment_protocol: The freshly sorted PSC-ATO derived SP4 population was pelleted for RNA extraction


title: B66-B223


title: B73


title: B64


title: B59


title: BS.154_B2_Post-SAM_MAM1


title: ESC-ATO CD4SP sample 1


title: ESC-ATO CD8SP sample 3

growth_protocol:
PSC-ATOs were generated using purified hEMPs and followed a two-step procedure including a hematopoietic induction phase and a T-cell differentiation phase.  MS5-hDLL4 cells were harvested by trypsinization and resuspended in hematopoietic induction medium composed of EGM2 (Lonza Ref CC-4176) supplemented with ROCK inhibitor Y-27632 dihydrochloride (10uM) (Tocris Bioscience, Cat. 1254) and TGF-βRI inhibitor SB-431542 (“SB blocker”) (10 uM) (Tocris Bioscience, Cat. 1614). 5x105 MS5-hDLL4 cells were combined with 0.5-1x104 purified hEMP per PSC-ATO in 1.5 ml Eppendorf tubes and centrifuged at 300 g for 5 min at 4˚C in a swinging bucket centrifuge. Multiple (up to 12) PSC-ATO cell volumes were prepared per tube. Supernatants were carefully removed and the cell pellet was resuspended by brief vortexing, and resuspended in hematopoietic induction medium at a volume of 6 µl per PSC-ATO. 6 µl of cells were plated as PSC-ATOs on a 0.4 µm Millicell transwell insert (EMD Millipore, Billerica, MA; Cat. PICM0RG50) (up to 2 PSC-ATOs per insert were plated) and placed in 6-well plates containing 1 ml hematopoietic induction medium per well. Medium was changed completely every 2-3 days for 1 weekM with medium composed of EGM2 with SB blocker (10uM). At day 7, medium was change to EGM2 + SB blocker (10uM) with the hematopoietic cytokines TPO 5ng/ml (Peprotech, Cat. 300-18), FLT3L 5ng/ml (Peprotech, Cat. 300-19), and SCF 50ng/ml (Peprotech, Cat. 300-07). This medium was changed every 2-3 days for an additional 7 days. At day 14, T cell differentiation was induced by changing the medium to “RB27” (described above) supplemented with 10 ng/ml SCF, 5 ng/ml FLT3L, and 5 ng/ml IL-7 (Peprotech, Cat. 200-07). Medium was changed completely every 3-4 days. PSC-ATO cells were harvested by adding MACS buffer (PBS/0.5% bovine serum album/2mM EDTA) to each well and briefly disaggregating the ATO by pipetting with a 1 ml “P1000” pipet, followed by passage through a 50 µm nylon strainer. Flow cytometry cell sorting of PSC-ATO derived SP8 T cell population used the following surface phenotypes: CD8 single positive (CD8SP; TCRαβ+CD3+CD8α+CD4-CD45RA+)
The hESC line H1 (WiCell, Madison, WI) was maintained on Matrigel coated plates (growth factor reduced, no phenol red; BD Biosciences, San Jose, CA), and cultured in mTESR medium (Stem Cell Technologies, Vancouver, BC).  PSC-ATOs were generated using purified hEMPs and followed a two-step procedure including a hematopoietic induction phase and a T-cell differentiation phase.  MS5-hDLL4 cells were harvested by trypsinization and resuspended in hematopoietic induction medium composed of EGM2 (Lonza Ref CC-4176) supplemented with ROCK inhibitor Y-27632 dihydrochloride (10uM) (Tocris Bioscience, Cat. 1254) and TGF-βRI inhibitor SB-431542 (“SB blocker”) (10 uM) (Tocris Bioscience, Cat. 1614). 5x105 MS5-hDLL4 cells were combined with 0.5-1x104 purified hEMP per PSC-ATO in 1.5 ml Eppendorf tubes and centrifuged at 300 g for 5 min at 4˚C in a swinging bucket centrifuge. Multiple (up to 12) PSC-ATO cell volumes were prepared per tube. Supernatants were carefully removed and the cell pellet was resuspended by brief vortexing, and resuspended in hematopoietic induction medium at a volume of 6 µl per PSC-ATO. 6 µl of cells were plated as PSC-ATOs on a 0.4 µm Millicell transwell insert (EMD Millipore, Billerica, MA; Cat. PICM0RG50) (up to 2 PSC-ATOs per insert were plated) and placed in 6-well plates containing 1 ml hematopoietic induction medium per well. Medium was changed completely every 2-3 days for 1 weekM with medium composed of EGM2 with SB blocker (10uM). At day 7, medium was change to EGM2 + SB blocker (10uM) with the hematopoietic cytokines TPO 5ng/ml (Peprotech, Cat. 300-18), FLT3L 5ng/ml (Peprotech, Cat. 300-19), and SCF 50ng/ml (Peprotech, Cat. 300-07). This medium was changed every 2-3 days for an additional 7 days. At day 14, T cell differentiation was induced by changing the medium to “RB27” (described above) supplemented with 10 ng/ml SCF, 5 ng/ml FLT3L, and 5 ng/ml IL-7 (Peprotech, Cat. 200-07). Medium was changed completely every 3-4 days. PSC-ATO cells were harvested by adding MACS buffer (PBS/0.5% bovine serum album/2mM EDTA) to each well and briefly disaggregating the ATO by pipetting with a 1 ml “P1000” pipet, followed by passage through a 50 µm nylon strainer. Flow cytometry cell sorting of ESC-ATO derived  double positive cell population used the following surface phenotypes: DP; CD4+CD8+CD3-. Cord blood ATOs were generated as previously described (Seet et al, 2017, Protocol exchange). MS5-hDLL1  cells were harvested by trypsinization and resuspended in serum free ATO culture medium (“RB27”) composed of RPMI 1640 (Corning, Manassas, VA), 4% B27 supplement (ThermoFisher Scientific, Grand Island, NY), 30 µM L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate (Sigma-Aldrich, St. Louis, MO) reconstituted in PBS, 1% penicillin/streptomycin (Gemini Bio-Products, West Sacramento, CA), 1% Glutamax (ThermoFisher Scientific, Grand Island, NY), 5 ng/ml rhFLT3L and 5 ng/ml rhIL-7 (Peprotech, Rocky Hill, NJ). RB27 was made fresh weekly. 1.5x105 MS5-hDLL1 cells were combined with 5x103 purified CD34+CD3- cells per ATO in 1.5 ml Eppendorf tubes and centrifuged at 300 g for 5 min at 4˚C in a swinging bucket centrifuge. Supernatants were carefully removed, and the cell pellet was resuspended by brief vortexing. ATOs were plated on a 0.4 µm Millicell transwell insert (EMD Millipore, Billerica, MA; Cat. PICM0RG50) placed in a 6-well plate containing 1 ml RB27 per well. Medium was changed completely every 3-4 days by aspiration from around the cell insert followed by replacement with 1 ml with fresh RB27/cytokines. ATO cells were harvested by adding FACS buffer (PBS/0.5% bovine serum album/2mM EDTA) to each well and briefly disaggregating the ATO by pipetting with a 1 ml “P1000” pipet, followed by passage through a 50 µm nylon strainer. Flow cytometry cell sorting of CB-ATO derived double positive cell population used the following surface phenotypes: DP; CD4+CD8+CD3-. Postnatal human thymi were obtained under IRB exemption as discarded waste from patients undergoing cardiac surgery at Children’s Hospital Los Angeles (CHLA). Thymic fragments were finely dissected in RPMI and disrupted by pipetting to release thymocytes into suspension, followed by passage through a 70 µm nylon strainer. Cells were analyzed fresh on the same or following day. Flow cytometry cell sorting of thymic SP4 T cell population used the following surface phenotypes: CD4 single positive (CD4SP; TCRαβ+CD3+CD8+CD4-CD45RA+).

treatment_protocol: The freshly sorted PSC-ATO derived SP8 population was pelleted for RNA extraction


title: ESC (TCR)-ATO CD8SP sample 2


title: BS.154_B1_SAM


title: BS.158_B1_SAM


title: BS.154_B3_Post-SAM_MAM2


title: ESC-ATO CD8SP sample 2


title: ESC-ATO CD4SP sample 2


title: B50


title: ESC-ATO CD8SP sample 1


title: B5


title: ESC-ATO DP early sample 3

description:
immature T cell population generated in ATOs from hematopoietic differentiation of human embryonic stem cells
GCAMhsExp.GEO.txt

growth_protocol:
PSC-ATOs were generated using purified hEMPs and followed a two-step procedure including a hematopoietic induction phase and a T-cell differentiation phase.  MS5-hDLL4 cells were harvested by trypsinization and resuspended in hematopoietic induction medium composed of EGM2 (Lonza Ref CC-4176) supplemented with ROCK inhibitor Y-27632 dihydrochloride (10uM) (Tocris Bioscience, Cat. 1254) and TGF-βRI inhibitor SB-431542 (“SB blocker”) (10 uM) (Tocris Bioscience, Cat. 1614). 5x105 MS5-hDLL4 cells were combined with 0.5-1x104 purified hEMP per PSC-ATO in 1.5 ml Eppendorf tubes and centrifuged at 300 g for 5 min at 4˚C in a swinging bucket centrifuge. Multiple (up to 12) PSC-ATO cell volumes were prepared per tube. Supernatants were carefully removed and the cell pellet was resuspended by brief vortexing, and resuspended in hematopoietic induction medium at a volume of 6 µl per PSC-ATO. 6 µl of cells were plated as PSC-ATOs on a 0.4 µm Millicell transwell insert (EMD Millipore, Billerica, MA; Cat. PICM0RG50) (up to 2 PSC-ATOs per insert were plated) and placed in 6-well plates containing 1 ml hematopoietic induction medium per well. Medium was changed completely every 2-3 days for 1 weekM with medium composed of EGM2 with SB blocker (10uM). At day 7, medium was change to EGM2 + SB blocker (10uM) with the hematopoietic cytokines TPO 5ng/ml (Peprotech, Cat. 300-18), FLT3L 5ng/ml (Peprotech, Cat. 300-19), and SCF 50ng/ml (Peprotech, Cat. 300-07). This medium was changed every 2-3 days for an additional 7 days. At day 14, T cell differentiation was induced by changing the medium to “RB27” (described above) supplemented with 10 ng/ml SCF, 5 ng/ml FLT3L, and 5 ng/ml IL-7 (Peprotech, Cat. 200-07). Medium was changed completely every 3-4 days. PSC-ATO cells were harvested by adding MACS buffer (PBS/0.5% bovine serum album/2mM EDTA) to each well and briefly disaggregating the ATO by pipetting with a 1 ml “P1000” pipet, followed by passage through a 50 µm nylon strainer. Flow cytometry cell sorting of ESC-ATO derived  double positive cell population used the following surface phenotypes: DP; CD4+CD8+CD3-.
The hESC line H1 (WiCell, Madison, WI) was maintained on Matrigel coated plates (growth factor reduced, no phenol red; BD Biosciences, San Jose, CA), and cultured in mTESR medium (Stem Cell Technologies, Vancouver, BC).  PSC-ATOs were generated using purified hEMPs and followed a two-step procedure including a hematopoietic induction phase and a T-cell differentiation phase.  MS5-hDLL4 cells were harvested by trypsinization and resuspended in hematopoietic induction medium composed of EGM2 (Lonza Ref CC-4176) supplemented with ROCK inhibitor Y-27632 dihydrochloride (10uM) (Tocris Bioscience, Cat. 1254) and TGF-βRI inhibitor SB-431542 (“SB blocker”) (10 uM) (Tocris Bioscience, Cat. 1614). 5x105 MS5-hDLL4 cells were combined with 0.5-1x104 purified hEMP per PSC-ATO in 1.5 ml Eppendorf tubes and centrifuged at 300 g for 5 min at 4˚C in a swinging bucket centrifuge. Multiple (up to 12) PSC-ATO cell volumes were prepared per tube. Supernatants were carefully removed and the cell pellet was resuspended by brief vortexing, and resuspended in hematopoietic induction medium at a volume of 6 µl per PSC-ATO. 6 µl of cells were plated as PSC-ATOs on a 0.4 µm Millicell transwell insert (EMD Millipore, Billerica, MA; Cat. PICM0RG50) (up to 2 PSC-ATOs per insert were plated) and placed in 6-well plates containing 1 ml hematopoietic induction medium per well. Medium was changed completely every 2-3 days for 1 weekM with medium composed of EGM2 with SB blocker (10uM). At day 7, medium was change to EGM2 + SB blocker (10uM) with the hematopoietic cytokines TPO 5ng/ml (Peprotech, Cat. 300-18), FLT3L 5ng/ml (Peprotech, Cat. 300-19), and SCF 50ng/ml (Peprotech, Cat. 300-07). This medium was changed every 2-3 days for an additional 7 days. At day 14, T cell differentiation was induced by changing the medium to “RB27” (described above) supplemented with 10 ng/ml SCF, 5 ng/ml FLT3L, and 5 ng/ml IL-7 (Peprotech, Cat. 200-07). Medium was changed completely every 3-4 days. PSC-ATO cells were harvested by adding MACS buffer (PBS/0.5% bovine serum album/2mM EDTA) to each well and briefly disaggregating the ATO by pipetting with a 1 ml “P1000” pipet, followed by passage through a 50 µm nylon strainer. Flow cytometry cell sorting of ESC-ATO derived  double positive cell population used the following surface phenotypes: DP; CD4+CD8+CD3-. Cord blood ATOs were generated as previously described (Seet et al, 2017, Protocol exchange). MS5-hDLL1  cells were harvested by trypsinization and resuspended in serum free ATO culture medium (“RB27”) composed of RPMI 1640 (Corning, Manassas, VA), 4% B27 supplement (ThermoFisher Scientific, Grand Island, NY), 30 µM L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate (Sigma-Aldrich, St. Louis, MO) reconstituted in PBS, 1% penicillin/streptomycin (Gemini Bio-Products, West Sacramento, CA), 1% Glutamax (ThermoFisher Scientific, Grand Island, NY), 5 ng/ml rhFLT3L and 5 ng/ml rhIL-7 (Peprotech, Rocky Hill, NJ). RB27 was made fresh weekly. 1.5x105 MS5-hDLL1 cells were combined with 5x103 purified CD34+CD3- cells per ATO in 1.5 ml Eppendorf tubes and centrifuged at 300 g for 5 min at 4˚C in a swinging bucket centrifuge. Supernatants were carefully removed, and the cell pellet was resuspended by brief vortexing. ATOs were plated on a 0.4 µm Millicell transwell insert (EMD Millipore, Billerica, MA; Cat. PICM0RG50) placed in a 6-well plate containing 1 ml RB27 per well. Medium was changed completely every 3-4 days by aspiration from around the cell insert followed by replacement with 1 ml with fresh RB27/cytokines. ATO cells were harvested by adding FACS buffer (PBS/0.5% bovine serum album/2mM EDTA) to each well and briefly disaggregating the ATO by pipetting with a 1 ml “P1000” pipet, followed by passage through a 50 µm nylon strainer. Flow cytometry cell sorting of CB-ATO derived double positive cell population used the following surface phenotypes: DP; CD4+CD8+CD3-. Postnatal human thymi were obtained under IRB exemption as discarded waste from patients undergoing cardiac surgery at Children’s Hospital Los Angeles (CHLA). Thymic fragments were finely dissected in RPMI and disrupted by pipetting to release thymocytes into suspension, followed by passage through a 70 µm nylon strainer. Cells were analyzed fresh on the same or following day. Flow cytometry cell sorting of thymic SP4 T cell population used the following surface phenotypes: CD4 single positive (CD4SP; TCRαβ+CD3+CD8+CD4-CD45RA+).

treatment_protocol: The freshly sorted PSC-ATO derived DP population was pelleted for RNA extraction


title: BS.135_B1_SAM


title: BS.139_B1_SAM


title: BS.129_B1_SAM


title: BS.132_B1_SAM


title: B49


title: ESC-ATO DP early sample 2


title: BS.120_B1_SAM


title: B48


title: BS.121_B1_SAM


title: B46


title: EMP sample 2

description:
Mesodermal progenitor population derived from mesodermal differentiation of human embryonic stem cells
GCAMhsExp.GEO.txt

growth_protocol:
The hESC line H1 (WiCell, Madison, WI) was maintained on Matrigel coated plates (growth factor reduced, no phenol red; BD Biosciences, San Jose, CA), and cultured in mTESR medium (Stem Cell Technologies, Vancouver, BC). To initiate mesoderm differentiation, mTESR medium was replaced with basal induction medium X-Vivo 15 (Lonza, Walkersville, MD). Basal induction medium was supplemented with human growth factors activin A, BMP4, VEGF and bFGF for 3.5 days (all at 10 ng/mL; R&D Systems, Minneapolis, MN), with the inclusion of activin A only on day 1 (“A-BVF” condition published in Evseenko et al. PNAS. 2010). After mesoderm induction, human embryonic mesodermal progenitor (hEMP) was isolated as CD326−CD56+ cells by fluorescence activated cell sorting (FACS) at day 3.5 of differentiation.
The hESC line H1 (WiCell, Madison, WI) was maintained on Matrigel coated plates (growth factor reduced, no phenol red; BD Biosciences, San Jose, CA), and cultured in mTESR medium (Stem Cell Technologies, Vancouver, BC).  PSC-ATOs were generated using purified hEMPs and followed a two-step procedure including a hematopoietic induction phase and a T-cell differentiation phase.  MS5-hDLL4 cells were harvested by trypsinization and resuspended in hematopoietic induction medium composed of EGM2 (Lonza Ref CC-4176) supplemented with ROCK inhibitor Y-27632 dihydrochloride (10uM) (Tocris Bioscience, Cat. 1254) and TGF-βRI inhibitor SB-431542 (“SB blocker”) (10 uM) (Tocris Bioscience, Cat. 1614). 5x105 MS5-hDLL4 cells were combined with 0.5-1x104 purified hEMP per PSC-ATO in 1.5 ml Eppendorf tubes and centrifuged at 300 g for 5 min at 4˚C in a swinging bucket centrifuge. Multiple (up to 12) PSC-ATO cell volumes were prepared per tube. Supernatants were carefully removed and the cell pellet was resuspended by brief vortexing, and resuspended in hematopoietic induction medium at a volume of 6 µl per PSC-ATO. 6 µl of cells were plated as PSC-ATOs on a 0.4 µm Millicell transwell insert (EMD Millipore, Billerica, MA; Cat. PICM0RG50) (up to 2 PSC-ATOs per insert were plated) and placed in 6-well plates containing 1 ml hematopoietic induction medium per well. Medium was changed completely every 2-3 days for 1 weekM with medium composed of EGM2 with SB blocker (10uM). At day 7, medium was change to EGM2 + SB blocker (10uM) with the hematopoietic cytokines TPO 5ng/ml (Peprotech, Cat. 300-18), FLT3L 5ng/ml (Peprotech, Cat. 300-19), and SCF 50ng/ml (Peprotech, Cat. 300-07). This medium was changed every 2-3 days for an additional 7 days. At day 14, T cell differentiation was induced by changing the medium to “RB27” (described above) supplemented with 10 ng/ml SCF, 5 ng/ml FLT3L, and 5 ng/ml IL-7 (Peprotech, Cat. 200-07). Medium was changed completely every 3-4 days. PSC-ATO cells were harvested by adding MACS buffer (PBS/0.5% bovine serum album/2mM EDTA) to each well and briefly disaggregating the ATO by pipetting with a 1 ml “P1000” pipet, followed by passage through a 50 µm nylon strainer. Flow cytometry cell sorting of ESC-ATO derived  double positive cell population used the following surface phenotypes: DP; CD4+CD8+CD3-. Cord blood ATOs were generated as previously described (Seet et al, 2017, Protocol exchange). MS5-hDLL1  cells were harvested by trypsinization and resuspended in serum free ATO culture medium (“RB27”) composed of RPMI 1640 (Corning, Manassas, VA), 4% B27 supplement (ThermoFisher Scientific, Grand Island, NY), 30 µM L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate (Sigma-Aldrich, St. Louis, MO) reconstituted in PBS, 1% penicillin/streptomycin (Gemini Bio-Products, West Sacramento, CA), 1% Glutamax (ThermoFisher Scientific, Grand Island, NY), 5 ng/ml rhFLT3L and 5 ng/ml rhIL-7 (Peprotech, Rocky Hill, NJ). RB27 was made fresh weekly. 1.5x105 MS5-hDLL1 cells were combined with 5x103 purified CD34+CD3- cells per ATO in 1.5 ml Eppendorf tubes and centrifuged at 300 g for 5 min at 4˚C in a swinging bucket centrifuge. Supernatants were carefully removed, and the cell pellet was resuspended by brief vortexing. ATOs were plated on a 0.4 µm Millicell transwell insert (EMD Millipore, Billerica, MA; Cat. PICM0RG50) placed in a 6-well plate containing 1 ml RB27 per well. Medium was changed completely every 3-4 days by aspiration from around the cell insert followed by replacement with 1 ml with fresh RB27/cytokines. ATO cells were harvested by adding FACS buffer (PBS/0.5% bovine serum album/2mM EDTA) to each well and briefly disaggregating the ATO by pipetting with a 1 ml “P1000” pipet, followed by passage through a 50 µm nylon strainer. Flow cytometry cell sorting of CB-ATO derived double positive cell population used the following surface phenotypes: DP; CD4+CD8+CD3-. Postnatal human thymi were obtained under IRB exemption as discarded waste from patients undergoing cardiac surgery at Children’s Hospital Los Angeles (CHLA). Thymic fragments were finely dissected in RPMI and disrupted by pipetting to release thymocytes into suspension, followed by passage through a 70 µm nylon strainer. Cells were analyzed fresh on the same or following day. Flow cytometry cell sorting of thymic SP4 T cell population used the following surface phenotypes: CD4 single positive (CD4SP; TCRαβ+CD3+CD8+CD4-CD45RA+).

treatment_protocol: The freshly sorted hEMP population was pelleted for RNA extraction

value: CD56+ Epcam-


title: EMP sample 3


title: BS.117_B1_SAM


title: B53


title: ESC-ATO DP early sample 1


title: B44


title: BS.096_B1_SAM


title: BS.100_B1_SAM


title: B45


title: B4


title: BS.092_B1_SAM


title: B41


title: B52


title: B39


title: PSC (H1 ESC) sample 1

description:
human embryonic stem cells
GCAMhsExp.GEO.txt

source_name: human embryonic stem cells  (H1 line)

growth_protocol:
The hESC line H1 (WiCell, Madison, WI) was maintained on Matrigel coated plates (growth factor reduced, no phenol red; BD Biosciences, San Jose, CA), and cultured in mTESR medium (Stem Cell Technologies, Vancouver, BC).
The hESC line H1 (WiCell, Madison, WI) was maintained on Matrigel coated plates (growth factor reduced, no phenol red; BD Biosciences, San Jose, CA), and cultured in mTESR medium (Stem Cell Technologies, Vancouver, BC).  PSC-ATOs were generated using purified hEMPs and followed a two-step procedure including a hematopoietic induction phase and a T-cell differentiation phase.  MS5-hDLL4 cells were harvested by trypsinization and resuspended in hematopoietic induction medium composed of EGM2 (Lonza Ref CC-4176) supplemented with ROCK inhibitor Y-27632 dihydrochloride (10uM) (Tocris Bioscience, Cat. 1254) and TGF-βRI inhibitor SB-431542 (“SB blocker”) (10 uM) (Tocris Bioscience, Cat. 1614). 5x105 MS5-hDLL4 cells were combined with 0.5-1x104 purified hEMP per PSC-ATO in 1.5 ml Eppendorf tubes and centrifuged at 300 g for 5 min at 4˚C in a swinging bucket centrifuge. Multiple (up to 12) PSC-ATO cell volumes were prepared per tube. Supernatants were carefully removed and the cell pellet was resuspended by brief vortexing, and resuspended in hematopoietic induction medium at a volume of 6 µl per PSC-ATO. 6 µl of cells were plated as PSC-ATOs on a 0.4 µm Millicell transwell insert (EMD Millipore, Billerica, MA; Cat. PICM0RG50) (up to 2 PSC-ATOs per insert were plated) and placed in 6-well plates containing 1 ml hematopoietic induction medium per well. Medium was changed completely every 2-3 days for 1 weekM with medium composed of EGM2 with SB blocker (10uM). At day 7, medium was change to EGM2 + SB blocker (10uM) with the hematopoietic cytokines TPO 5ng/ml (Peprotech, Cat. 300-18), FLT3L 5ng/ml (Peprotech, Cat. 300-19), and SCF 50ng/ml (Peprotech, Cat. 300-07). This medium was changed every 2-3 days for an additional 7 days. At day 14, T cell differentiation was induced by changing the medium to “RB27” (described above) supplemented with 10 ng/ml SCF, 5 ng/ml FLT3L, and 5 ng/ml IL-7 (Peprotech, Cat. 200-07). Medium was changed completely every 3-4 days. PSC-ATO cells were harvested by adding MACS buffer (PBS/0.5% bovine serum album/2mM EDTA) to each well and briefly disaggregating the ATO by pipetting with a 1 ml “P1000” pipet, followed by passage through a 50 µm nylon strainer. Flow cytometry cell sorting of ESC-ATO derived  double positive cell population used the following surface phenotypes: DP; CD4+CD8+CD3-. Cord blood ATOs were generated as previously described (Seet et al, 2017, Protocol exchange). MS5-hDLL1  cells were harvested by trypsinization and resuspended in serum free ATO culture medium (“RB27”) composed of RPMI 1640 (Corning, Manassas, VA), 4% B27 supplement (ThermoFisher Scientific, Grand Island, NY), 30 µM L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate (Sigma-Aldrich, St. Louis, MO) reconstituted in PBS, 1% penicillin/streptomycin (Gemini Bio-Products, West Sacramento, CA), 1% Glutamax (ThermoFisher Scientific, Grand Island, NY), 5 ng/ml rhFLT3L and 5 ng/ml rhIL-7 (Peprotech, Rocky Hill, NJ). RB27 was made fresh weekly. 1.5x105 MS5-hDLL1 cells were combined with 5x103 purified CD34+CD3- cells per ATO in 1.5 ml Eppendorf tubes and centrifuged at 300 g for 5 min at 4˚C in a swinging bucket centrifuge. Supernatants were carefully removed, and the cell pellet was resuspended by brief vortexing. ATOs were plated on a 0.4 µm Millicell transwell insert (EMD Millipore, Billerica, MA; Cat. PICM0RG50) placed in a 6-well plate containing 1 ml RB27 per well. Medium was changed completely every 3-4 days by aspiration from around the cell insert followed by replacement with 1 ml with fresh RB27/cytokines. ATO cells were harvested by adding FACS buffer (PBS/0.5% bovine serum album/2mM EDTA) to each well and briefly disaggregating the ATO by pipetting with a 1 ml “P1000” pipet, followed by passage through a 50 µm nylon strainer. Flow cytometry cell sorting of CB-ATO derived double positive cell population used the following surface phenotypes: DP; CD4+CD8+CD3-. Postnatal human thymi were obtained under IRB exemption as discarded waste from patients undergoing cardiac surgery at Children’s Hospital Los Angeles (CHLA). Thymic fragments were finely dissected in RPMI and disrupted by pipetting to release thymocytes into suspension, followed by passage through a 70 µm nylon strainer. Cells were analyzed fresh on the same or following day. Flow cytometry cell sorting of thymic SP4 T cell population used the following surface phenotypes: CD4 single positive (CD4SP; TCRαβ+CD3+CD8+CD4-CD45RA+).

treatment_protocol: The freshly sorted H1 ESCs were pelleted for RNA extraction


title: EMP sample 1


title: PSC (H1 ESC) sample 2


title: L40D2

city: Wuhan

first: Hong

last: Meiyan

state: Hubei

address: Xudong 2nd Road

department: Oil Crops Research Institute

country: China

institute: Chinese Academy of Agricultural Sciences

data_processing:
Sequencing-received raw image data were transformed by base calling into sequence data. Low quality reads and reads with adaptors or N% more than 5% were removed before data analysis using the FastQC tool
After filtering, the remaining high-quality reads “clean reads” stored in FASTQ were used for statistical analyses. The clean reads were then mapped to the Zea mays genome using the TopHat2 tool
The genomic localization results of all sequencing reads data were assembled with the cufflinks package, and then were compared to the known genetic models in order to identify any new genes and alternative splicing events using the cuffcompare package
New genes were aligned by BLASTX (E-value <1e−5) to the NCBI nonredundant, GO (http://www.geneontology.org/), Swiss-Prot (http://www.ebi.ac.uk/uniprot/), KOG (http://www.ncbi.nlm.nih.gov/COG/), and KEGG (Kyoto Encyclopedia of Genes and Genomes; http://www.genome.jp/kegg/) protein databases to annotate the functions.
The gene expression level was determined according to the number of fragments per kilobase of exon per million fragments mapped (RPKM).
Genome_build: B73_RefGen_v4
Supplementary_files_format_and_content: excel file include RPKM values for all Samples

organism: Zea mays

source_name: embryo

extract_protocol:
developing seeds were collected manually at 15-, 20-, 25-, 30-, 35- and 40-DAP in triplicate. Seeds were soaked in water overnight at room temperature and embryos were then dissected from their endosperm and frozen immediately using liquid nitrogen and stored at -80 °C until use.
Thirty six libraries representing the embryos from the two genotypes collected at six developmental stages and in three replicates were constructed for transcriptome sequencing. Total RNA was extracted using a TIANGEN RNA Prep Pure Plant kit (Tiangen Biotech Co. Ltd, Beijing, China), and purified with the Dynabeads® Oligo (dT)25 kit (Life, USA). RNA concentration was measured using nanodrop spectrophotometer (Nanodrop Technologies, USA) and integrity was evaluated with the Agilent 2100 Bioanalyzer (Agilent Technologies, USA). The cDNA library was constructed by using a NEBNext® Ultra RNA Library Prep Kit, the quality of retrieved cDNA was checked using the Agilent 2100 Bioanalyzer (Agilent Technologies, USA). After adenylation of 3’ ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 250~300 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then, 3 μl USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37°C for 15 min followed by 5 min at 95 °C before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. Finally, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system. Sequencing was perfomed using an Illumina HiSeq™ 2500 paired-end sequencing system.

tag: line

value: low oil content

tag: developmental stage

value: 40 days after pollination

value: embryo


title: B37


title: BS.079_B3_Post-SAM_MAM2


title: TG84_RUL

type: genomic

city: Lebanon

first: Youdinghuan

state: NH

address: Hinman Box 7650

department: Epidemiology

institute: Dartmouth College

description: BAL sample from healthy, right upper lobe (RUL)

hyb_protocol: The Illumina MethylationEPIC array hybridization and scanning were performed at the University of Southern California Molecular Genomics Core.

data_processing: The minfi R/Bioconductor package (version 1.21) was used to normalize and extract the data.

label: Cy3, Cy5

molecule: genomic DNA

source_name: Healthy, right upper lobe (RUL)

label_protocol: Bisulfite conversion of DNA was performed using the Zymo EZ DNA methylation kit.

extract_protocol: DNA was extracted from lung macrophages via Qiagen DNeasy Blood and Tissue Kit.  DNA was quantitated on a Qubit 3.0 Fluorometer.

value: F

tag: disease status

value: Healthy


title: SAT108_RLL

description: BAL sample from healthy, right lower lobe (RLL)

scan_protocol: The Illumina MethylationEPIC array hybridization and scanning were performed at the University of Southern California Molecular Genomics Core.

source_name: Healthy, right lower lobe (RLL)

value: M


title: TG84_RLL


title: BS.085_B1_SAM

source_name: non-fasted plasma

value: Male


title: RB95_RLL


title: L40D1


title: B36


title: L40D3


title: L35D3

value: 35 days after pollination


title: BS.086_B1_SAM

value: Female


title: B35

value: Biliary Atresia

value: Liver


title: BS.079_B1_SAM


title: Dex-treated (24hrs) X2 (duplicate)

city: Newark

first: Danish

last: Sayed

state: NJ

address: 185 south orange avenue

department: CBMM

institute: Rutgers-NJMS

data_processing:
RNAseq data analysis was done by Active Motif. Data analysis report is attached as Pdf
Directional Poly-A RNA-seq libraries were prepared and sequenced as PE75 (75-bp paired-end reads) on  Illumina NextSeq 500 to a depth of 47-53M read pairs. The “TopHat” algorithm was used to align the reads to the  rn5 genome:  http://ccb.jhu.edu/software/tophat/manual.shtml
The alignments in the BAM files were further analyzed using the Cufflinks suite of programs (running  consecutively: Cufflinks à Cuffcompare à Cuffdiff)  http://cole-trapnell-lab.github.io/cufflinks/manual/
Cufflinks was run using the rn5-genes as a reference database (gtf file). By doing so, the output shows the known  genes with their RNA-Seq metrics (as FPKM = Fragments Per Kilobase of exon model per Million mapped  fragments), but novel transcripts are not identified. The 6 (3 x 2 replicates) cufflinks outputs were compared using  cuffdiff. All cuffdiff output files can be opened and converted to Excel tables. This was done for the most important  table: “gene_exp.diff”: “3514Rutgers_allcomparisons_gene_exp_diff.xlsx”. By default, genes with q-values <0.05  are designated as “significant” (yes in last column), and in this assay with replicates the q- and p-values are now  more meaningful.
The gene_exp.diff table contains all 3 possible pair-wise comparisons. To simplify this table, we generated a  derived table that only contains the 2 comparisons between the DEX-treated samples and the Control sample,  and with corresponding columns arranged next to each other. This file is called  “3514Rutgers_DEXvsCONT_gene_exp_diff.xlsx”. Note that in these tables derived from the gene_exp.diff cuffdiff  output, it is best to ignore entries with “NOTEST” in column “status”, as well as short genes (e.g. noncoding small  RNA genes). Noncoding RNAs are often located inside mRNA genes and the signals are likely derived from the  mRNA gene expression and do not reflect the transcription of the noncoding small RNA gene.
A filtered table with the most differential genes is again included. This time the “significant” columns were used to  only keep genes that have “yes” for at least 1 time point. 737 genes are on this list.
Selected graphics generated from the cuffdiff output with the R bioconductor package cummeRbund are included.  In the Volcano plots, the genes with q-values <0.05 are indicated with red dots. Please consult online  documentation for an interpretation of these plots:  http://compbio.mit.edu/cummeRbund/manual_2_0.html
Complete tophat, cufflinks, cuffcompare and cuffdiff outputs are provided for completeness. Please consult the  manuals in the links above to find descriptions of all output files. Many of the files are not of immediate interest.
Genome_build: rn5

organism: Rattus norvegicus

source_name: Neonatal Rat Ventricular Myocytes

growth_protocol: Neonatal Rat Ventricle myocytes are cultured from 1day old SD rat pups by colageanase and DNAse digestion. Cardiomyocytes are enriched by Percoll gradient and preplating, which removes non-cardiomyocytes. Cardiomyocytes are plated in medium (DMEM-F12, containing high glucose, L-glutamine and HEPES) with 10% FBS. Cardiomyocytes are changed to serum-free medium after 24hrs.

extract_protocol: Cardiomyocytes are scraped and snapped frozen in liquid nitrogen and stored at -140degreeC. Cells were sent to Active Motif for  RNAseq.

treatment_protocol: Cardiomyocytes are treated with Dexamethasone (Sigma) at concentration of 100nM or Ethanol (control)

tag: strain

value: Sprague Dawley

tag: cell type

value: Neonatal Rat Ventricular Myocytes

tag: treatment

value: Dex-treated (24hrs)


title: SAA87_RUL

tag: age

tag: gender


title: DC93_RLL


title: RAH72_RLL


title: B31


title: RB95_RUL


title: SAT108_RUL


title: BS.079_B2_Post-SAM_MAM1

tag: group

tag: wlz

tag: waz


title: SAA87_RLL


title: B3

tag: tissue


title: SH96_RUL


title: L35D1


title: L35D2


title: BS.064_B3_Post-SAM_MAM2

tag: laz


title: B29


title: L30D3

value: 30 days after pollination


title: SH96_RLL


title: CP83_RLL


title: B28


title: BS.064_B2_Post-SAM_MAM1


title: MAP88_RUL


title: MAP88_RLL


title: L30D2


title: BS.058_B1_SAM

value: SAM


title: L30D1


title: input

library_source: genomic

data_processing:
Seq Analysis - 75-nt sequence reads generated by Illumina Seq are mapped to the rn5 genome suing BWA algorithm and stored into BAM format.
Determination of Fragment Density - 5'ends of the aligned reads (=tags) are extended in silico at their 3'ends to length of 150-250bp. To identify the density of fragments along the genome, the genome is divided into 32nt bins and the number of fragments in each bin is determined and stored in bigWig file, which can be used to visualize data on genome browsers.
Peak Finding - Local enrichments of tags is termed intervals. MACS is used to identify the binding sites and signinfncant enrichments of GR to discrete genomic regions
Active Regions - to compare control and Dex tretaed samples, overlapping GR inetrvals are grouped into active regions. In locations where only one sample has an interval, this interval defines the active region.
ChIP-Seq analysis done by Active Motif (Data Explanation file attached in Pdf format)
Genome_build: rn5

extract_protocol:
Cardiomyocytes are scraped and snapped frozen in liquid nitrogen and stored at -140degreeC. Cells were sent to Active Motif for GR-ChIP-seq and RNAseq.
ChIP, Library Construction and Illumina sequencing performed by Active Motif

tag: assay performed

value: Gr-ChIP-Seq

tag: antibody


title: CK90_RUL


title: RAH72_RUL


title: BS.064_B1_SAM


title: Dex-treated (1hr)

value: Anti-GR (Santa Cruz, cat# sc8992)


title: BS.018_B3_Post-SAM_MAM2


title: Control (Ethanol) X2 (duplicate)

value: Control (Ethanol)


title: L25D3

value: low oil content

value: 25 days after pollination

value: embryo


title: B247

tag: disease state


title: L25D2


title: CP83_RUL

value: M

value: Healthy


title: L25D1


title: BS.018_B1_SAM

value: Male


title: CK90_RLL

value: F


title: CRFK cells_C02

city: Muang

first: Phongsakorn

last: Chuammitri

state: Chiang Mai

address: Cholprathan Road

department: Veterinary Biosciences

country: Thailand

institute: Chiang Mai Universtiy

description: 50 bp single-end reads

data_processing:
Raw data (raw reads) of fastq format were firstly processed through custom perl and python scripts. In this step, clean data (clean reads) were obtained by removing reads containing poly-N, with 5ʹ adapter contaminants, without 3ʹ adapter or the insert tag, containing ploy A or T or G or C and low quality reads from raw data. The Q20, Q30, and GC-content of the raw data were calculated
The small RNA tags were mapped to reference sequence (Mus musculus mm10) by Bowtie without mismatch to analyze their expression and distribution on the reference.
Mapped small RNA tags were used to looking for known miRNA. miRBase 21.0 was used as reference, modified software miRDeep2 and sRNA-tools-cli was used to obtain the potential miRNA and draw the secondary structures.
To remove tags originating from protein-coding genes, repeat sequences, rRNA, tRNA, snRNA, and snoRNA, small RNA tags were mapped to RepeatMasker, Rfam database or those types of data from the specified species itself.
The characteristics of the hairpin structure of miRNA precursor can be used to predict novel miRNA. The available software miREvo (Wen et al., 2012) and miRDeep2 (Friedländer et al., 2011) were integrated to predict novel miRNA through exploring the secondary structure, the Dicer cleavage site and the minimum free energy (MFE) of the small RNA tags unannotated in the former steps. At the same time, custom scripts were used to obtain the identified miRNA counts as well as base bias on the first position with a certain length and on each position of all identified miRNA, respectively.
To make every unique small RNA mapped to only one annotation, we follow the following priority rule: known miRNA > rRNA > tRNA > snRNA > snoRNA > repeat > gene > NAT-siRNA > gene > novel miRNA > ta-siRNA. The total rRNA proportion was used a marker as sample quality indicator. Usually it should be less than 40% in animal samples as high quality.
In our analysis pipeline, miRNAs, which might have base edited could be detected by aligning all the sRNA tags to mature miRNA, allowing one mismatch.
Exploring the occurrence of miRNA families identified from the samples in other species. In our analysis pipeline, known miRNA used miFam.dat (http://www.mirbase.org/ftp.shtml) to look for families; novel miRNA precursor was submitted to Rfam (http://rfam. sanger.ac.uk/search/) to look for Rfam families.
Predicting the target gene of miRNA was performed by miRanda.
miRNA expression levels were estimated by TPM (transcript per million) through the following criteria. Normalization formula: Normalized expression = mapped read count/Total reads x 10^6
Differential expression analysis of two conditions/groups (two biological replicates per condition) were performed using the DESeq R package (1.20.0).
Gene Ontology (GO) enrichment analysis of differentially expressed miRNA genes were implemented by the iDEP.90, PANTHER GO Enrichment Analysis, g:Profiler/g:GOSt/g:Convert, ClueGO and CluePedia Cytoscape plug-in, KEGG, and GOseq R package, in which gene length bias was corrected. GO terms with corrected p-value less than 0.05 were considered significantly enriched by differential expressed genes. KOBAS software was used to test the statistical enrichment of differential expression genes in the KEGG pathways.

organism: Felis catus

source_name: CRFK Feline Kidney Cell line

growth_protocol: CRFK monolayer is cultivated in 25 cm^2 tissue culture flasks (T-25) containing DMEM supplemented with 5% FBS, antibiotic and antifungal in 5% CO2 incubator at 37°C until 80% confluent.

extract_protocol:
After 72 h incubation, total RNA of CRFK infected with CPV-2c Laotian stains (CPV) or control was isolated using RNAzol® RT reagent (Sigma-Aldrich, St. Louis, MO, USA) plus Direct-zol™ RNA Miniprep kit (Zymo Research, Irvine, CA, U.S.A.) as manufacture’s protocol.
A total amount of 3 μg total RNA per sample was used as input material for the small RNA library. Sequencing libraries were generated using NEBNext® Multiplex Small RNA Library Prep Set for Illumina® (New England Biolabs, Ipswich, MA, USA)
The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq SR Cluster Kit v3-cBot-HS (Illumina, San Diego, CA, USA)

treatment_protocol: The 80% confluent CRFK cells were infected with CPV isolated (CPV-2c Laotial isolates) at MOI of 0.1 or PBS. After adsorbed for 1 h at 37°C, the inoculum was removed, and DMEM with 5% FBS was added. Cells were again incubated at 37°C , 5% CO2 for 72 h.

value: Kidney epithelial cells

tag: treatment group

value: Control

tag: virus isolate


title: L20D3

tag: line

tag: developmental stage

value: 20 days after pollination


title: CRFK cells_T01

description: 50 bp single-end reads

value: CPV-2c infection


title: BS.018_B2_Post-SAM_MAM1


title: CRFK cells_T02

description: 50 bp single-end reads


title: BS.011_B3_Post-SAM_MAM2

value: Female


title: BS.011_B2_Post-SAM_MAM1


title: B243

source_name: Liver

value: Biliary Atresia

value: Liver


title: CRFK cells_C01

description: 50 bp single-end reads


title: TMUV_24hpi_3

city: Xi'an

first: Hai

last: Li

address: No.76 Yanta West Road

department: Department of Pathogenic Microbiology and Immunology

institute: Xi'an Jiaotong University

data_processing:
Useful Perl script was used to filter the original raw data, to trim Smart_seq2 public primer sequence from the reads , remove the contaminated reads for adapters, remove the low quality reads and remove the reads whose N base more than 5% for total bases .
Bowtie2 v2.2.3 was used for building the genome index, and clean data was mapped to the reference genome using TopHat v2.0.12.
Transcript assemble, difference expression anlysis was  finished by Cufflinks 2.2.1.
Genome_build: Gallus_gallus-5.0 (Ensembl release 94); Tembusu virus strain Du/CH/LSD/110128 (GenBank: KC136210.1)
Supplementary_files_format_and_content: The BAM (Binary Alignment/Map) file format is a generic and highly compressed format for storing alignments.txt file could use as BAMs in Cuffquant to quantifies gene and transcript expression levels.

organism: Gallus gallus

growth_protocol: HD11 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 units/ml penicillin, 100 μg/ml streptomycin and 2 mM L_glutamine. Cell cultures were incubated at 37°C/5% CO2.

extract_protocol:
Seperated single_cell samples were treated by Smart_Seq2 protocol to get amplified cDNA.
RNA libraries were prepared for sequencing using standard Illumina protocols

treatment_protocol: HD11 cells were infected with TMUV at MOI 1

value: Chicken macrophage


title: B24


title: BS.011_B1_SAM

value: SAM


title: B244


title: B241


title: TMUV_24hpi_2


title: L20D2


title: L20D1


title: B242


title: AE106_RUL

tag: disease status


title: B231


title: L15D3

value: 15 days after pollination


title: TMUV_24hpi_1

tag: cell type


title: L15D2

value: low oil content


title: BS.004_B3_Post-SAM_MAM2


title: L15D1


title: B227


title: BS.004_B1_SAM


title: BS.004_B2_Post-SAM_MAM1


title: B228


title: TMUV_12hpi_3


title: TMUV_12hpi_2


title: B225


title: BS.003_B3_Post-SAM_MAM2


title: TMUV_12hpi_1


title: AE106_RLL


title: Control_24hpi_3


title: H40D2

value: high oil content

value: 40 days after pollination


title: BRW110_RLL

value: M


title: B22


title: B214


title: DC93_RUL

value: Healthy


title: H35D3

value: 35 days after pollination

value: embryo


title: BS.003_B2_Post-SAM_MAM1

value: Male


title: H40D1


title: Control_24hpi_2


title: B212


title: H40D3


title: B1274202G_MDCF1_Treatment


title: H35D2


title: B21


title: B1274201G_MDCF1_Baseline


title: Control_24hpi_1


title: B1273202G_MDCF1_Treatment


title: B208


title: Control_12hpi_3


title: H35D1


title: BS.003_B1_SAM


title: B207


title: Control_12hpi_2


title: H30D3

value: 30 days after pollination


title: B1273201G_MDCF1_Baseline


title: Control_12hpi_1


title: Normal_435_TCF7_input

city: Boston

first: Kornelia

last: Polyak

state: MA

address: 450 Brookline Ave

department: Medical Oncology

institute: Dana-Farber Cancer Institute

description: Input for TCF7 ChIPseq of breast organoids from Normal

data_processing:
ChIPseq aligher, bowtie
ChIPseq peak call, macs2 2.1.1.20160309
motif, homer
CNV analysis, true
Genome_build: hg19
Supplementary_files_format_and_content: raw read counts matrix, BED format

source_name: breast organoids

extract_protocol:
phenol-chloroform extraction for ChIP DNA
Rubicon ThruPLEX DNA-seq Kit

value: female

value: breast organoids

tag: genotype/variation

value: Normal (control)

value: input


title: Normal_309_TCF7_input


title: H25D3

value: 25 days after pollination


title: Dex-treated (1hr) X2 (duplicate)

tag: strain

value: Sprague Dawley

value: Neonatal Rat Ventricular Myocytes

tag: treatment

value: Dex-treated (1hr)


title: H30D2


title: B203

value: Biliary Atresia

value: Liver


title: B206


title: BRCA2_393_TCF7_input

description: Input for TCF7 ChIPseq of breast organoids from BRCA2 mutation carrier

tag: gender

value: BRCA2 mutation


title: Control (Ethanol)

value: Gr-ChIP-Seq

value: Anti-GR (Santa Cruz, cat# sc8992)


title: B20


title: B202


title: BRW110_RUL

tag: age

value: M

value: Healthy


title: BRCA2_156_TCF7_input


title: B250


title: H25D2

value: high oil content

value: embryo


title: B2


title: B1266201G_MDCF1_Baseline

tag: group

value: Female

tag: wlz

tag: waz


title: B1266202G_MDCF1_Treatment

tag: laz


title: B1264202G_MDCF1_Treatment


title: H25D1

tag: tissue


title: B1264201G_MDCF1_Baseline


title: BRCA1_448_TCF7_input

description: Input for TCF7 ChIPseq of breast organoids from BRCA1 mutation carrier

value: female

value: breast organoids

value: BRCA1 mutation

tag: antibody


title: B1253201G_MDCF1_Baseline


title: H30D1

value: 30 days after pollination


title: H20D3

value: 20 days after pollination


title: B191

tag: disease state


title: BRCA1_201_TCF7_input

value: input


title: B190c


title: H20D2


title: B1248202G_MDCF1_Treatment


title: B1253202G_MDCF1_Treatment


title: B19


title: BRCA2_393_TCF7

description: TCF7 ChIPseq of breast organoids from BRCA2 mutation carrier


title: H20D1


title: Normal_435_TCF7

description: TCF7 ChIPseq of breast organoids from Normal

value: Normal (control)


title: B189c

value: Biliary Atresia


title: Normal_309_TCF7


title: H15D1

value: 15 days after pollination


title: B1248201G_MDCF1_Baseline