title: Mettl3 cKO LSK SON rep3

address: 417 68th street

institute: Mamorial Sloan Kettering Cancer Center

description: KS_3_counts columns in the processed data files include its raw gene read counts, raw TE read counts, and alternative splicing event percentages.

data_processing:
For the gene expression analysis, reads were aligned onto the Mouse reference genome (mm10) and the LENTI-SON construct by the tophat software.
For the transposable elements (Tes) expression analysis, adapters were trimmed by Cutadapt v4.1. Reads were aligned onto the Mouse reference genome (mm10) and the LENTI-SON construct by STAR v2.7.10b.
Precomputed RepeatMasker annotations v4.0.5 for all TEs were downloaded from RepeatMasker, which were then used to reconstruct full-length LTR copies with 'one code to find them all v1.0' with the default parameters.
The reconstructed TEs annotation and the mm10 basic genes annotation from GENCODE v25 were merged and was used as the reference database for gene quantification in HTSeq v2.0.2.
For the alternative splicing analysis, adapters were trimmed using Cutadapt v4.1. Reads were analyzed by VAST-TOOLS v2.5.1 with the database Mm2 using the default parameters.
Assembly: mm10
Supplementary files format and content: gene_expression.csv: Tab-delimited text file including the raw gene read counts for each sample.
Supplementary files format and content: te_expression.csv: Tab-delimited text file including the raw TE read counts for each sample.
Supplementary files format and content: as.csv: Tab-delimited text file including the alternative splicing event percentages for each sample.

source_name: mouse bone marrow

growth_protocol: WT and mettl3 cKO mice were used to harvest bone marrow. LSK (Lin-cKit+Sca1+) cells were FACS sorted and placed in SFEM media supplemented with cytokines (50 ng/ml SCF, 10 ng/ml IL-3, and 10 ng/ml IL-6, 10ng/ml TPO and 20 ng/ml FLT3L).

extract_protocol:
Sorted cells were harvested and frozen in TRIzol reagent. RNA was extracted using Phenol-Chloroform.
After PicoGreen quantification and quality control by Agilent BioAnalyzer, library preparation was performed using the SMART-Seq v4 Ultra Low Input RNA Kit.

treatment_protocol: Cells were infected with SON overexpressing lentivirus or empty vector 48hr post infection. GFP+ cells were sorted.

value: mouse bone marrow

value: hematopoietic stem and progenitor cell

value: SON overexpressing


title: mcherry-late-2C_1

status: Public on Dec 07 2023

type: SRA

city: Bad Nauheim

first: Carsten

last: Kuenne

address: Ludwigstrasse 43

department: Bioinformatics

country: Germany

institute: Max Planck Institute for Heart and Lung Research

library_source: transcriptomic

data_processing:
Trimmomatic version 0.39 was employed to trim reads after a quality drop below a mean of Q15 in a window of 5 nucleotides and keeping only filtered reads longer than 15 nucleotides (Bolger et al., Trimmomatic: a flexible trimmer for Illumina sequence data).
Reads were aligned versus Ensembl mouse genome version mm39 (Ensembl release 104) with STAR 2.7.10a (Dobin et al., STAR: ultrafast universal RNA-seq aligner).
Aligned reads were filtered to remove: duplicates with Picard 3.0.0 (Picard: A set of tools (in Java) for working with next generation sequencing data in the BAM format), multi-mapping, ribosomal, or mitochondrial reads.
Gene counts were established with featureCounts 2.0.4 by aggregating reads overlapping exons excluding those overlapping multiple genes (Liao et al., featureCounts: an efficient general purpose program for assigning sequence reads to genomic features).
The raw count matrix was normalized with DESeq2 version 1.36.0 (Love et al., Moderated estimation of fold change and dispersion for RNA-Seq data with DESeq2).
The Ensemble annotation was enriched with UniProt data (Activities at the Universal Protein Resource (UniProt)).
Assembly: mm39
Supplementary files format and content: The tab-delimited matrix shows DESeq normalized gene counts.

molecule: total RNA

organism: Mus musculus

source_name: 2-cell mouse embryo

extract_protocol:
Five embryos were pooled and dispensed into tubes.
SMART-Seq® v4 Ultra® Low Input RNA Kit (Takara Bio) was used following manufacturer’s protocol without RNA extraction and the Nextera XT kit with v2 Full Set indexes (N7-S5) was used for library preparation.Sequencingwas performed on a Nova-seq instrument (Illumina) with2x75bp paired end setup.

tag: strain

tag: cell type

value: 2-cell mouse embryo

tag: genotype

value: WT

tag: treatment

value: injected with 200 ng/µl NLS-mCherry mRNA


title: Early-Resolving COVID associated Pulmonary Fibrosis 6

city: Charlottesville

first: Anwaruddin

last: Mohammad

state: VA

address: Pinn Hall

department: Bioinformatics Core

country: USA

institute: University of Virginia

library_source: transcriptomic single cell

data_processing:
The demultiplexing, barcoded processing and gene counting were made using the Cell Ranger software v6.0 except for ipf1 which uses v4.0
Supplementary files format and content: Tab-separated values files and matrix files

organism: Homo sapiens

source_name: 55 yo Male

extract_protocol:
Briefly, around 10,000 cells were targeted per sample and loaded onto each well of a Chromium Single Cell G Chip to generate single cell emulsions primed for reverse transcription.  After breaking the emulsion, the single cell specific barcoded DNAs were subjected to cDNA amplification and QC on the Agilent 4200 TapeStation Instrument, using the Agilent D5000 kit.  Each sample cDNA was used to prepare indexed libraries that were pooled prior to sequencing.  A quality control run was performed on the Illumina Miseq using the nano 300 cycle kit (1.4 Million reads/run), to estimate the number of targeted cells per sample. The cell estimate enabled the core to re-balance the pooled sample prior to deep sequencing.
The generation of single cell indexed libraries, from the thawed COVID PF and IPF PBMCs, was performed by the School of Medicine Genome Analysis and Technology Core, RRID:SCR_018883, using the 10X Genomics chromium controller platform and the Chromium Single Cell 5′ Library & Gel Bead Kit v1.1 reagent.  Deep sequencing was performed on either the NGS Illumina NextSeq 500 using the 150 cycle kit or NextSeq 2000 using the P3-100 cycle kit.  Control samples were sequenced on the MGISEQ-2000.

tag: tissue

value: blood

value: peripheral blood mononuclear cells

tag: phenotype


title: Established myelodysplastic CMML, sample 11

city: Munich

first: Stephan

last: Hutter

address: Max-Lebsche-Platz 31

institute: MLL Munich Leukemia Laboratory

data_processing:
Data processing was performed within Illumina's BaseSpace plattform using the RNA-seq Alignment app (v2.0.1)
Reads were mapped with STAR aligner (v2.5.0a) to the human reference genome hg19 (RefSeq annotation)
Gene specific read abundance was calculated with Cufflinks (v2.2.1)
Assembly: hg19
Supplementary files format and content: tab-delimited text files contain gene specific read count values for each sample

source_name: Bone marrow aspirate

extract_protocol:
Total RNA was extracted using the MagNA Pure 96 Instrument and the MagNA Pure 96 Cellular RNA LV Kit (Roche LifeScience, Mannheim, Germany)
The TruSeq Total Stranded RNA kit was used, starting with 250ng of total RNA, to generate RNA libraries following the manufacturer’s recommendations (Illumina, San Diego, CA, USA)

value: Bone marrow aspirate

tag: disease state

value: myelodysplastic CMML


title: Early-Resolving COVID associated Pulmonary Fibrosis 5

source_name: 58 yo Male


title: Established myelodysplastic CMML, sample 100


title: mcherry-early-2C_7


title: Mettl3 cKO LSK SON rep1

city: New York

first: Michael

last: Kharas

state: New York

department: Molecular Pharmacology Program

description: KS_1_counts columns in the processed data files include its raw gene read counts, raw TE read counts, and alternative splicing event percentages.

tag: cell line

value: primary cells


title: Early-Resolving COVID associated Pulmonary Fibrosis 4

source_name: 63 yo Male


title: mcherry-early-2C_5


title: Mettl3 cKO LSK GFP rep3

description: KC_3_counts columns in the processed data files include its raw gene read counts, raw TE read counts, and alternative splicing event percentages.

value: empty vector


title: mcherry-early-2C_4


title: Mettl3 cKO LSK GFP rep2

description: KC_2_counts columns in the processed data files include its raw gene read counts, raw TE read counts, and alternative splicing event percentages.


title: Established myelodysplastic CMML, sample 10


title: Late-Resolving COVID associated Pulmonary Fibrosis 5


title: Mettl3 cKO LSK GFP rep1

description: KC_1_counts columns in the processed data files include its raw gene read counts, raw TE read counts, and alternative splicing event percentages.


title: Established myelodysplastic CMML, sample 1


title: Late-Resolving COVID associated Pulmonary Fibrosis 4

source_name: 67 yo Male


title: Established myelodysplastic CMML, sample 13


title: Healthy Control, sample 9

value: healthy


title: CMU1, PBS 24h, 1

city: Genève

first: Clément

last: David

state: Canton de Genève

address: Rue Michel-Servet 1

department: PATIM

country: Switzerland

institute: Université de Genève

description:
CD32_Kpn_Counts.txt
CD32_mouse_counts.txt

data_processing:
Raw sequence reads were filtered using Trimmomatic (v.0.39).
Raw sequence reads were sequentially aligned to the mouse genome to remove mouse-derived sequences and then to the Kp genome using bowtie2
BAM files obtained were analysed using R (v.4.0.3): Reads were assigned to genes using Rsubread package (v.2.4.2)
Reads were then analysed using ‘DeSeq2’ package (v.1.30.0)
GSEA was performed using the package ‘clusterProfiler’ (v.4.6.0)
Assembly: mm10 and
Supplementary files format and content: tab-delimited tables include raw counts affiliated to each sample
Supplementary files format and content: tab-delimited tables include DESeq2 statistical output for each experiment

organism: Klebsiella pneumoniae

source_name: cecum/bacteria

growth_protocol: CR-Kp ST-258 contained in feces samples was frozen at-80°C until RNA extraction. Murine cecal tissues were put at -20°C in RNAlater.

extract_protocol:
CR-Kp ST-258 RNA and murine RNA were isolated from faecal pellets or caecal contents using the RNeasy PowerMicrobiome Kit (Qiagen).
CR-KP ST-258 RNA sample libraries and murine RNA sample libraries  were prepared using the Illumina Stranded Total RNA Prep kit with the Ribo-zero Plus, and they were sequenced using the Illumina NovaSeq 6000 with single read 100bp.

value: cecum/bacteria

value: Klebsiella pneumoniae ST258/intestinal cells

value: gram-negative bacteria/murine cells

tag: agent

value: PBS

tag: time point


title: WT LSK SON rep3

description: WS_3_counts columns in the processed data files include its raw gene read counts, raw TE read counts, and alternative splicing event percentages.

value: mouse bone marrow


title: Mettl3 cKO LSK SON rep2

description: KS_2_counts columns in the processed data files include its raw gene read counts, raw TE read counts, and alternative splicing event percentages.

value: hematopoietic stem and progenitor cell


title: Early-Resolving COVID associated Pulmonary Fibrosis 3

source_name: 66 yo Male


title: WT LSK SON rep2

description: WS_2_counts columns in the processed data files include its raw gene read counts, raw TE read counts, and alternative splicing event percentages.

value: SON overexpressing


title: mcherry-late-1C_7

source_name: 1-cell mouse embryo

value: 1-cell mouse embryo


title: mcherry-early-2C_6

value: 2-cell mouse embryo

value: WT


title: Late-Resolving COVID associated Pulmonary Fibrosis 3

source_name: 54 yo Male

value: blood


title: WT LSK SON rep1

description: WS_1_counts columns in the processed data files include its raw gene read counts, raw TE read counts, and alternative splicing event percentages.


title: mcherry-late-1C_6

value: injected with 200 ng/µl NLS-mCherry mRNA


title: WT LSK GFP rep3

description: WC_3_counts columns in the processed data files include its raw gene read counts, raw TE read counts, and alternative splicing event percentages.


title: Healthy Control, sample 64

value: Bone marrow aspirate


title: mcherry-late-1C_5


title: Healthy Control, sample 63


title: WT LSK GFP rep2

description: WC_2_counts columns in the processed data files include its raw gene read counts, raw TE read counts, and alternative splicing event percentages.


title: mcherry-late-1C_4


title: Healthy Control, sample 62


title: Idiopathic Pulmonary Fibrosis 2

source_name: 56 yo Male

value: peripheral blood mononuclear cells

value: Idiopathic Pulmonary Fibrosis


title: WT LSK GFP rep1

description: WC_1_counts columns in the processed data files include its raw gene read counts, raw TE read counts, and alternative splicing event percentages.

value: primary cells

value: empty vector


title: mcherry-early-2C_3


title: Early-Resolving COVID associated Pulmonary Fibrosis 1

source_name: 48 yo Female


title: Healthy Control, sample 61

value: healthy


title: mcherry-early-2C_1


title: mcherry-late-1C_3


title: mcherry-late-1C_1


title: BL6.O.rep2 (DISE-023)

city: Chicago

first: Marcus

last: Peter

state: IL

address: 303 East Superior Street, Lurie 6-123

institute: Northwestern University Feinberg School of Medicine

data_processing:
Processing was done with the SPOROS pipeline: https://github.com/ebartom/SPOROS
Reads were trimmed with Trim_galore to remove all standard Illumina adapters and trimmed reads less than 6 bp in length
Reads were demultiplexed according to adaptor barcodes to separate into the individual samples listed below.
6 bp UMIs were removed with Trim_galore (4N before pull-down sequence and 2N after).  This resulted in the deposited fastq files.
Cleaned reads were sorted and uniq -c used to identify the total counts of all unique sequences
Reads were blasted against a set of processed miRNAs and the top blast hit retained as a putative source for the read.
Reads with hits against artificial and adapter sequences are removed from the table, as well as reads with a summed count < total number of samples
Supplementary files format and content: Tab-delimited text file with read, seed (bp 2-7), cell viability based on seed, raw read counts for each sample, and putative source based on blast

source_name: 18 months old BL6, brain, cortex and hippoc.

extract_protocol:
Cell lysates were subjected to pull down with a GW182 peptide bound to anti-FLAG M2 Magnetic beads RNA at a size of 19-35 nt was gel extracted and used to generate library
The quality and quantity of the RNA samples was checked using an Agilent bio-analyzer. Small RNA-SEQ libraries were generated using Illumina small RNA SEQ kits using the Illumina provided protocol containing library fragments 140-160 bp in size.
RNA-Seq; sequenced on an Illumina HiSEQ4000 using Illumina provided reagents and protocols

treatment_protocol: RNA was isolated from 250 mg of cortex and hippocampus combined

tag: age

value: 18 months old

value: cortex and hippoc.

tag: fraction

value: Ago bound RNA


title: Late-Resolving COVID associated Pulmonary Fibrosis 2

source_name: 68 yo Male


title: BL6.O.rep1 (DISE-023)


title: Healthy Control, sample 8


title: Late-Resolving COVID associated Pulmonary Fibrosis 1

source_name: 50 yo Male


title: uninjected-late-2C_7


title: Healthy Control, sample 7


title: Healthy Control, sample 60


title: BL6.Y.rep2 (DISE-023)

source_name: 2 months old BL6, brain, cortex and hippoc.

value: 2 months old


title: Healthy Control, sample 6


title: uninjected-late-2C_6


title: Idiopathic Pulmonary Fibrosis 3

source_name: 41 yo Female


title: Early-Resolving COVID associated Pulmonary Fibrosis 2

source_name: 60 yo Male


title: Healthy Control, sample 59


title: BL6.Y.rep1 (DISE-023)


title: Old.18M.mouse.cortexNhippo.rep3 (DISE-022)


title: HeLa-siNSUN2-2-INP

city: anderlecht

first: Haili

last: Ma

address: route de lennik 808

country: Belgium

institute: ULB

data_processing:
Raw sequencing data were first analysed with "FastQC". Low-complexity reads were removed with the "AfterQC" tool with default parameters. To get rid of reads originating from rRNA or tRNA, the reads were mapped to human tRNA and rRNA sequences with "Bowtie2". Reads that did not map to tRNA or rRNA sequences were then further processed with "Trimmomatic" using default parameters to remove adapter sequences. The resulting fastq data were again analysed with "FastQC" to ensure that no further processing was needed.
The processed fastq reads were mapped to the human reference genome (GRCh37/hg19) using the "STAR" algorithm with reference transcriptome includes Ensembl v85 and long noncoding from LNCipedia v5.2. HPB normalised coverage profiles (bedgraph) were generated with the bamTobw tool (https://github.com/YangLab/bamTobw).
Assembly: hg19
Supplementary files format and content: bedgraph
Library strategy: m5C MeRIP-seq

molecule: polyA RNA

source_name: Human HeLa cell line

growth_protocol: HeLa cells were maintained in DMEM (Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin and streptomycin (Pen Strep, Gibco). All cells were cultured at 37°C in a humidified atmosphere containing 5% CO2.

extract_protocol:
(RNA purification) Total RNA was first extracted from siCtrl and siNSUN2 HeLa cells with the RNeasy kit (Qiagen), and then 1000 µg DNA-free total RNA was subjected to poly(A)+RNA enrichment through oligo-dT selection using GenElute™ mRNA Miniprep Kit (Sigma). (m5C-immunoprecipitation) mRNA was fragmented into range 200-300 nt nucleotide-long fragments in RNA fragmentation buffer at 94°C for 18s. Fragmented RNA was then precipitated with ethanol and resuspended in RNase-free water. The amount and size of fragmented RNA was tested using an Invitrogen Qubit Fluorometer and an Agilent 2100 Bioanalyzer with the Qubit RNA Assay kit (Thermo Fisher) and RNA 6000 Nano kit (Agilent), respectively. Then 10-50 ng of fragmented RNA was stored at -80°C to serve as input. The remaining RNA was first denatured at 70°C for 5 min and then incubated overnight at 4°C with 0.5 mg/ml anti-m5C monoclonal antibody (Diagenode #C15200003) on a rotating wheel in IP buffer (50 mM Tris-HCl, 750 mM NaCl, 0.5% Igepal CA-630, RNasin 400 U/ml and ribonucleoside vanadyl complex 2 mM) supplemented with protease inhibitors (cOmplete, Mini, EDTA-free, Roche). The next day, 50 µl of Dynabeads™ Protein G (Invitrogen) were washed three times with IP buffer and blocked by incubating with 0.5 mg/ml BSA in IP buffer for 1 h on a rotating wheel.  The blocked beads were washed twice, then added to the IP mix and incubated for 2 h at 4°C with gentle rotation.  After extensive washing with IP buffer, bound RNA was purified by TriPure Isolation Reagent (Roche) and resuspended in RNase-free water.
The sequence library was prepared with SMARTer® Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian (Takara) for both input and IP samples.

treatment_protocol: HeLa cells were transfected by electroporation with siRNA (control or siNSUN2 siRNA).

value: Human HeLa cell line

tag: knockeddown

tag: library type

value: INPUT

tag: replicate


title: HeLa-siCTRL-1-INP

value: siCtrl


title: Healthy Control, sample 57

source_name: Bone marrow aspirate


title: uninjected-late-2C_3

source_name: 2-cell mouse embryo

value: 2-cell mouse embryo


title: HeLa-siCTRL-2-INP


title: Healthy Control, sample 58


title: HeLa-siNSUN2-1-INP


title: Old.18M.mouse.cortexNhippo.rep2 (DISE-022)


title: uninjected-late-2C_1

value: WT


title: Healthy Control, sample 56


title: HeLa-siCTRL-2-m5C

value: IP


title: Old.18M.mouse.cortexNhippo.rep1 (DISE-022)


title: Healthy Control, sample 55


title: Young.2M.mouse.cortexNhippo.rep3 (DISE-022)


title: HeLa-siNSUN2-2-m5C


title: uninjected-early-2C_6


title: uninjected-early-2C_7


title: HeLa-siCTRL-1-m5C


title: Healthy Control, sample 54


title: Young.2M.mouse.cortexNhippo.rep2 (DISE-022)


title: uninjected-late-2C_5


title: Young.2M.mouse.cortexNhippo.rep1 (DISE-022)


title: Healthy Control, sample 53


title: uninjected-late-2C_4


title: uninjected-early-2C_5


title: HeLa-siNSUN2-1-m5C


title: uninjected-early-2C_4


title: Healthy Control, sample 52


title: uninjected-early-2C_3


title: Healthy Control, sample 50


title: K562 cells, SRSF2P95H, rep2

data_processing:
Paired-end RNA-Seq raw reads were analyzed with FastQC (v0.11.5) and then filtered by AfterQC (v0.9.6) with the default parameters to remove low-complexity reads. The Trimmomatic tool (v0.33) was then used to trim adapter sequences and filter reads smaller than 35 bp. The resulting fastq data were again analysed with FastQC to ensure that no further processing was needed.
The clean reads were aligned to the hg19 genome with the STAR algorithm (v2.6.1d) using the reference transcriptome based on Ensembl v85 and LNCipedia v5.2.
Read count extraction was performed using HTseq tool.
Assembly: hg19
Supplementary files format and content: tab-delimited text files include raw read count for each sample

growth_protocol: HeLa cells were maintained in DMEM (Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin and streptomycin (Pen Strep, Gibco). K562 cells were cultured in IMDM (Gibco) supplemented with 10% FBS and 1% Pen Strep. All cells were cultured at 37 °C in a humidified atmosphere containing 5% CO2.

extract_protocol:
Total RNA was purified with the RNeasy kit (Qiagen) and treated with the RNase-Free DNase Set (Qiagen) to remove the residual DNA. Total RNA samples from HeLa cells was then subjected to rRNA depletion using Ribominus Human/Mouse Transcriptome Isolation Kit (Invitrogen). Enrichment of mRNA from total RNA sample in K562 cells was performed using GenElute™ mRNA Miniprep Kit (Sigma-Aldrich).
RNA libraries for RNA-seq were prepared using KAPA Stranded mRNA-Seq kit according to the manufacturer’s instructions.

treatment_protocol: For transient gene knockdown, HeLa cells were cultured to 50-60% confluency. The cells were then transfected by electroporation with control siRNA (universal negative control) or NSUN2 siRNA (5’-CAC GTG TTC ACT AAA CCC TAT-3’), using the LONZA Kit (VCA-1001, Lonza, Germany) according to the manufacturer’s instructions. After 48 h, the transfected cells were washed in PBS and RNA was extracted.

value: human erythroleukemic cell line

value: SRSF2P95H heterozygous mutant


title: K562 cells, SRSF2P95H, rep1

value: SRSF2P95H heterozygous mutant  SRSF2 P95H heterozygous mutant  P95H heterozygous mutant


title: Healthy Control, sample 5

value: Bone marrow aspirate


title: Healthy Control, sample 49


title: TgTau.rep3 (DISE-031)

source_name: Tau fragment tg brain, mouse #1198, hippoc.

extract_protocol:
Cell lysates were subjected to pull down with a GW182 peptide bound to anti-FLAG M2 Magnetic beads RNA at a size of 19-35 nt was gel extracted and used to generate library
The quality and quantity of the RNA samples was checked using an Agilent bio-analyzer. Small RNA-SEQ libraries were generated using Illumina small RNA SEQ kits using the Illumina provided protocol containing library fragments 140-160 bp in size.
RNA-Seq; sequenced on an Illumina HiSEQ 4000 using Illumina provided reagents and protocols

value: Tau fragment tg

value: brain


title: K562 cells, shNSUN2, rep2

value: NSUN2 knockdown


title: uninjected-late-1C_7

value: 1-cell mouse embryo


title: Ctrl.WT.rep2 (DISE-031)

source_name: WT brain, hippoc.


title: K562 cells, shNSUN2, rep1


title: Ctrl.WT.rep3 (DISE-031)


title: Healthy Control, sample 48

value: healthy


title: K562 cells, Ctrl, rep1


title: Healthy Control, sample 47


title: RubiconKO.rep4 (DISE-033)

source_name: 5XFADtg/Rubicon ko, brain, mouse #282

value: 5XFADtg/Rubicon ko

value: Ago bound RNA


title: Ctrl.WT.rep1 (DISE-031)


title: K562 cells, Ctrl, rep2


title: uninjected-late-1C_6


title: Healthy Control, sample 46


title: SRSF2 PAR-CLIP Seq in SRSF2(P95H) K562 cell rep2

data_processing:
The raw sequencing data were first analyzed using FastQC (v0.11.5, default parameters). Subsequently, reads were stripped of adaptor sequences (cutadapt v1.9.1) with parameters cutadapt -a AGATCGGAAGAG and cutadapt -m 15 -u 4 -a AAAAAAAAAA (only for libraries prepared by the SMARTer smRNA-seq kit), and then low-quality bases were removed with Trimmomatic (v0.33, parameters: LEADING:20, TRAILING:20, SLIDINGWINDOW:4:15, MINLEN:15). Processed reads more than 15 nt long were defined as clean reads. The resulting fastq data was again analyzed using FastQC to ensure no further processing was needed.
The remaining reads were mapped on hg19 genome using Bowtie (v1.1.2, parameters: -v 2 -m 10 --best --strata).
PARalyzer (v1.5) software was used to define the cluster of SRSF2 binding sites with the default parameters.
Assembly: hg19
Supplementary files format and content: csv file of SRSF2 binding clusters

growth_protocol: HeLa cells were maintained in DMEM (Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin and streptomycin (Pen Strep, Gibco). K562 cells were cultured in IMDM (Gibco) supplemented with 10% FBS and 1% Pen Strep. For RNA interference maintenance, K562 cells were kept under selection with 1.5μg/ml puromycin. All cells were cultured at 37°C in a humidified atmosphere containing 5% CO2.

extract_protocol:
The cells were lysed, digested with 1 U/μl RNase T1 at 22 °C for 8 min, and immunoprecipitated with anti-Flag M2 magnetic beads (Sigma). The protein-RNA-bead complex was digested with 10 U/ μl RNase T1 again at 22°C for 8 min and incubated with 0.5 U/μl Alkaline Phosphatase Calf Intestinal (CIP, NEB) for 10 min at 37°C. The beads were washed and then incubated with 0.5 U/μl T4 Polynucleotide Kinase (NEB) for 15 min at 37°C. After washing, one-sixth of the beads were resuspended in LDS loading buffer (Invitrogen), boiled at 95°C for 10 min, and the mixture was resolved by SDS-PAGE to detect the immunoprecipitation efficiency. One-sixth of the beads were labeled with biotin using the RNA 3′ End Biotinylation kit (Thermo), following the manufacturer's instructions for PAR-CLIP-biotin chemiluminescent nucleic acid detection. The rest of the beads were also boiled and the mixture electrophoresed through a NuPAGE Bis-Tris protein gel. Parts containing target protein-RNA complexes were cut from the gel according to the protein-RNA-biotin signal. The protein-bound RNA in the gel pieces was recovered by D-Tube™ Dialyzer Midi (Merck-Millipore), digested with proteinase K (Roche), and extracted with phenol-chloroform.
Library preparation was performed using the Takara SMARTer smRNA-seq kit (PAR-CLIP in HeLa cells) or the NEBNext® Multiplex Small RNA Library Prep kit (PAR-CLIP in K562 cells) for Illumina, following the manufacturer's instructions.

treatment_protocol:
HeLa cells were co-transfected by electroporation with siRNA (control or siNSUN2 siRNA) and the pCMV-Flag-SRSF2 plasmid. Control and NSUN2-knockdown K562 cells were co-transfected with the pCMV-Flag-SRSF2 and pCMV-Myc-SRSF2 plasmids. SRSF2P95H mutant K562 cells were co-transfected with the pCMV-Flag-SRSF2P95H and pCMV-Myc-SRSF2P95H plasmids.
Transfected cells were cultured in a medium supplemented with 200 μM 4-thiouridine (4-SU) (Sigma) for 14 h and then irradiated once with 400 mJ/cm2 at 365 nm before harvest.

tag: replicates

tag: srsf2 genotype

value: P95H heterozygous mutant

tag: rna interference

value: shRNA-Ctrl


title: TgTau.rep1 (DISE-031)

source_name: Tau fragment tg brain, mouse #1196, hippoc.


title: TgTau.rep2 (DISE-031)

source_name: Tau fragment tg brain, mouse #1197, hippoc.


title: SRSF2 PAR-CLIP Seq in SRSF2(P95H) K562 cell rep1

tag: cell type


title: RubiconKO.rep3 (DISE-033)

source_name: 5XFADtg/Rubicon ko, brain, mouse #277

tag: genotype


title: uninjected-late-1C_5

source_name: 1-cell mouse embryo

tag: strain

tag: treatment


title: uninjected-late-1C_4


title: uninjected-early-2C_1


title: Healthy Control, sample 45

tag: tissue


title: RubiconKO.rep2 (DISE-033)

source_name: 5XFADtg/Rubicon ko, brain, mouse #273


title: uninjected-late-1C_3


title: SRSF2 PAR-CLIP Seq in shNSUN2 K562 cell rep2

value: Wildtype


title: RubiconKO.rep1 (DISE-033)

source_name: 5XFADtg/Rubicon ko, brain, mouse #271


title: Healthy Control, sample 43

tag: disease state


title: Healthy Control, sample 44


title: uninjected-late-1C_1


title: Healthy Control, sample 42


title: input_E-G

city: Copenhagen

first: Lars

middle: Roed

last: Ingerslev

address: Blegdamsvej 3B

department: NNF Center for Basic Metabolic Research

country: Denmark

institute: Copenhagen University

library_source: genomic

data_processing:
Reads were aligned to the ENSEMBL GRCm38 release 102 primary assembly using bwa-mem2 version 2.2.1 with default settings.
PCR duplicates were removed using UMI-tools version 1.1.1 in --paired mode
Conversion to bam format as well as intermediary sorting steps were done using samtools version 1.13
Reads with both ends mapped at most 1000 bp apart and a minimum mapping quality score of 20 were counted into bins of width 150, 300, 600, 900, 1200 and 10000 bp
Assembly: GRCm38
Supplementary files format and content: csv file with fragment counts, genomic bins in the rows and samples in the columns

molecule: genomic DNA

source_name: Pancreatic islet

growth_protocol: Islets were isolated from 10-12-week-old C57BL/6NRj mice (Janvier, Le Genest-Saint-Isle, France) as previously described23. The day after isolation, the islets were used for experiments in RPMI 1640 media with 1 % Penicillin/Streptomycin (P/S) and 2 % human serum (Biowest, Nuaillé, France).

extract_protocol:
After 10 days exposure to IL-1β and or butyrate, the islets (500-600 per condition) were washed in PBS. Cross-linking was performed in 0.5 % formaldehyde/PBS solution at room temperature for 7.5 min and quenched with 0.125 M glycine for 5 min. The islets were washed with PBS before resuspension in 1 mL of IP buffer (67 mM TrisHCl (pH 8), 100 mM NaCl, 5 mM EDTA (pH 8), 0.2 % NaN3, 0.33 % SDS, 1.67 % Triton X100, supplemented with protease inhibitor). Homogenization was performed by passing the islets through a 25 g needle. Chromatin was sonicated to an average length of 200-400 bp (~30 cycles, high intensity on Diagenode, Biorupter). Chromatin was cleared by centrifugation at 20,000 g for 30 min. For each ChIP, 700 ng DNA was incubated with 4 μg H3K27ac antibody (cat# Ab4729, Abcam) overnight rotating at 4 °C. Chromatin-antibody complexes were captured using Protein G Sepharose 4 Fast Flow beads (GE healthcare, Broendby, Denmark) for 4 hours followed by three washes with low-salt buffer (20 mM TrisHCl (pH 8), 2 mM EDTA, 1 % Triton X-100, 0.1 % SDS, 150 mM NaCl) and a final wash with high-salt buffer (20 mM TrisHCl (pH 8), 2 mM EDTA, 1% Triton X-100, 0.1 % SDS, 500 mM NaCl). Chromatin was decrosslinked in 1 % SDS and 0.1 M NaHCO3 for 6 hours at 65 °C. DNA was purified using Qiagen MinElute PCR purification kit (Qiagen, Aarhus, Denmark) and the concentration was determined using Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific)
DNA libraries were prepared with 7-14 ng of DNA usingthe NEBNext Ultra DNA library prep kit for Illumina (New England Biolabs, Hitchin, UK) and Unique Dual Index UMI Adaptors DNA set 1 (New England Biolabs) for Illumina sequencing according to the manufacturer’s protocol. For each library preparation, the optimal number of PCR cycles was determined by running a real-time PCR reaction using Brilliant III Ultra-fast SYBR Green QPCR Master Mix (Agilent, Santa Clara, CA, USA) and a C1000 Thermal cycler (Bio-Rad). The quality and the quantity of the libraries were checked on Bioanalyzer 2100 instrument (High Sensitivity DNA kit, Agilent) and DNA quantified by Qubit (Thermo Fisher Scientific) prior to sequencing.

treatment_protocol: The islets were exposed to IL-1β (50 pg/ml) (cat# 55457, BD Biosciences, Lyngby, Denmark) and/or butyrate (0.2 mM) (cat# B5887, Sigma-Aldrich, Soeborg, Denmark) for 10 days at 37 °C.

value: Pancreatic islet

tag: Sex

value: male

tag: yield after_ip_(ng)

value: E+G

value: Input


title: SRSF2 PAR-CLIP Seq in shNSUN2 K562 cell rep1

tag: cell line

value: human erythroleukemic cell line


title: Healthy Control, sample 51


title: input_B-D

value: B+D


title: SRSF2 PAR-CLIP Seq in Ctrl K562 cell rep2


title: RubiconHet.rep4 (DISE-033)

source_name: 5XFADtg/Rubicon het, brain, mouse #297

value: 5XFADtg/Rubicon het


title: RubiconHet.rep3 (DISE-033)

source_name: 5XFADtg/Rubicon het, brain, mouse #293


title: SRSF2 PAR-CLIP Seq in Ctrl K562 cell rep1


title: sample_G_Il1b+Butyrate

value: G

value: Il1b + Butyrate


title: 3N, replicate 3, scRNAseq

city: shenyang

first: sun

middle: yuan

last: yuan

address: 沈北新区蒲河路77号

country: China

institute: China Medical University

description: 10x Genomics

data_processing:
Reads were processed using the Cell Ranger 2.1.0 pipeline with default and recommended parameters. FASTQs generated from Illumina sequencing output were aligned to the human genome, version GRCH38, using the STAR algorithm.Next, Gene-Barcode matrices were generated for each individual sample by counting UMIs and filtering non-cell associated barcodes. Finally, we generate a gene-barcode matrix containing the barcoded cells and gene expression counts. This output was then imported into the Seurat (v2.3.0) R toolkit for quality control and downstream analysis of our single cell RNAseq data. All functions were run with default parameters, unless specified otherwise. We excluded cells with fewer than 200 or more than 6000 detected genes (where each gene had to have at least one UMI aligned in at least three cells). The expression of mitochondria genes was calculated using PercentageFeatureSet function of the seurat package.To remove low activity cells, cells with more than 10 percent expression of mitochondria genes were excluded. The normalized data (NormalizeData function in Seurat package) was performed for extracting a subset of variable genes. Variable genes were identified while controlling for the strong relationship between variability and average expression. Next, we integrated data from different samples after identifying ‘anchors’ between datasets using FindIntegrationAnchors and IntegrateData in the seurat package.Then we performed principal component analysis (PCA) and reduced the data to the top 30 PCA components after scaled the data. We visualized the clusters on a 2D map produced with t-distributed stochastic neighbor embedding (t-SNE).
Assembly: GRCH38
Supplementary files format and content: Cell Ranger output files (barcodes.tsv, features.tsv, matrix.mtx)

source_name: larynx

extract_protocol:
Tissues were transported in sterile culture dish with 10 ml 1x Dulbecco's Phosphate-Buffered Saline (DPBS; Thermo Fisher, Cat. no. 14190144) on ice to remove the residual tissue storage solution, then minced on ice. We used dissociation enzyme 0.25% Trypsin (Thermo Fisher, Cat. no. 25200-072) and 10 ug/mL lDNase I (Sigma, Cat. no. 11284932001) dissolved in PBS with 5% Fetal Bovine Serum (FBS; Thermo Fisher, Cat. no. SV30087.02) to digest the tissues. Tissues were dissociated at 37 ℃ with a shaking speed of 50 r.p.m for about 40 min.We repeatedly collected the dissociated cells at interval of 20 min to increase cell yield and viability. Cell suspensions were filtered using a 40 um nylon cell strainer and red blood cells were removed by 1X Red Blood Cell Lysis Solution (Thermo Fisher, Cat. no. 00-4333-57) . Dissociated cells were washed with 1x DPBS containing 2% FBS. Cells were stained with 0.4% Trypan blue (Thermo Fisher, Cat. no. 14190144) to check the viability on Countess® II Automated Cell Counter (Thermo Fisher)
Beads with unique molecular identifier (UMI) and cell barcodes were loaded close to saturation, so that each cell was paired with a bead in a Gel Beads-in-emulsion(GEM). After exposure to cell lysis buffer, polyadenylated RNA molecules hybridized to the beads. Beads were retrieved into a single tube for reverse transcription. On cDNA synthesis, each cDNA molecule was tagged on the 5’end (that is, the 3’end of a messenger RNA transcript) with UMI and cell label indicating its cell of origin. Briefly, 10× beads that were then subject to second-strand cDNA synthesis, adaptor ligation, and universal amplification.Sequencing libraries were prepared using randomly interrupted whole-transcriptome amplification products to enrich the 3’ end of the transcripts linked with the cell barcode and UMI. All the remaining procedures including the library construction were performed according to the standard manufacturer’s protocol (CG000206 Rev D).

value: larynx

tag: tissue type

value: N

tag: age

value: 60-65 years old

tag: individual

value: patient 3


title: sample_G_Butyrate

value: Butyrate


title: RubiconHet.rep2 (DISE-033)

source_name: 5XFADtg/Rubicon het, brain, mouse #285

value: brain

tag: fraction

value: Ago bound RNA


title: Healthy Control, sample 40

value: Bone marrow aspirate

value: healthy


title: RubiconHet.rep1 (DISE-033)

source_name: 5XFADtg/Rubicon het, brain, mouse #284


title: C24 SCR01*1887 Ah20mirS3 bio rep 10

city: Villeneuve d'Ascq

first: Vincent

last: Castric

address: Batiment SN2, bureau 207

country: France

institute: CNRS / Universite de Lille - Sciences et Technologies

data_processing:
Reads were trimmed, quality-filtered, and mapped to the Ah01 allele using methlypy (Schultz et al., 2015), following the paired-end pipeline with it's default settings and specifying AT2G20610 as the unmethylated control.
Assembly: TAIR10 (AT2G20610 - BSAS control) and Arabidopsis halleri Ah01 S locus
Supplementary files format and content: allc_merged.tsv columns: sample/chr/position/strand/sequence context/number of unmethylated reads/number of methylated reads/methylation status outputed by methylpy (0 - unmethylated; 1 - methylated)
Supplementary files format and content: conversion_eff.txt: bisulfite conversion efficiency for each replicate, as determined by methylpy based on the level of methylation in the control region AT2G20610 (BSAS)
Supplementary files format and content: primers_BSAS.txt:  primers used for amplification of genomic regions to be sequenced

organism: Arabidopsis thaliana

source_name: immature floral buds

growth_protocol: Arabidopsis thaliana plants used in this study were grown in a greenhouse, using standard growth conditions (16 h light/8 h dark; 110 μmol/s/m2; 21°C; 70% humidity)

extract_protocol:
Genomic DNA was extracted from floral buds using the NucleoSpin Plant II kit (Macherey-Nagel), and converted with the EZ DNA Methylation Kit (Zymo Research), including the modifications A and B, as described by the supplier, to optimize the conversion efficiency. Eight to ten biological replicates were extracted and converted for each genotype. Converted DNA was then amplified using primers flanking the Ah04mir1887 or Ah20mirS3 target sites in the intron and promoter regions of SCR01, respectively (table S1). For each sRNA precursor, seven amplicons were generated, which amplify the top and bottom strands of the ~800 bp that flank the target site. As a negative control, we also amplified AT2G20610, which has been previously described to be an unmethylated negative control in A. thaliana Col plants (Sigman et al., 2021). Amplification of bisulfite converted DNA was performed using the TaKaRa EpiTaqTM HS (Takara), and the resulting PCR reactions were cleaned-up using the NucleoSpin Gel and PCR Clean‑up XS kit (Macherey-Nagel).
All amplicons obtained from a single biological replicate were pooled in equimolar amounts and a total of 100 ng were used for library preparation following the modified Illumina Nextera Flex library preparation protocol described before (Gaio et al., 2021; Sigman et al., 2021).

value: immature floral buds


title: 3L, replicate 2, scRNAseq

description: 10x Genomics

source_name: lymph gland

value: lymph gland

value: L

value: male


title: sample_G_Il1b

value: Pancreatic islet

tag: replicate

value: Wildtype


title: Healthy Control, sample 4

source_name: Bone marrow aspirate


title: 3R, replicate 2, scRNAseq

description: 10x Genomics

value: R


title: sample_G_Control

value: G

value: Control


title: Healthy Control, sample 39


title: C24 SCR01*1887 Ah20mirS3 bio rep 9


title: sample_E_Butyrate

value: E


title: 3T, replicate 3, scRNAseq

description: 10x Genomics

value: larynx

value: T

value: 60-65 years old


title: Healthy Control, sample 38


title: C24 SCR01*1887 Ah20mirS3 bio rep 8


title: C24 SCR01*1887 Ah20mirS3 bio rep 7


title: 2N, replicate 4, scRNAseq

description: 10x Genomics

value: N

value: patient 2


title: Healthy Control, sample 37

value: Bone marrow aspirate


title: Healthy Control, sample 41

value: healthy


title: sample_E_Il1b+Butyrate

value: Il1b + Butyrate


title: Healthy Control, sample 36


title: sample_E_Control


title: 2L, replicate 3, scRNAseq

description: 10x Genomics

value: lymph gland


title: 2T, replicate 2, scRNAseq

description: 10x Genomics


title: sample_E_Il1b


title: Healthy Control, sample 35


title: 1N, replicate 1, scRNAseq

description: 10x Genomics

value: patient 1


title: C24 SCR01*1887 Ah20mirS3 bio rep 5

value: immature floral buds


title: C24 SCR01*1887 Ah20mirS3 bio rep 6


title: sample_D_Butyrate

value: Pancreatic islet

value: male

value: D

value: Butyrate


title: Healthy Control, sample 34


title: sample_D_Il1b+Butyrate

value: Wildtype


title: sample_D_Il1b


title: 1R, replicate 1, scRNAseq

description: 10x Genomics

value: R


title: C24 SCR01*1887 Ah20mirS3 bio rep 4


title: C24 SCR01*1887 Ah20mirS3 bio rep 3


title: Healthy Control, sample 33


title: 1T, replicate 1, scRNAseq

description: 10x Genomics

value: larynx


title: Healthy Control, sample 32


title: C24 SCR01*1887 Ah20mirS3 bio rep 2


title: sample_D_Control

value: Control


title: Healthy Control, sample 31


title: Healthy Control, sample 30


title: C24 SCR01*1887 Ah20mirS3 bio rep 1


title: sample_B_Il1b+Butyrate

value: B


title: sample_B_Il1b


title: sample_B_Butyrate


title: Healthy Control, sample 3


title: SCR01*1887 bio rep 10


title: Healthy Control, sample 29


title: SCR01*1887 bio rep 9


title: sample_B_Control


title: Healthy Control, sample 28


title: SCR01*1887 bio rep 7


title: SCR01*1887 bio rep 6


title: Healthy Control, sample 27


title: Healthy Control, sample 26


title: WT7_2

city: Beijing

first: Jiacheng

last: Wu

address: 5 Dongdansantiao, Dongcheng District

institute: PUMC  & CAMS

data_processing:
Illumina Casava 1.8 software used for basecalling
Raw data (raw reads) of fastp format were firstly processed through in-house perl scripts..In this step, clean data (clean reads) were obtained by removing reads containing adapter, reads containing ploy-N and low quality reads from raw data. At the same time, Q20, Q30 and GC content the clean data were calculated. All the downstream analyses were based on the clean data with high quality.
Reference genome and gene model annotation files were downloaded from genome website directly. Index of the reference genome was built using Hisat2 v2.0.5 and paired-end clean reads were aligned to the reference genome using Hisat2 v2.0.5.
featureCounts v1.5.0-p3 was used to count the reads numbers mapped to each gene.
Differential expression analysis of two conditions/groups (two biological replicates per condition) was performed using the DESeq2 R package (1.20.0).
Assembly: GRCm38 (mm10)
Supplementary files format and content: tab-delimited text files include FPKM values and gene count for each Sample

source_name: colon

extract_protocol:
Total RNA was isolated using TRIzol (Invitrogen) according to the manufacturer’s instructions.
RNA libraries for RNA-seq were prepared using NEBNext® Ultra™ RNA Library Prep Kit for Illumina® following manufacturer's protocols.

value: colon

value: intestinal epithelial cell

value: seven days after DSS administration


title: WT0_2

value: without DSS administration


title: SCR01*1887 bio rep 5


title: WT7_1


title: Healthy Control, sample 25

value: Bone marrow aspirate

value: healthy


title: SCR01*1887 bio rep 4


title: SCR01*1887 bio rep 8


title: KO7_2


title: proximal femur periosteum, Ctrl5

type: RNA

city: Wuhan

first: Xin

last: Tang

address: 1277 jiefang avenue

institute: Union Hospital, Tongji Medical College, Huazhong University of Science and Technology

description: Gene expression data from proximal femur periosteum of a 14-year old boy with coxa valga

hyb_protocol: Following fragmentation, 10 ug of cDNA were hybridized for 16h at 48℃ on Affymetrix HTA2.0 Array.Chips were washed and stained in the Affymetrix Fluidics Station 400.

scan_protocol: Chips were scanned using the Affymatrix GeneArray Scanner  3000.

data_processing: The data were analyzed with RMA (Robust Multiarray Average) using Affymetrix default analysis settings and global scaling as quantile normalization. The summarization mean target intensity of each array was arbitrarily set to 100, Pvalue≤0.05

label: biotin

source_name: control_periosteum from proximal femur

label_protocol: Biotinylated cDNA were prepared according to the standard Affymetrix protocol from 1 ug total RNA.

extract_protocol: Extraction of total RNA was performed according to the Qiagen's instructions

treatment_protocol: Periosteum specimens were biopsied from the proximal femur of each patient during surgery and transferred to liquid nitrogen immediately after being washed with saline

tag: subject status

value: Control patients (with femoral fracture/coxa valga)

tag: gender

value: periosteum from proximal femur


title: SCR01*1887 bio rep 3

value: immature floral buds


title: Healthy Control, sample 24


title: proximal femur periosteum, Ctrl3

description: Gene expression data from proximal femur periosteum of a 14-year old boy with femoral fracture

value: male


title: proximal femur periosteum, Ctrl4

description: Gene expression data from proximal femur periosteum of a 9-year old boy with femoral fracture


title: proximal femur periosteum, Ctrl1


title: proximal femur periosteum, Ctrl2

description: Gene expression data from proximal femur periosteum of a 6-year old boy with femoral fracture


title: proximal femur periosteum, LCPD8

description: Gene expression data from proximal femur periosteum of a 6-year old boy with LCPD

source_name: LCPD_periosteum from proximal femur

value: patient with Legg-Calve-Perthes disease (LCPD)


title: proximal femur periosteum, Ctrl6

description: Gene expression data from proximal femur periosteum of a 9-year old boy with coxa valga


title: proximal femur periosteum, LCPD9

description: Gene expression data from proximal femur periosteum of a 5-year old girl with LCPD

value: female


title: Healthy Control, sample 23


title: KO7_1


title: proximal femur periosteum, LCPD7

description: Gene expression data from proximal femur periosteum of a 9-year old boy with LCPD


title: SCR01*1887 bio rep 2


title: SCR01*1887 bio rep 1


title: Healthy Control, sample 21


title: KO0_1


title: WT0_1

value: Wildtype


title: SCR01 Ah04mir1887 bio rep 9


title: Healthy Control, sample 20


title: Gcontrol_3

city: Pamplona

first: Marta

middle: Maria

last: Alonso Roldán

state: Navarra

address: CIMA building , Pio XII 55

department: Dpt of Pediatrics

country: Spain

institute: University Hospital of Navarra

data_processing:
The quality of the samples was verified using FastQC v0.11.8 software.
FASTQ files were mapped to GRCm38 human assembly using STAR v2.6.1c with default parameters.
featureCounts v1.6.0 software and Gencode vM25 release were used to obtain read counts at gene level considering only those reads aligned to exonic regions
Obtained gene counts were normalized using edgeR and voom function of limma
Assembly: GRCm38
Supplementary files format and content: tab-delimited text files include gene counts obtained with featureCounts for each sample.

growth_protocol: NP53 DIPG cell line were maintained in DMEM supplemented with 10% fetal bovine serum (FBS) and antibiotics in humidified atmosphere with 5% CO2 at 37C

extract_protocol:
The mRNA conditions used were 1µg total RNA, 20ng/µl concentration, RIN >7.0, rRNA ratio >1.0
Libraries for the sequencing were prepared using TruSeq stranded mRNA + NovaSeq 6000 100 PE (2 x 100 bp) 50 M total reads / sample (5 Gb / sample).

value: DMG tumor cell line

value: WT

tag: sample name

value: Gcontrol


title: proximal femur periosteum, LCPD6

description: Gene expression data from proximal femur periosteum of a 8-year old boy with LCPD


title: Healthy Control, sample 22


title: KO0_2

tag: cell type


title: proximal femur periosteum, LCPD5

description: Gene expression data from proximal femur periosteum of a 7-year old boy with LCPD


title: Healthy Control, sample 2


title: Gcontrol_2

tag: genotype


title: Gcontrol_1


title: SCR01 Ah04mir1887 bio rep 6


title: SCR01 Ah04mir1887 bio rep 8


title: G2C9_3

value: TIM-3 knockout


title: SCR01 Ah04mir1887 bio rep 10


title: Smad4_WT_A5628

city: Rochester

first: Aram

middle: F.

last: Hezel

address: 601 Elmwood Ave, Box 704

institute: Wilmot Cancer Institute

data_processing:
Sequence reads were cleaned and adapter trimmed using trimmomatic-0.36 before mapping each sample individually to the mouse reference genome (GRCm38.p5, primary assembly + GENCODE M12 annotation) with STAR2.5.2b.
Raw read counts were obtained using featurecounts from the subread1.5.0p3 package and GENCODE M12 mouse gene annotations using only uniquely aligned reads (default) and including multi-mapping reads (-M).
DESeq2-1.14.1 within R-3.3.2 was used to perform data normalization and differential expression analysis with an adjusted p-value threshold of 0.05 on each set of raw expression measures.
Assembly: mm9
Supplementary files format and content: tab-delimited text files include RPKM values for each sample

source_name: liver

growth_protocol: All cell lines were maintained in DMEM supplemented with 10% fetal bovine serum (FBS) in humidified atmosphere with 5% CO2 at 37°C.

extract_protocol:
RNA was isolated using RNeasy Plus Micro Kit (Qiagen).
Nextera cDNA library was prepared using the SMART-Seq v4 Ultra Low Input RNA Kit.

treatment_protocol: For liver injury studies, mice were fed 0.1% DDC (3,5-diethoxycarbonyl-1,4-dihydrocollidine) diet for 2 weeks. Reactive cholangiocytes were isolated from digested injured mouse liver via FACS followed by RNA amplification and sequencing analysis.

value: liver

value: reactive cholangiocytes


title: Smad4_WT_A5626


title: SCR01 Ah04mir1887 bio rep 7


title: Healthy Control, sample 19


title: G2C9_2


title: SCR01 Ah04mir1887 bio rep 5


title: proximal femur periosteum, LCPD3


title: proximal femur periosteum, LCPD4

description: Gene expression data from proximal femur periosteum of a 10-year old boy with LCPD


title: Healthy Control, sample 17


title: Healthy Control, sample 16


title: G2C9_1


title: proximal femur periosteum, LCPD2

description: Gene expression data from proximal femur periosteum of a 6-year old girl with LCPD


title: Healthy Control, sample 15


title: G1C7_3


title: SCR01 Ah04mir1887 bio rep 4


title: proximal femur periosteum, LCPD1


title: SCR01 Ah04mir1887 bio rep 3


title: Smad4_WT_A5221


title: Healthy Control, sample 14


title: Healthy Control, sample 18


title: Smad4_WT_A5219


title: Smad4_floxed_A5218


title: Smad4_floxed_A5624


title: Healthy Control, sample 13


title: Healthy Control, sample 12


title: SCR01 bio rep 10

value: C24 SCR01


title: Smad4_floxed_A5217


title: SCR01 Ah04mir1887 bio rep 2


title: Smad4_floxed_A5447


title: G1C7_1


title: MA9 BM, scRNAseq

city: Monrovia

first: Ling

last: Li

state: CA

address: 1218 S 5TH AVE

department: Hem Malignancies Trans Sci

institute: City of Hope National Medical Center

description: 10x Genomics

data_processing:
Raw sequencing data were processed using the 10x Genomics Cell Ranger pipeline to generate FASTQ files
Sequences were aligned to mm10 genome to gene expression count using CellRanger's default settings
Gene expression level of Refseq coding genes were quantified using CellRanger (V7.0.0)'s default settings
Assembly: mm10
Supplementary files format and content: text files containing the raw gene expression count

source_name: Bone marrow

extract_protocol:
BM cells in MA9 transplanted mice were collected for analysis. Cells were resuspended as single cells in the presence of 0.4% BSA and loaded onto a 10x Genomics Chromium instrument to generate an emulsion of single-cell gel beads (GEMs). Approximately 5,000-10,000 cells were loaded per channel.
scRNA-seq libraries were prepared using the Chromium™ Single Cell 3′ Library & Gel Bead Kit v2 (PN-120237), Single Cell 3′ Chip Kit v2 (PN-120236) and i7 Multiplex Kit (PN-120262) (10x Genomics, Pleasanton, CA, USA), following the Single Cell 3′ Reagent Kits v2 User Guide (Manual Part # CG00052 Rev A).
Libraries were sequencedon an Illumina NovaSeq system as 2 × 150 paired-end reads, one full lane per sample, for >90% sequencing saturation.

value: Bone marrow cells

value: Bone marrow

tag: strain

value: 6-8 weeks old


title: Healthy Control, sample 10

value: Bone marrow aspirate

value: healthy


title: SCR01 bio rep 8

value: immature floral buds


title: Sorted p16- Stromal cells (CD45-CD31-CD29+)3

city: Baltimore

first: Christopher

last: Cherry

state: MD

address: 1800 Orleans St

institute: Johns Hopkins University

data_processing:
Demux with bcl2fastq
Alignment with STAR 2.7.3a using GENCODE release M23 GRCm38.p6
Assembly: GENCODE release M23 GRCm38.p6
Supplementary files format and content: Read counts from STAR

source_name: tdTomato p16 reporter on C57Bl/6

extract_protocol:
After collection of tissue samples, samples were submerged in TRIzol reagent, and finely minced and grinded. For in vitro cultures, media was aspirated from tissue culture plates and TRIzol added according to the manufacturers recommended volume. The TRIzol was repeated pipetted across the dish. RNA isolation for both tissue and culture samples was then performed using Qiagen RNeasy Mini Kits following TRIzol extraction.
After isolation of RNA as described previously, cDNA synthesis and library preparation were performed using the TruSeq RNA Library Prep Kit V2. The Agilent BioAnalyzer system was used to perform quality control on samples before and during library preparation. RNA samples with RIN scores below 7 post-cDNA synthesis were removed from analysis. Libraries were pooled and sequenced with paired-end sequencing to a depth of 50bp for each end at a targeted depth of 30M unique reads per sample.

treatment_protocol: All volumetric muscle loss surgeries were performed when animals were 10 weeks of age. Bilateral muscle resections of the quadriceps were performed as previously described. Defects were filled with 30 mg of (poly)caprolactone (particulate, Mn=50,000 g/mol, mean particle size < 600um, Polysciences), decellularized extracellular matrix (Matristem, Acell), or 50 uL of phosphate buffered saline as a control. All materials were UV sterilized prior to implantation and all animals given subcutaneous carprofen (Rimadyl Zoetis) at 5 mg/kg. Samples were harvested 1- or 6- weeks post-surgery. During harvest, the complete quadriceps together with any remaining implant material was harvested and processed for downstream experiments as described in the following sections.

tag: tdtomato

value: Negative


title: Smad4_WT_A5215

tag: tissue


title: Smad4_floxed_A5216


title: Healthy Control, sample 1

tag: disease state


title: Sorted p16- Stromal cells (CD45-CD31-CD29+)2


title: SCR01 bio rep 7


title: Sorted p16- Stromal cells (CD45-CD31-CD29+)1


title: SCR01 bio rep 5


title: Biopsy 1555

data_processing:
Adaptors and low-quality bases in the raw sequencing reads were removed with Cutadapt and Trimmomatic, respectively. Clean reads with lengths greater than 18 nt were mapped to the hg19 bisulfite converted genome using meRanGh from meRanTK with parameters: -fmo -mmr 0.01. Only samples and also their luciferase spike-in RNA showinged more than 99.5% of C to T conversion rate in PBMC as well as luciferase spike-in RNA was were used for further analysis. The m5C sites were called using meRanCall from meRanTK with parameters: -mBQ 20 -mr 0. The high confidence m5C sites with a coverage depth ≥ 30, methylation level ≥ 0.1 and methylated cytosine depth ≥ 5 were reported
Assembly: hg19
Supplementary files format and content: m5C site
Library strategy: RNA bisulfite seq

source_name: sorted peripheral blood CD14+ mononuclear cells

extract_protocol:
Peripheral blood mononuclear cells (PBMC) were sorted using density centrifugation Pancoll (Pan-Biotech) and CD14+ monocytes were sorted by negative selection with magnetic beads and the AutoMacs system (Miltenyi Biotech). Total RNA was extracted using RNA Purification Plus Kit (Norgen Biotek). All total RNA samples had RIN values > 7. Ribo-off rRNA Depletion Kit (Vazyme) was used to remove ribosomal RNA.
Around 500 ng ribo-depleted RNA was mixed with 1.5 ng of in vitro transcribed firefly luciferase spike-in RNA and fragmented into approximately 150 nucleotides in length with RNA fragmentation reagent (Ambion).  Bisulfite treatment was performed using the EZ RNA methylation Kit (Zymo Research) with some modifications. In brief, fragmented RNA was converted using two cycles of 5 min at 70 °C followed by 45 min at 64 °C. RNA desulphonation and purification were also performed using this kit. RNA quantity was determined by Qubit. cDNA libraries were constructed using the KAPA Stranded mRNA-Seq Kit (KK8421) according to the manufacturer’s instructions.

tag: nsun2 category

value: Low

tag: Sex

value: M


title: Sorted p16+ Stromal cells (CD45-CD31-CD29+)3

value: Positive


title: G1C7_2

tag: cell line

value: DMG tumor cell line


title: SCR01 Ah04mir1887 bio rep 1


title: Biopsy 1745

value: sorted peripheral blood CD14+ mononuclear cells

value: High

value: F


title: SCR01 bio rep 4


title: Sorted p16+ Stromal cells (CD45-CD31-CD29+)2


title: SCR01 bio rep 9


title: Healthy Control, sample 11

extract_protocol:
Total RNA was extracted using the MagNA Pure 96 Instrument and the MagNA Pure 96 Cellular RNA LV Kit (Roche LifeScience, Mannheim, Germany)
The TruSeq Total Stranded RNA kit was used, starting with 250ng of total RNA, to generate RNA libraries following the manufacturer’s recommendations (Illumina, San Diego, CA, USA)


title: Biopsy 1504


title: Sorted p16+ Stromal cells (CD45-CD31-CD29+)1


title: SCR01 bio rep 3


title: Biopsy 1925


title: SCR01 bio rep 2


title: Biopsy 2146


title: SCR01 bio rep 1


title: Biopsy 2042


title: CD8 cells treated with Vorinostat under dose 2 uM, replicate 2

city: Tampa

first: Amer

last: Beg

state: FL

address: 12902 Magnolia Drive

institute: Moffitt Cancer Center

description: Mouse splenic CD8 cells treated with Vorinostat under dose 2 uM

hyb_protocol: The labeled RNA was hybridized to Affymetrix U133 Plus 2.0 microarrays and the staining and scanning of the chips followed the prescribed procedure outlined in the Affymetrix technical manual.

scan_protocol: GeneChips were scanned using the Hewlett-Packard GeneArray Scanner G2500A.

data_processing: Data were analyzed and annotated using the Bioconductor R packages: affy, gcrma and hgu133plus2.db

source_name: Mus musculus splenic CD8 cells

label_protocol: RNA was converted to cDNA and then amplified and labeled with biotin using the Ambion Message Amp Premier RNA Amplification Kit (Life Technologies, Grand Island, NY) following the manufacturer’s protocol.

growth_protocol: Splenic CD4+ and CD8+ T cells were isolated by negative selection using the MojoSort Mouse CD4+ and CD8+ T Cell Isolation Kit (Biolegend) following the manufacturer’s instructions. Isolated cells were plated at 2x106 per mL in RPMI 1640 containing 2mM L-glutamine and 25 mM HEPES, supplemented with 10 mM Sodium Pyruvate, nonessential amino acids, 100 U/ml penicillin/streptomycin, 55 μM β-ME, and 10% fetal calf serum (complete media).

extract_protocol: RNA extraction using RNeasy Plant Mini Kit (QIAGEN) following the manufacturer’s instructions

treatment_protocol: T cells were stimulated with plate-bound 3ug/ml anti-CD3 and solubilized 5ug/ml anti-CD28 antibodies (eBioscience) for 2 days. Activated T cells were treated with 0.5, 2, 10uM Vorinostat or 2, 10, 30nM Romidepsin for 1 day

value: splenic

value: CD8 cell

tag: treatment

value: Vorinostat, 2 uM


title: UPS sample 7 female

city: NEW HAVEN

first: Arie

last: Kaffman

state: CT

address: 300 GEORGE ST. SUITE 901, SUITE 901

institute: YALE UNIVERSITY

data_processing:
Reads were trimmed for quality using customized scripts with a minimum accepted length of 45 bases.  The trimmed reads were aligned to the mm10 reference genome using gencode annotation (Frankish, A., et al. Nucleic Acids Res, 2019) and the HISAT2 alignment software (Kim, D et al. Nat Methods, 2015).
The StringTie software (Kim, D et al. Nat Methods, 2015) was used to determine transcript abundance estimation (TPM).
Low expressing gene (TPM < 3 in all three groups) were removed and the remaining 13,245 genes were analyzed using DESeq2 script in R (Love et al., 2014) to identify genes that are differentially expressed between the three groups.
Genome_build: mm10
Supplementary_files_format_and_content: TPM values for all samples, Summary of differentially regulate genes of 3 comparisons (CTL-LB, CTL-UPS, LB-UPS)

source_name: P17, left hippocampus

extract_protocol:
RNA was isolated from the left hippocampus using the AllPrepâ DNA/RNA/Protein Mini Kit (QIAGEN, Cat# Cat# 80004) per manufacturer’s instructions and stored at -80°C.
Total RNA samples were quantified and checked for quality (all RINs>9.0) using the 2100 Bioanalyzer on a RNA 6000 Nano Assay (Agilent Technologies, Inc). Samples concentrations were normalized to 500ng total RNA and libraries were constructed using the KAPA mRNA HyperPrep Kit for Illumina Platforms (KAPA Biosystems) according to the manufacture’s protocol using 9 PCR cycles for library amplification. cDNA libraries were validated using the Bioanalyzer 2100 on a High Sensitivity DNA assay. Quantification was done using the KAPA Library Quantification Kit for Illumina Platforms on a Roche Lightcycler 480.  Sequencing was done on an Illumina NovaSeq 6000 using the S4 XP workflow 2x100 with each library using 1.25% of the S4 lane.

treatment_protocol: Pups were exposed to either control, LB or UPS conditions as described in see Johnson FK. et al. Translational psychiatry (2018). At postnatal day 17 (P17) pups anesthetized with chloral hydrate and immediately processed to collect the hippocampi. Dissected hippocampi were snap frozen in liquid nitrogen and stored at -80C.

value: Balb/cByj

value: left hippocampus

tag: early life stress

value: UPS

value: Female

tag: age


title: Biopsy 1633


title: UPS sample 7 male

value: Male


title: UPS sample 8 male


title: UPS sample 6 male


title: T98G cell,sh-CHASERR3

city: wuhu

first: Hui

last: Yang

state: Anhui

address: No.22 Wenchang West Road,Wuhu,Anhui 241002,P.R.China

institute: wannan medical college

data_processing:
The Illumina platform was used for the acquisition of a large number of samples with double-ended sequencing data. In view of the influence of the data error rate on the results, we used Trimmomatic software to perform quality pre-processing on the raw data. The quality pre-processing steps included splice removal; removal of low-quality reads; removal of low-quality bases from the 3' and 5' ends in different ways; counting of raw sequencing quantity, effective sequencing quantity, Q30, GC content and comprehensive evaluation. A statistical summary was also made of the number of reads in the entire QC process.
We used hisat2 to sequence Clean Reads against a specified reference genome to obtain positional information on the reference genome or gene, as well as information on sequence features specific to the sequenced samples
In transcriptome sequencing analysis, the expression level of a protein-coding gene is estimated by counting the number of sequenced sequences (reads) that are located in the genomic region of the protein-coding gene or in the exonic region of the protein-coding gene. We used known reference gene sequences and annotation files as a database. We used a sequence similarity comparison approach to determine the expression abundance of each protein-coding gene in each sample. The htseq-count software was used to obtain the number of reads compared to the protein-coding genes in each sample. The cufflinks software was used to calculate the FPKM values of the expression of the protein-coding genes.
Assembly: mm10
Supplementary files format and content: Supplementary files format and contnet:processed data.xls

growth_protocol: T98G glioma cell were maintained in DMEM supplemented with 10% fetal bovine serum(FBS) and antibiotics in humidified atmosphere with 5% CO2 at 37℃

extract_protocol:
RNA was collected and extracted from glioma cell T98G using the Rneasy mini plus kit(Qiagen), and 2ug of total RNA was used to construct the sequencing library
We used Trimmomatic software to pre-process raw data and statistically aggregate reads throughout the quality control process.RNA libraries for RNA-seq were prepared using the SMARTER mRNA-seq library preparation kit according to the manufacturer's protocol. The construction of the libraries was subject to quality control on an Agilent 2100 bioanalyzer.

treatment_protocol: Treatment of T98G glioma cells with sh-NC and sh-lnc CHASERR for 48 hours knocked down lnc CHASERR expression in glioma cell lines.

value: glioma cells

value: CHASERR knockdown


title: T98G cell,sh-CHASERR2


title: CD8 cells treated with Vorinostat under dose 2 uM, replicate 1


title: T98G cell,sh-CHASERR1


title: CD8 cells treated with Vorinostat under dose 10 uM, replicate 2

description: Mouse splenic CD8 cells treated with Vorinostat under dose 10 uM

value: Vorinostat, 10 uM


title: Biopsy 1954

value: M


title: UPS sample 6 female


title: T98G cell,sh-NC3

value: NC


title: CD8 cells treated with Vorinostat under dose 0.5 uM, replicate 1

description: Mouse splenic CD8 cells treated with Vorinostat under dose 0.5 uM

value: Vorinostat, 0.5 uM


title: UPS sample 5 male


title: CD8 cells, stimulated control, replicate 2

description: Mouse splenic CD8 cells, stimulated control

value: stimulated control


title: ESI017 WT+1G4TCR SP8 (hDLL4 Stroma), replicate 2, VDJ scRNA-seq

city: Los Angeles

first: Gay

middle: M

last: Crooks

address: 610 CHARLES E. YOUNG DRIVE EAST

department: Department of Pathology & Lab Medicine

institute: University of California, Los Angeles (UCLA)

description: 10X Genomics

library_source: other

data_processing:
Sequenced libraries were demultiplexed and used FASTQ were genereated by the UCLA Technology Center for Bioinformatics and Genomics core. Sequenced libraries for 5' Gene Expression. (5' GEX) and VDJ libraries were processed  and count matrices were generated using the CellRanger software v7.0.0 Multi pipeline (https://support.10xgenomics.com/single-cell-vdj/software/pipelines/latest/using/multi).
Count matrices for 5' GEX and ADT libraries are provided, and fully reconstructed TCR (VDJ) chains are included as filtered_contig_annotations.csv
The 3 TKO VDJ had no aligned results.
Assembly: hg38
Supplementary files format and content: Tab-separated values files and matrix files

molecule: other

source_name: ESI017 WT ESO hPSC

growth_protocol:
PSCs were maintained as single-cell cultures on Matrigel-coated 6-well plates in mTeSRTM Plus complete medium. PSCs were harvested as single cell suspension after treatment with TrypLETM Express (ThermoFisher Scientific, Cat. 12604-013) for 6 minutes at 37C, washed, and counted. Cells were resuspended directly in X-VIVO15 medium (Lonza, Cat. 04-418Q) supplemented with rhActivin A (10ng/mL) (R&D Systems, Cat. 338-AC-0101), rhBMP4 (10ng/mL) (R&D Systems, Cat. 314-BP-010), rhVEGF (10ng/mL) (R&D Systems, Cat. 298-VS-005), rhFGF (10ng/mL) (R&D Systems, Cat. 233-FB-025), and ROCK inhibitor Y-27632 dihydrochloride (10µM) (Tocris, Cat. 1254). Cells were plated on Matrigel coated 6-well plates at 3.3 x 106 cells per well in 3mL. Media was then changed daily with X-VIVO 15 supplemented with rhBMP4 (10ng/mL), rhVEGF (10ng/mL), and rhFGF (10ng/mL). At day 3.5, cells were washed 3 times with PBS and incubated with Accutase (Innovative Cell Technologies, Cat. AT-104), 1mL per well for 10m at 37C. Cells were harvested by dilution with MACS buffer (PBS, 0.5% bovine serum albumin, 2mM EDTA) followed by depletion of CD326+ (EPCAM) cells by magnetic cell sorting (MACS) using CD326 (EPCAM) MicroBeads (Miltenyi, Auburn, CA, Cat. 130-061-101). In addition, EMPs derived from RAG1-/-RAG2-/-B2M-/- triple knockout PSCs were stained with PE-conjugated hB2M antibody (Biolegend) and also depleted of B2M+ cells using MACS Anti-PE MicroBeads (Miltenyi, Auburn, CA, Cat. 130-048-801) in addition to CD326 MicroBeads.
Embryonic mesodermal organoids (EMOs) and ATOs were generated as previously described12. Briefly, sequential generation of EMOs and then ATOs was accomplished in 3D aggregates through changing media (Fig. 1A and Supplementary Fig. 2A). EMOs were established by aggregating purified EMPs with MS5-hDLL4 cells. MS5-hDLL4 cells were harvested by trypsinization and resuspended in “hematopoietic induction media” comprised of EGM2 (Lonza, Cat. CC-4176) supplemented with ROCK inhibitor Y-27632 dihydrochloride (10µM) (Tocris Bioscience, Cat. 1254) and TGF-bRI inhibitor SB-431542 (“SB Blocker”) (10µM) (Tocris Bioscience, Cat. 1614). At Day -14, 5 x 105 MS5-hDLL4 cells were combined with 5 x 104 (H1) or 1 x 105 (ESI017) purified EMPs per ATO in 1.5mL Eppendorf tubes and centrifugated at 300 x g for 5m at 4C in a swinging bucket centrifuge. Multiple (up to 180) EMOs 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 5µL per EMO. 3 EMOs were individually plated (5µL/EMO) on a 0.4mm Millicell transwell insert (EMD Millipore, Billerica, MA, Cat. PIMC0R5G50) and then placed in a 6-well plate containing 1mL of hematopoietic induction medium per well. Medium was changed every 2-3 days for 1 week with medium composed of EGM2 with SB Blocker (10mM). At Day -7, medium was changed to EGM2 + SB Blocker (10mM) with the hematopoietic cytokines, rhTPO 5ng/mL (R&D Systems, Cat. 288-TPN-025), rhFLT3L 5ng/mL (R&D Systems, Cat. 308-FK-025), and rhSCF 50ng/mL (R&D Systems, Cat. 255-SC-200). This medium was changed every 2-3 days for an additional 7 days. At Day 0, ATOs were initiated by simply changing to ATO culture medium (“RB27”) composed of RPMI 1640 (Corning, Manassas, VA, Cat. 10-040-CV), 4% B27 supplement (ThermoFisher Scientific, Cat. 17504044), 30 mM L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate (Sigma-Aldrich, St. Louis, MO, Cat. A8960-5G) reconstituted in PBS, 1% Penicillin-Streptomycin (ThermoFisher Scientific, Cat. 15140122), 1% Glutamax (ThermoFisher Scientific, Cat. 35050061), 10ng/mL rhSCF (R&D Systems), 5ng/mL rhFLT3L (R&D Systems), and 5ng/mL rhIL-7 (R&D Systems, Cat. 207-IL-200). Medium was completely changed every 3-4 days.
For experiments involving the use of engineered stromal lines for ATO T cell differentiation (MS5-hDLL4-A*0201, MS5-hDLL4-A*0201-hB2M, MS5-hDLL4-A*0201-hB2M-ICAM), EMOs were harvested in bulk after 14 days (Day 0) by adding 1mL MACS buffer (PBS, 0.5% bovine serum albumin, 2mM EDTA) to each filter, briefly disaggregating the ATO by scraping with a 1mL “P1000” pipette, and then passed through a 50µm nylon strainer. Harvested cells were counted, and then 2 x 104 cells were combined with 2.5 x 105 engineered MS5-hDLL4 stromal cell lines per ATO in 1.5mL Eppendorf tubes. Cell mixtures were centrifuged at 300 x g for 5m at 4C in a swinging bucket centrifuge prior to formation 3 ATO aggregates (5µL/aggregate) per 0.4mm Millicell transwell insert (EMD Millipore, Cat. PIMC0R5G50), similar to EMO aggregate formation described above.

extract_protocol:
Isolation of ATO-derived SP8: ATOs were harvested by adding MACS buffer (PBS, 0.5% bovine serum albumin, 2mM EDTA) to each filter, briefly disaggregating the ATO by pipetting with a 1mL “P1000” pipette, and then passed through a 50µm nylon strainer; SP8 T cells were harvested after 6 Weeks of T cell differentiation, and DP precursors were harvested after 3 Weeks. For bulk-scale collection of ATOs, aggregates were harvested in a similar fashion; however, up to 150 aggregates were collected in a well filled with MACS buffer and then transferred onto a 50µm nylon filter, where aggregates were physically dissociated on top of the filter using the back end of a sterile 1mL syringe. After dissociation, the filter was washed with MACS buffer, and cell mixtures were centrifuged at 300 x g for 5m at 4C in a swinging bucket centrifuge.
Single cell RNA library preparation and sequencing: ATO-derived T cells were isolated as described above and then FACS-sorted for ATO-derived SP8 T cells from WT (mCD29-CD45+CD8a+CD4-) and 1G4 TCR-transduced (mCD29-mTagBFP2+CD45+CD8a+CD4-) PSCs ATOs into PBS + 0.04% bovine serum albumin. For ESI017 WT PSCs differentiated in MS5-hDLL4 stroma ATOs, SP8 and CD45+ fractions were both collected. Cells were counted and resuspended at a concentration of ~1000 cells/µL and provided to the Technology Center for Genomics and Bioinformatics (TCGB) core for unique molecular identifier (UMI) tagging and generation of gene expression (GEX) and human TCR repertoire (VDJ) libraries using the 10X Chromium Next GEM Single Cell V(D)J Reagent Kit v1.1 (10X Genomics, Pleasanton, CA). Similarly, peripheral blood (PB) SP8s were FACS-isolated (CD45+CD8A+CD8B+CD4-TCRab+CD3+) from PB mononuclear cells obtained from healthy donors, and provided to the TCGB core for UMI tagging and library generation. Fully constructed libraries for all samples were run in one S4 flowcell on the Illumina Novaseq Platform.
5' Gene Expression (GEX), VDJ, and Feature Barcoding libraries were constructed according to the manufacturer's instructions (Chromium Next GEM Single Cell 5' Reagent Kits v2, 10X Genomics).

treatment_protocol: Cell Lines: The MS5-hDLL4 cell line was generated in our lab as previously described12. Briefly, MS5 cells45 were transduced with a lentiviral vector encoding full-length human DLL4 (hDLL4). The highest 5% DLL4-expressing cells were isolated by FACS using an anti-hDLL4 antibody and passaged in DMEM with 10% fetal calf serum (FCS). Stable expression was confirmed by flow cytometry for hDLL4 expression after several weeks of culture, as well as qRT-PCR and RNA sequencing. For the cell lines including Class I MHC and scaffolding proteins, the previously-derived MS5-hDLL4 line was transduced with varying combinations of individual lentiviruses encoding human HLA-A*0201, hB2M, and hICAM1. The highest 5% of transduced cells were isolated by FACS using antibodies detecting HLA-A*0201, hB2M, and hICAM1 and passaged in DMEM with 10% FCS for expansion and cryopreservation. Stable expression was also confirmed by flow cytometry for hDLL4, HLA-A*0201, hB2M, and ICAM1.  Gene editing of human pluripotent stem cell lines. CRISPR/Cas9 gene editing of PSCs was performed with ribonucleoprotein complexes57 (RNPs) of purified spCas9-NLS (QB3 MacroLab, UC Berkeley, Berkeley, CA) and custom synthesized single guide RNAs (gRNAs) (Synthego, Mountain View, CA). spCas9-NLS and gRNAs were stored at -80C prior to use for gene editing of PSCs. Briefly, PSCs were allowed to grow in wells of a Matrigel-coated 6-well plate until reaching ~70% confluency before being dissociated with TrypLETM Express (ThermoFisher Scientific, Cat. 12604-013) and resuspended in single cell solution. Prior to nucleofection, 60pmol spCas9 and individual 84pmol gRNA were complexed together at a ratio of 1pmol spCas9:1.4pmol gRNA for 15m at room temperature. PSCs were resuspended at a concentration of 2 x 105 cells in 14µL of P4 Primary Cell Nucleofector® Solution (P4 Primary Cell 4D-NucleofectorTM X Kit S, Lonza, Cat. V4XP-4032).   For single gRNA reactions, fully complexed RNPs (60pmol spCas9:84pmol gRNA) were added to resuspended cells and the volume was brought up to 20µL using P4 Primary Cell Nucleofector® Solution (Lonza). For dual gRNA reactions, RNPs were complexed individually and then added (2 x 60pmol spCas9 total) into the cell suspension with a custom-synthesized single stranded oligo donor template (ssODN, 100bp, resuspended at 100µM) (Ultramer® DNA Oligo, Integrated DNA Technologies, Skokie, IA) to a final concentration of 3µM in solution. 20µL of combined PSC, RNP, and/or ssODN solutions were added into individual wells of the Lonza 16-well Nucleocuvette® Strip (Lonza). Nucleofection was performed on the Lonza 4D-Nucleofector® Core and X Unit (Lonza, Cat. AAF1003B and AAF-1003X) using pulse and frequency code “CB-150”. Cells were allowed to rest in the cuvette for 10m before transferring into 1.5mL mTeSRTM Plus and ROCK inhibitor Y-27632 dihydrochloride (10µM), and then plated in 1 well of a Matrigel-coated 12-well plate. Culture medium was changed daily, and ROCK inhibitor Y-27632 dihydrochloride (10µM) was removed from medium after 48hr. Edited PSCs were allowed to reach ~70% confluency before being expanded for single cell cloning and cryopreservation.  Knockout of RAG1 and RAG2 in both H1 and ESI017 parent lines was achieved using a gene ablation strategy that excised the ~30KB region on Chromosome 11 in which their coding regions were located using both RAG1 CRISPR #4 and RAG2 CRISPR #14 gRNAs (Supplementary Fig. 1C-D), and a 100bp ssODN HDR template (5’-TTTTCATTGTTCTCAGGTACCTCAGCCAGCATGGCAGCCTCTTTCCCAC CCCCTGGTATTGCTGGAGCCTCTCCTGGGGACTTTTGAACAGGTGACCCGA-3’) (Supplementary Fig. 1G). Complete ablation of RAG1 and RAG2 genes was confirmed by PCR amplification of a 693bp sequence within the excised region in PSC clones (RAG PCR #1, FWD: 5’- tgtatactgggacccttggggag-3’, REV: 5’- agaattcccacagatgcggcagag-3’); proper rejoining of the chromosomal ends was confirmed with PCR amplification of a novel 609bp product using primers that targeted sequences outside of the ~30KB region excised by gene-editing in PSC clones (RAG PCR #2, FWD: 5’-tgtatactgggacccttggggag-3’, REV: 5’-gtcacggcttttgtaacctcgg-3’) (Supplementary Fig. 1G-H). Knockout of B2M in DKO lines was achieved by introducing INDELs at the beginning of its coding sequence (Exon 1 with B2M gRNA #3, and polyclonal knockout lines were purified via FACS (Supplementary Fig. 4C-F).   Transduction of human pluripotent stem cell lines: NYESO TCR-transduced PSC lines were generated by transduction of unedited or knockout H1 or ESI017 PSCs with either lentiviral vectors encoding the 1G4 TCR (HLA-A*0201-restricted recognizing NYESO157-165 peptide) or the F5 TCR (HLA-A*0201-restricted recognizing MART126-35 peptide) and the fluorescent marker mTagBFP2. Briefly, PSCs were dissociated into single cell suspension and plated at a density of 2 x 105 cells/well of a Matrigel-coated 6-well plate in mTeSRTM Plus culture medium with ROCK inhibitor Y27632 dihydrochloride (10µM). The following day, culture medium was changed to 1mL of mTeSRTM, and concentrated lentiviral supernatant was added directly into the wells. Medium was changed each day until cells reached ~70% confluency, when cells were dissociated with TypLETM Express (ThermoFisher Scientific, Cat. 12604-013) and purified via FACS sorting using the following phenotype: TRA1-81+mTagBFP2+. Isolated cells were returned to culture on Matrigel-coated 6-well plates and mTeSRTM Plus culture medium for expansion and cryopreservation.

value: ESI017 WT ESO hPSC

value: WT, transduced to express Opt1G4 TCR

value: Total single positive CD8+ (SP8+, including CD45RO and CD45RA) T cells, derived in vitro from PSCs

value: Unedited

value: TCR-transduced

value: artificial thymic organoid

tag: time

value: 6 Weeks

tag: library type

value: VDJ


title: CD8 cells treated with Vorinostat under dose 0.5 uM, replicate 2

value: splenic

value: CD8 cell


title: CD8 cells, stimulated control, replicate 1


title: T98G cell,sh-NC2

value: glioma cells


title: ESI017 WT+1G4TCR SP8 (hDLL4 Stroma), replicate 2, GEX scRNA-seq

description: 10X Genomics

value: 5' GEX


title: CD8 cells treated with Vorinostat under dose 10 uM, replicate 1

value: Vorinostat, 10 uM


title: UPS sample 4 male

value: Balb/cByj

value: left hippocampus

value: UPS

value: Male


title: Healthy Human PBMC SP8, replicate 1, GEX scRNA-seq

description: 10X Genomics

source_name: Peripheral Blood Mononuclear Cells (PBMCs)

value: Healthy Donor

value: Total single positive CD8+ (SP8+) T cells

value: No TCR Tranduction


title: UPS sample 5 female

value: Female


title: ESI017 WT+1G4TCR SP8 (A02BI Stroma), replicate 3, VDJ scRNA-seq

description: 10X Genomics

value: ESI017 WT ESO hPSC

value: WT, transduced to express Opt1G4 TCR

value: Total single positive CD8+ (SP8+, including CD45RO and CD45RA) T cells, derived in vitro from PSCs

value: Unedited

value: TCR-transduced


title: p10 PP CINI 2

city: Dresden

first: Anthony

last: Gavalas

address: Fetscherstr. 105

institute: TU Dresden

data_processing:
bcl2fastq2:  version 2.17.1.14
alignment:  Sequencing data was aligned to the human reference hg38 (obtained from Ensembl) with the aligner gsnap (2017-06-20). In order to detect splices sites Ensembl gene annotation version 87 was used during the alignment step (gsnap.sse42 -d hg38 --gunzip -A sam -t 10 --use-sarray=1 --input-buffer-size=500000 --output-buffer-size=500000 -B 5 -N 1 -n 1 -s EnsemblGene-87.ss.hg38.iit)
gene counts: Uniquely mapped reads were assigned to gene with the tool featureCounts (1.5.2) (featureCounts -a EnsemblGene-87.hg38.TR.gtf -s 2 -o genecount/bfx783.hg38.e87.txt --primary -Q 1 -T 8)
Assembly: hg38
Supplementary files format and content: bfx783.counts.tsv.gz (This is a tab separated file. Each row is a gene with shows the number of counts assigned to samples. Each column is a sample and several columns provide gene information (e.g. gene ID, chromosome, start, end, strand, biotype))

source_name: H1 (W01) hES cell line

extract_protocol:
Qiagen RNeasy
Total RNA with an integrity number of ≥ 8.8 was used. mRNA was isolated from 1 ug total RNA by poly-dT enrichment using the NEBNext Poly(A) mRNA Magnetic Isolation Module according to the manufacturer’s instructions. Final elution was done in 15ul 2x first strand cDNA synthesis buffer (NEBnext, NEB). After chemical fragmentation by incubating for 15 min at 94°C the sample was directly subjected to the workflow for strand specific RNA-Seq library preparation (Ultra Directional RNA Library Prep, NEB). For ligation custom adaptors were used 1: (Adaptor-Oligo 5'-ACA CTC TTT CCC TAC ACG ACG CTC TTC CGA TCT-3', Adaptor-Oligo 2: 5'-P-GAT CGG AAG AGC ACA CGT CTG AAC TCC AGT CAC-3'). After ligaton adapters were depleted by an XP bead purification (Beckman Coulter) adding bead in a ratio of 1:1. Indexing was done during the following PCR enrichment (15 cycles) using custom amplification primers carring the index sequence indicated with ‘NNNNNN’. (Primer1: Oligo_Seq AAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCG ATC T, primer2: GTG ACT GGA GTT CAG ACG TGT GCT CTT CCG ATC T, primer3: CAA GCA GAA GAC GGC ATA CGA GAT NNNNNN GTG ACT GGA GTT. After two more XP beads purifications (1:1) libraries were quantified. For Illumina flowcell production, samples were equimolarly pooled and distributed on all lanes used for 75bp single read sequencing on Illumina HiSeq 2500.

treatment_protocol: as described in publication

value: H1 (W01) hES cell line

value: pancreas progenitors

value: wild type

value: CINI

tag: passage


title: p10 PP CINI 3


title: p10 PP CINI 1


title: ESI017 TKO8+1G4TCR SP8 (A02BI Stroma), replicate 3, VDJ scRNA-seq

description: 10X Genomics

source_name: ESI017 TKO8 ESO hPSC

value: ESI017 TKO8 ESO hPSC

value: RAG/RAG2/B2M Triple Knockout (TKO), transduced to express Opt1G4 TCR

value: RAG1/RAG2/B2M Knockout

value: artificial thymic organoid

value: 6 Weeks

value: VDJ


title: ESI017 WT+1G4TCR SP8 (A02BI Stroma), replicate 3, GEX scRNA-seq

description: 10X Genomics

value: 5' GEX


title: CD8 cells treated with Romidepsin under dose 30 nM, replicate 2

description: Mouse splenic CD8 cells treated with Romidepsin under dose 30 nM

value: splenic

value: CD8 cell

value: Romidepsin, 30 nM


title: CD8 cells treated with Romidepsin under dose 30 nM, replicate 1


title: CD8 cells treated with Romidepsin under dose 2 nM, replicate 2

description: Mouse splenic CD8 cells treated with Romidepsin under dose 2 nM

value: Romidepsin, 2 nM


title: CD8 cells treated with Romidepsin under dose 2 nM, replicate 1


title: UPS sample 3 female

value: Balb/cByj

value: left hippocampus

value: UPS

value: Female


title: ESI017 TKO8+1G4TCR SP8 (A02BI Stroma), replicate 3, GEX scRNA-seq

description: 10X Genomics

value: Total single positive CD8+ (SP8+, including CD45RO and CD45RA) T cells, derived in vitro from PSCs

value: TCR-transduced


title: UPS sample 2 male

value: Male


title: UPS sample 2 female


title: ESI017 WT SP8 (hDLL4 Stroma), replicate 1, VDJ scRNA-seq

description: 10X Genomics

source_name: ESI017 WT hPSC

value: ESI017 WT hPSC

value: WT hPSC

value: Unedited

value: No TCR Transduction


title: T98G cell,sh-NC1

value: glioma cells

value: NC


title: UPS sample 4 female


title: ESI017 WT Total 45+ (hDLL4 Stroma), replicate 1, VDJ scRNA-seq

description: 10X Genomics

value: Total CD45+ cells, derived from in vitro PSCs

value: artificial thymic organoid

value: 6 Weeks

value: VDJ


title: p5 PP CINI 3

value: H1 (W01) hES cell line

value: pancreas progenitors

value: wild type

value: CINI


title: UPS sample 3 male

value: Balb/cByj

value: left hippocampus

value: UPS


title: UPS sample 1 male

value: Male


title: ESI017 WT Total 45+ (hDLL4 Stroma), replicate 1, GEX scRNA-seq

description: 10X Genomics

value: ESI017 WT hPSC

value: WT hPSC

value: 5' GEX


title: CD8 cells treated with Romidepsin under dose 10 nM, replicate 1

description: Mouse splenic CD8 cells treated with Romidepsin under dose 10 nM

value: splenic

value: CD8 cell

value: Romidepsin, 10 nM


title: CD4 cells treated with Vorinostat under dose 10 uM, replicate 2

description: Mouse splenic CD4 cells treated with Vorinostat under dose 10 uM

source_name: Mus musculus splenic CD4 cells

value: CD4 cell

value: Vorinostat, 10 uM


title: UPS sample 1 female

value: LB

value: Female


title: p5 PP CINI 1

value: H1 (W01) hES cell line

value: pancreas progenitors


title: CD4 cells treated with Vorinostat under dose 2 uM, replicate 2

description: Mouse splenic CD4 cells treated with Vorinostat under dose 2 uM

value: Vorinostat, 2 uM


title: CD4 cells treated with Vorinostat under dose 2 uM, replicate 1


title: CD8 cells treated with Romidepsin under dose 10 nM, replicate 2


title: p0 PP CINI 3

value: wild type

value: CINI


title: ESI017 WT SP8 (hDLL4 Stroma), replicate 1, GEX scRNA-seq

description: 10X Genomics

value: Total single positive CD8+ (SP8+, including CD45RO and CD45RA) T cells, derived in vitro from PSCs

value: Unedited

value: No TCR Transduction

value: artificial thymic organoid

value: 6 Weeks


title: CD4 cells treated with Vorinostat under dose 10 uM, replicate 1

value: splenic


title: LB sample 8 male

value: Balb/cByj

value: left hippocampus

value: Male


title: p0 PP CINI 2


title: p5 PP CINI 2


title: ESI017 WT+1G4TCR SP8 (A02BI Stroma), replicate 2, VDJ scRNA-seq

description: 10X Genomics

value: ESI017 WT ESO hPSC

value: WT, transduced to express Opt1G4 TCR

value: TCR-transduced

value: VDJ


title: p0 PP CINI 1

value: H1 (W01) hES cell line

value: pancreas progenitors


title: ESI017 TKO8+1G4TCR SP8 (A02BI Stroma), replicate 2, VDJ scRNA-seq

description: 10X Genomics

value: ESI017 TKO8 ESO hPSC

value: RAG/RAG2/B2M Triple Knockout (TKO), transduced to express Opt1G4 TCR

value: RAG1/RAG2/B2M Knockout


title: ESI017 WT+1G4TCR SP8 (A02BI Stroma), replicate 2, GEX scRNA-seq

description: 10X Genomics

value: Total single positive CD8+ (SP8+, including CD45RO and CD45RA) T cells, derived in vitro from PSCs

value: Unedited

value: 5' GEX


title: LB sample 7 male

value: LB


title: HeLa cells, siSRSF2, rep2

data_processing:
Paired-end RNA-Seq raw reads were analyzed with FastQC (v0.11.5) and then filtered by AfterQC (v0.9.6) with the default parameters to remove low-complexity reads. The Trimmomatic tool (v0.33) was then used to trim adapter sequences and filter reads smaller than 35 bp. The resulting fastq data were again analysed with FastQC to ensure that no further processing was needed.
The clean reads were aligned to the hg19 genome with the STAR algorithm (v2.6.1d) using the reference transcriptome based on Ensembl v85 and LNCipedia v5.2.
Read count extraction was performed using HTseq tool.
Assembly: hg19
Supplementary files format and content: tab-delimited text files include raw read count for each sample

source_name: HeLa

extract_protocol:
Total RNA was purified with the RNeasy kit (Qiagen) and treated with the RNase-Free DNase Set (Qiagen) to remove the residual DNA. Total RNA samples from HeLa cells was then subjected to rRNA depletion using Ribominus Human/Mouse Transcriptome Isolation Kit (Invitrogen). Enrichment of mRNA from total RNA sample in K562 cells was performed using GenElute™ mRNA Miniprep Kit (Sigma-Aldrich).
RNA libraries for RNA-seq were prepared using KAPA Stranded mRNA-Seq kit according to the manufacturer’s instructions.

value: HeLa

value: human cervical cancer cell line

value: SRSF2 knockdown


title: ESI017 TKO8+1G4TCR SP8 (A02BI Stroma), replicate 2, GEX scRNA-seq

description: 10X Genomics

value: artificial thymic organoid

value: 6 Weeks


title: LB sample 7 female

value: Balb/cByj

value: left hippocampus

value: Female


title: HeLa cells, siSRSF2, rep1


title: p0 PP CINI 4

value: H1 (W01) hES cell line

value: wild type

value: CINI


title: CD4 cells treated with Vorinostat under dose 0.5 uM, replicate 1

description: Mouse splenic CD4 cells treated with Vorinostat under dose 0.5 uM

value: splenic

value: CD4 cell

value: Vorinostat, 0.5 uM


title: CD4 cells, stimulated control, replicate 2

description: Mouse splenic CD4 cells, stimulated control

value: stimulated control


title: HeLa cells, siNSUN2, rep2

value: NSUN2 knockdown


title: LB sample 9 female

value: LB


title: LB sample 6 female


title: CD4 cells treated with Vorinostat under dose 0.5 uM, replicate 2


title: LB sample 8 female


title: CD4 cells treated with Romidepsin under dose 30 nM, replicate 2

description: Mouse splenic CD4 cells treated with Romidepsin under dose 30 nM

value: Romidepsin, 30 nM


title: HeLa cells, siNSUN2, rep1

value: HeLa

value: human cervical cancer cell line


title: ESI017 WT+1G4TCR SP8 (hDLL4 Stroma), replicate 1, VDJ scRNA-seq

description: 10X Genomics

value: ESI017 WT ESO hPSC

value: WT, transduced to express Opt1G4 TCR

value: Total single positive CD8+ (SP8+, including CD45RO and CD45RA) T cells, derived in vitro from PSCs

value: Unedited

value: TCR-transduced

value: artificial thymic organoid

value: VDJ


title: LB sample 5 male

value: Balb/cByj

value: left hippocampus

value: Male


title: HeLa cells, siCtrl, rep2

value: WT


title: LB sample 5 female

value: Female


title: CD4 cells treated with Romidepsin under dose 30 nM, replicate 1

value: splenic

value: CD4 cell


title: ESI017 WT+1G4TCR SP8 (A02BI Stroma), replicate 1, GEX scRNA-seq

description: 10X Genomics

value: 6 Weeks

value: 5' GEX


title: HeLa cells, siCtrl, rep1

value: HeLa

value: human cervical cancer cell line


title: LB sample 4 male

value: LB


title: Morex8DAP, Input, rep1A

city: Olomouc

first: Šimon

last: Pavlů

address: Šlechtitelů 31

department: Centre of Plant Structural and Functional Genomics

country: Czech Republic

institute: Institute of Experimental Botany of the Czech Academy of Sciences

data_processing:
Reads from the ChIP-seq pipeline went through qualitative trimming by Trim Galore (v. 0.6.2).
Mapping to the MorexV3 reference genome was performed using the Bowtie 2 (v. 2.4.2).
Duplicated reads were then removed using Picard tools (v. 2.9.0).
Peaks were called using the MACS2 software.
Assembly: MorexV3
Supplementary files format and content: bigWig

organism: Hordeum vulgare

source_name: 8DAP embryo

extract_protocol: NEBNextUltraII library preparation kit

value: embryo

tag: developmental stage

value: morex

tag: chip antibody


title: ESI017 WT+1G4TCR SP8 (hDLL4 Stroma), replicate 1, GEX scRNA-seq

description: 10X Genomics

value: ESI017 WT ESO hPSC

value: WT, transduced to express Opt1G4 TCR

tag: cell type

value: Total single positive CD8+ (SP8+, including CD45RO and CD45RA) T cells, derived in vitro from PSCs

value: Unedited

value: TCR-transduced


title: CD4 cells treated with Romidepsin under dose 2 nM, replicate 2

description: Mouse splenic CD4 cells treated with Romidepsin under dose 2 nM

value: Romidepsin, 2 nM


title: CD4 cells, stimulated control, replicate 1

value: stimulated control


title: LB sample 4 female

value: Balb/cByj

value: left hippocampus

value: Female


title: LB sample 3 male

value: Male


title: Morex8DAP, Input, rep2


title: ESI017 WT+1G4TCR SP8 (A02BI Stroma), replicate 1, VDJ scRNA-seq

description: 10X Genomics

value: artificial thymic organoid

value: 6 Weeks

value: VDJ


title: CD4 cells treated with Romidepsin under dose 2 nM, replicate 1

value: splenic

value: CD4 cell


title: CD4 cells treated with Romidepsin under dose 10 nM, replicate 2

description: Mouse splenic CD4 cells treated with Romidepsin under dose 10 nM

value: Romidepsin, 10 nM


title: CD4 cells treated with Romidepsin under dose 10 nM, replicate 1


title: Morex8DAP, Input, rep1

value: embryo

value: morex


title: ESI017 TKO8+1G4TCR SP8 (A02BI Stroma), replicate 1, GEX scRNA-seq

description: 10X Genomics

value: ESI017 TKO8 ESO hPSC

value: RAG/RAG2/B2M Triple Knockout (TKO), transduced to express Opt1G4 TCR

value: Total single positive CD8+ (SP8+, including CD45RO and CD45RA) T cells, derived in vitro from PSCs

value: RAG1/RAG2/B2M Knockout

value: 5' GEX


title: Jurkat cells, 1 uM Brequinar, 24 hours, replicate 3

city: Cambridge

first: Gabriela

last: Alexe

state: MA

address: 415 Main St.

department: Computational Biology and Bioinformatics

institute: Broad Institute

data_processing:
RNA-Seq data analysis was performed in alignment with the ENCODE Consortium standards (https://www.encodeproject.org/chip-seq/).
Quality control tests for unmapped reads were performed based on the FastQC v.0.11.9 software (Babraham Bioinformatics, http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and summarized with multiQC v1.9.30 The human reads were mapped to hg19/gencode v19 genome  using STAR v2.7.2b31  with standard parameters  --outSAMtype BAM SortedByCoordinate --outSAMunmapped None      --outSAMattributes NH HI NM MD AS XS --outReadsUnmapped Fastx --outSAMstrandField intronMotif --quantMode TranscriptomeSAM GeneCounts --quantTranscriptomeBan IndelSoftclipSingleend. Quality control for the mapped reads and for replicate reproducibility were performed using SARTools v1.7.3.32
Gene level reads were summarized by counting the reads that overlapped the hg19/gencode v19 gene exons by using the featureCounts v1.6.3 method implemented in the Subread v2.0.0 package (http://subread.sourceforge.net).33 Gene counts were normalized and used to quantify the differential genes between the experimental and control conditions using DESeq2 v1.32.0.34
Gene expression was estimated based on the log2(TPM+1) scores for normalized reads.35 The expressed genes were identified as the genes with  maximum log2(TPM+1)  expression ≥ 1 across all conditions. The gene differentiability was assessed with DESeq2 based on the robust shrunken log2 fold change scores  and  the approximate posterior estimation for GLM coefficients (apeglm v1.6)  method for effect size.34 The cutoffs for gene differentiability were |fold change expression| ≥ 1.5 and adj P-value ≤ 0.10. Heatmaps for the visualization of transcriptional changes induced by treatment were created by using the Morpheus software platform (https://software.broadinstitute.org/morpheus/).
Assembly: hg19 (gencode v19)
Supplementary files format and content: tab-delimited text files include counts and log2(TPM+1) values for each sample

source_name: Jurkat

extract_protocol:
Total RNA was harvested from 5x106 Jurkat cells either treated with 1uM Brequinar or with DMSO, over 8 hours and 24 hours, using an RNeasy-Plus Mini Kit (Qiagen).
Following RNA isolation, samples were sent to Novogene for library preparation and sequencing on Illumina NovaSeq 6000.

value: Jurkat

value: immortalized line of T lymphocyte cells

tag: genotype

value: WT

value: 1 uM Brequinar

value: 24 hours


title: ESI017 TKO8+1G4TCR SP8 (A02BI Stroma), replicate 1, VDJ scRNA-seq

description: 10X Genomics

value: TCR-transduced

value: artificial thymic organoid

value: 6 Weeks


title: LB sample 3 female

tag: strain

value: Balb/cByj

value: left hippocampus

value: LB

value: Female


title: Jurkat cells, 1 uM Brequinar, 24 hours, replicate 2


title: LB sample 2 male

value: Male


title: Morex8DAP, H3K27me3, rep2

value: embryo

value: morex


title: Jurkat cells, 1 uM Brequinar, 24 hours, replicate 1