title: iPSC-derived OPCs, control_1, CT30, differtiation 3, excluded from analysis

city: Palo Alto

first: Erin

middle: M

last: Gibson

address: 3165 Porter Drive

department: Psychiatry and Behavioral Sciences

institute: Stanford University

description: Library name: TDR72Z_30_CT30_300_3

data_processing:
Data quality control and comparisons were conducted by the Plasmidsaurus online analysis platform in November 2025.  In short, quality of the fastq files was assessed using FastQC v0.12.1. Reads were then quality filtered using fastp v0.24.0 with poly-X tail trimming, 3' quality-based tail trimming, a minimum Phred quality score of 15, and a minimum length requirement of 50 bp. Quality-filtered reads were aligned to the reference genome using STAR aligner v2.7.11 with non-canonical splice junction removal and output of unmapped reads, followed by coordinate sorting using samtools v1.22.1. PCR. Optical duplicates were removed using UMI-based deduplication with UMIcollapse v1.1.0. Alignment quality metrics, strand specificity, and read distribution across genomic features were assessed using RSeQC v5.0.4 and Qualimap v2.3. Gene-level expression quantification was performed using featureCounts (subread package v2.1.1) with strand-specific counting, multi-mapping read fractional assignment, exons and three prime UTR as the feature identifiers, and grouped by gene_id. Final gene counts were annotated with gene biotype and other metadata extracted from the reference GTF file. Differential expression was done with edgeR v4.0.16 using standard practice including filtering for low-expressed genes with edgeR::filterByExprwith default values and FDR values were used for further analysis. Samples CT24_300_3, CT24_021_3, CT30_300_3, CT30_021_3, CT36_300_3, CT36_021_3, CT42_300_3, CT42_021_3, CT42_008_3 were excluded from the analysis.
Assembly: GRCh38
Supplementary files format and content: Expression_matrix.tsv includes counts and CPM values for every sample

growth_protocol: Human inducible pluripotent stem cells (hiPSC) from RRMS (relapsing remitting, early-stage MS, n=3) and SPMS (secondary progressive, late-stage MS, n=3) MS patients, and age/sex-matched healthy controls (n=3 control) were acquired from the New York Stem Cell Foundation (see Suppl. Fig. 12A for line details) and differentiated towards OPCs, as previously described132. In short, iPSCs were cultured in 6-well plates coated with matrigel (Corning 354230, 1:100) in iPSC culture media comprising Miltenyi StemMACS iPS-brew XF with supplement (Miltenyi 130-104-368) and 1% Antibiotic Antimycotic Solution (Sigma). ROCK inhibitor Y-27632 (10 µM, Tocris) was added to the iPSC media for the first 24 hours. Once the iPSCs reached 70–80% confluency, cells were detached with pre-warmed EDTA (0.5 mM) for passaging, or pre-warmed accutase (SIGMA A6964) for embryoid bodies (EBs) formation. To form EBs, cells were resuspended in iPSC media supplemented with ROCK inhibitor and seeded (~ 1.5 × 10⁶ cells) in AggreWell™400 microwell culture plates (Stem Cell Technologies 34815) overnight. The EBs were then transferred to 10-cm petri dishes (Falcon 351029), placed on a rotary shaker, and cultured for 7 days in a chemically defined medium (CDM): 1:1 mix of F12 and Iscove’s modified Dulbecco’s medium (Invitrogen) with chemically defined Lipid concentrate 100 (Invitrogen), monothioglycerol (450 µM, Sigma), BSA (5 mg/ml, Sigma), transferrin (15 mg/ml, Roche), insulin (7 mg/ml, Roche), and 1% Antibiotic Antimycotic Solution. N-acetyl cysteine (1 mM, Sigma), Activin inhibitor SB 431542 (10 µM, Sigma), and LDN193189 (0.1 µM, Stratech) were added for neural induction. Next, the neurospheres formed from the EBs were caudalized for 7 days in CDM supplemented with N-acetyl cysteine (1 mM, Sigma), heparin (5 µg/ml, Sigma), retinoic acid (0.1 µM, Sigma), and basic fibroblast growth factor (FGF-2; 10 ng/ml, PeproTech). For morphological assessment of neural conversion, the cells were plated on plates coated with laminin (10 μg/ml, Sigma). Post-neural induction, the spheres were cultured in advanced DMEM medium containing 0.5% GlutaMAX (Invitrogen), 5 µg/ml heparin (Sigma), 1% N2 (Invitrogen), 1% B27 (Invitrogen), and 1% Antibiotic Antimycotic Solution. To initiate ventralization, purmorphamine (1 µM, Calbiochem), retinoic acid (1 µM, Sigma), and FGF-2 (10 ng/ml, PeproTech) were added to the media for 7 days, after which FGF-2 was removed for 14 days. hOPC proliferation was then promoted by supplementing the medium with FGF-2 (10 ng/mL), T3 (60 ng/mL, Sigma), PDGFα (20 ng/mL, PeproTech), SAG (1 µM, Calbiochem), purmorphamine (1 µM, Sigma), and IGF-1 (10 ng/mL, PeproTech). After 14 days, spheres were dissociated using the Worthington papain dissociation system (LK003150 per manufacturer’s instructions) and single cells were seeded in matrigel-coated T-75 Flasks (CELLTREAT 229341). After 24 hours, cells were purified for PDGFRα by MACS using PE-conjugated CD140a antibody (Miltenyi 130-115-238) and Anti-PE MicroBeads (Miltenyi 130-048-801) according to the manufacturer’s instructions.

extract_protocol:
Bulk RNA sequencing was performed by Plasmidsaurus using Illumina Sequencing Technology.
1.Poly-A mRNA capture and reverse transcription for cDNA synthesis: oligo-dT primer to capture just the mRNA (contains sample barcode, UMI, and Read 1 sequences). 2. Second strand synthesis and tagmentation: double stranded-cDNA is generated. Tagmentation creates fragments and incorporates Read 2 sequence. 3.Illumina library amplification: adds unique dual indices (UDI = i5, i7) and P5/P7 sequences.

treatment_protocol: PDGFRα+ cells were seeded in matrigel-coated 6-well plates and allowed to recover for 24 hours before synchronization with dexamethasone (100nM, 1h, Sigma-Aldrich D4902). Following complete media change, cells were harvested at 24h (CT24), 30h (CT30), 36h (CT36), or 42h (CT42). Cells lysates were preserved in Zymo DNA/RNA Shield (ZYMO RESEARCH R1100-50) for bulk RNA sequencing performed by Plasmidsaurus.

value: iPSC-derived OPCs

value: differentiation 3


title: TotalseqB_NEA6_cDNA_1

status: Public on Aug 07 2026

type: SRA

city: San Francisco

first: Gian Carlo

middle: L.

last: Baldonado

state: CA

address: 2340 Sutter Street

department: Dermatology

country: USA

institute: University of California San Francisco

description:
Library name: TotalseqB_NEA6_cDNA_1
TotalSeq-B CITE-seq cDNA gene expression library; pooled PBMCs; donors demultiplexed using Demuxlet

library_source: transcriptomic

data_processing:
Raw FASTQ files were processed using Cell Ranger v8.0 (10x Genomics).
Cell Ranger count was run with the GRCh38 reference genome (refdata-gex-GRCh38-2020-A) and a custom TotalSeq-B antibody feature reference for ADT.
Donor demultiplexing was performed using Demuxlet with matched genotyping data.
Quality filtering, normalization, dimensionality reduction, and clustering performed using Seurat v5 with Weighted Nearest Neighbor (WNN) analysis for joint RNA and protein modalities.
Cell type annotation performed manually based on canonical marker gene and protein expression.
Assembly: GRCh38 (hg38)
Supplementary files format and content: MEX format: barcodes.tsv.gz (cell barcodes), features.tsv.gz (gene and ADT features), matrix.mtx.gz (count matrix) output by Cell Ranger v8.0. Each file prefixed with sample name for unique identification.
Supplementary files format and content: feature_reference.csv: TotalSeq-B antibody panel feature reference used by Cell Ranger.

molecule: polyA RNA

organism: Homo sapiens

source_name: peripheral blood

extract_protocol:
Gene expression and ADT libraries were prepared using the 10x Genomics Chromium Single Cell 5' Library & Gel Bead Kit with Feature Barcode Technology. Libraries were sequenced on an Illumina NovaSeq 6000.
PBMCs from multiple donors were multiplexed per sequencing batch using hashtag oligonucleotides (HTOs) and loaded onto the 10x Genomics Chromium Controller.

treatment_protocol: PBMCs were isolated from whole blood using Ficoll density gradient centrifugation and cryopreserved. Cells were thawed, washed, and stained with a TotalSeq-B antibody panel (BioLegend) per manufacturer protocol, then loaded onto the 10x Genomics Chromium Controller. Donors were multiplexed per batch using hashtag oligonucleotides (HTOs).

tag: tissue

value: peripheral blood

tag: cell type

value: PBMC

tag: batch


title: Experiment 2_LV_P_29

status: Public on Aug 06 2026

city: Leiden

first: Sen

last: Zhang

address: Albinusdreef 2, Einthoven Laboratory, D4-29

country: Netherlands

institute: Leiden Univesity Medical Center

description:
Library name: LV_P_29
column X

data_processing:
The raw data end reads were trimmed and quality controlled by FASTQ with default parameters (https://github.com/OpenGene/fastp), and the clean reads were separately aligned to the reference genome (GRCm38) with orientation mode (5′ to 3′ orientation) using the HISAT2 software (http://ccb.jhu.edu/software/hisat2/index.shtml). The overall consistency of Q20% and GC% across all samples indicates a stable, consistent sequencing run. Q20 values ranged from 96.21–98.40% and GC content ranged from 46.18–48.39% across all samples, indicating consistent sequencing quality. The mapped reads of each sample were assembled by StringTie in a reference-based approach (https://ccb.jhu.edu/software/stringtie/). To identify differentially expressed genes (DEGs) between the vehicle and SXB groups, the expression levels of each gene were calculated according to the transcripts per million reads (TPM) method. RSEM (http://deweylab.biostat.wisc.edu/rsem/) was used to quantify gene abundance. Analysis of DEGs was performed using DESeq2, and the resulting P-values were adjusted for multiple comparisons using the Benjamini–Hochberg FDR method. DEGs are defined as fold changes > 2 and adjusted P-values (padj) P < 0.05. Volcano plots were generated using the EnhancedVolcano feature (https://github.com/kevinblighe/EnhancedVolcano). KEGG pathway enrichment analysis was carried out by KOBAS (http://kobas.cbi.pku.edu.cn/home.do). GSEA was conducted to identify gene sets that share common biological function and significantly enriched gene sets were annotated based on the Molecular Signatures Database.
Assembly: clean reads were separately aligned to the reference genome (GRCm38) with orientation mode (5′ to 3′ orientation) using the HISAT2 software (http://ccb.jhu.edu/software/hisat2/index.shtml)
Supplementary files format and content: Liver RNA-seq_processed data_P.xlsx
Supplementary files format and content: Liver RNA-seq_processed data_T.xlsx

molecule: total RNA

organism: Mus musculus

source_name: Liver

extract_protocol:
TRIzol RNA isolation reagent
The Illumina HiSeq 2500 platform was used to construct RNA libraries and generate reads of 2 × 150 bp long paired ends (Illumina, San Diego, CA, USA).

value: Liver

tag: genotype

tag: treatment


title: SW1463 50nM Soto 6 Days_3

city: Göttingen

first: Jonas

last: Buchloh

address: Robert-Koch-Straße 40

country: Germany

institute: University Medicine Göttingen

description: Library name: Soto_6d_3

data_processing:
Reads were aligned with the STAR aligner (version v2.7.11) based on the GRCh38 genome
Counts were quantified using Salmon (version v1.10.0)
Assembly: GRCh38
Supplementary files format and content: FASTQ format

source_name: Cell line

extract_protocol:
RNA isolation was conducted with Rneasy Mini Kit (Qiagen, #74106).
RNA sequencing library was prepared with PE150.

tag: cell line

value: Sotorasib


title: Experiment 2_LV_P_28

description:
Library name: LV_P_28
column W


title: SW1463 50nM Soto 6 Days_1

description: Library name: Soto_6d_1


title: Experiment 1_LV_T_10

description:
Library name: LV_T_10
column J


title: SW1463, 50nM Soto, 24 Hours_3

description: Library name: Soto_24h_3


title: SJRHB013758_C2 Rep 2_DYE6551

city: Memphis

first: Michael

last: Dyer

state: TN

address: 262 Danny Thomas

institute: St. Jude Children's Research Hospital

description: Library name: SJRHB013758_C2 Rep 2

library_source: transcriptomic single cell

data_processing:
Single-cell (scRNA-seq) and single-nucleus (snRNA-seq) RNA sequencing data were generated from patient tumors, patient-derived xenografts (PDXs), and in vitro culture models (2D, 3D, and Cancer Cell Line Factory [CCLF]) representing multiple pediatric solid tumor types. Analyses were conducted using consistent methodological principles within an evolving computational framework. Although individual datasets were processed with slightly different versions of analytical tools, all followed a unified workflow for alignment, quality control, normalization, integration, and downstream analysis as described below.
Raw sequencing reads in FASTQ format were first assessed for quality using FASTQC (v0.11.5). Alignment and gene quantification were performed using Cell Ranger (v7.0.0 or v8.0.1, 10x Genomics) against the appropriate genome references. Patient samples were aligned to the human GRCh38 reference genome, whereas xenograft and mixed-origin datasets (PDX, 3D) were aligned to a dual human–mouse reference (GRCh38 + mm10 or GRCh38 + GRCm39). In all cases, intronic reads were included during counting to enhance transcript detection sensitivity. For rhabdomyosarcoma samples, the cell count parameter in Cell Ranger was fixed at 10,000 to mitigate barcode overestimation.
Comprehensive quality control (QC) filtering was applied to remove debris, damaged cells, multiplets, and nonhuman contaminants. Cells were excluded based on a combination of criteria, including the number of detected genes, total UMI counts, and the proportion of mitochondrial reads. Typical thresholds included the retention of cells with at least 100–1,000 detected genes and ≥500 UMIs, and exclusion of cells with >5–25% mitochondrial transcripts, depending on dataset type (≤10% for most scRNA-seq, ≤1% for snRNA-seq). For select samples, Outlier cells exceeding three median absolute deviations (MADs) from the median number of detected genes or UMIs were also removed.For select samples, doublets were detected and filtered using demuxafy (PMID 38622708) and Seurat-based outlier identification. In xenograft and culture samples, potential mouse contamination was addressed by removing barcodes showing predominant alignment to the mouse genome. For select tumor datasets, background ambient RNA contamination was further reduced using CellBender (v0.2.0) (PMID 37550580).
Quality-controlled gene expression matrices were processed using Seurat (v4.4.0 or v5.0; Hao et al., 2021). Data were normalized using the NormalizeData() function (log-normalization with a scale factor of 10,000) and scaled via linear regression to account for differences in sequencing depth and mitochondrial content. Highly variable genes (typically 2,000–3,000 per dataset) were identified for downstream analysis. Dimensionality reduction was performed using principal component analysis (PCA), with the top 30 principal components retained for nonlinear projection via Uniform Manifold Approximation and Projection (UMAP). Graph-based clustering was implemented using the Leiden algorithm (Traag et al., 2019) through Seurat’s FindClusters() function at resolutions between 0.1 and 0.4.
To integrate datasets across experimental models, replicates, and biological conditions, multiple batch-correction frameworks were employed, including Harmony (Korsunsky et al., 2019), Reciprocal PCA (RPCA) (Stuart et al., 2019), fastMNN (Haghverdi et al., 2018), and scVI (Lopez et al., 2018). The choice of integration method was dataset-dependent and guided by visual inspection of inter-sample mixing and Local Inverse Simpson’s Index (LISI) metrics (Korsunsky et al., 2019). All integrated datasets were subsequently clustered and analyzed uniformly.
Automated annotation was performed using SingleR (Aran et al., 2019) by comparison with the Human Cell Atlas and the Database of Immune Cell Expression (DICE). Clusters identified as immune populations or doublets were excluded prior to reintegration. For select datasets, copy-number variation (CNV) inference was carried out using inferCNV (https://github.com/broadinstitute/inferCNV/wiki), supplemented by Numbat for single-nucleus variant calling (PMID 36163550). In some analyses, cell cycle scores were computed using Seurat’s CellCycleScoring() function to quantify the proportion of S- and G2/M-phase genes. Differential gene expression analysis was performed using Seurat’s FindMarkers() function (Wilcoxon rank-sum test) with thresholds of log₂ fold change ≥ 0.25, a minimum expression frequency of 0.1, and adjusted p < 0.01.Additionally, cells were annotated using known gene expression profiles from a reference dataset (Patel et al., 2022) using scRNA-seq reference label transfer method, FindTransferAnchors(), from Seurat, enabling the classification of cells in query datasets at specified clustering resolution.
For multi-tumor cohorts (Ewing sarcoma, neuroblastoma, osteosarcoma, and rhabdomyosarcoma), analyses were executed using the Snap workflow (https://github.com/stjude-dnb-binfcore/sc-rna-seq-snap), a containerized and reproducible pipeline maintained by the St. Jude Bioinformatics Core. All dependencies were configured for R v4.4.0 and Seurat v4.4.0, ensuring full computational reproducibility (see GitHub repository for versioning reports). Each cohort was subdivided into three analytical groups: 1. Dual-genome cohort (PDX and 3D samples): aligned to GRCh38 + GRCm39 and filtered for low-quality and mouse-derived cells. 2. Human-genome cohort (patient, 2D, CCLF samples): aligned to GRCh38 and processed using identical QC parameters. 3. Integrated cohort: merged from the two above and analyzed jointly after Harmony-based integration. Cells with fewer than 500 UMIs, fewer than 300–1,000 genes (depending on tumor type), or >10% mitochondrial reads were removed prior to normalization and scaling. Highly variable features (n = 3,000) were used for PCA, followed by clustering with Leiden at resolution 0.1. Marker genes were identified per cluster, and top-ranked genes were examined for biological interpretability.
Interactive visualization and exploratory analysis were facilitated using ShinyCell (v2.1.0) (Ouyang et al., 2021). Processed datasets and interactive R Shiny applications are available through institutional repositories and project-specific GitHub resources (URLs to be provided upon publication).
Assembly: hg38
Supplementary files format and content: barcodes, matrix, features

source_name: cells

growth_protocol: Cells dissociated from O-PDX tumors were seeded onto PrimeSurface 96-well low-adhesion U-bottom plates at 25,000-50,000 cells/well (S-Bio, MS-9096UZ). Plates were centrifuged at 300xg for 3 minutes to aggregate the cells in each well. After 5-7 days, organoids had formed and were transferred to 0.5% geltrex- (Thermo, A14133-02) coated 10cm standard tissue culture plates. Cells that migrate from the organoid were considered a 2D culture and referred to as passage 0. Additional organoids were collected for downstream applications and cryopreserved in 90% FBS and 10% DMSO. After 7-10 days, 2D cells were passaged to fresh geltrex-coated 10cm dishes for 5-7 days and harvested for downstream molecular assays and cryopreserved stocks.

extract_protocol:
Extract protocol: To dissociate organoids into single-cell suspension 40U papain was added to 400uL of papain buffer(1mM L-cystein, 0.5mM EDTA in PBS -/-) and incubated at 37°C for ~5 min. The organoids were added and incubating at 37°C for ~5 min with occasional flicking until the organoid dissociated to the point of floating. 40uL of DNase was added and the dissociated cells were incubated at 37 °C for an additional 5 min. The cell suspension was filtered with a 40-μm mesh cell strainer and washed with 2mL PBS−/−. The filtered cells were slowly added to a 5 ml BSA cushion medium (4% BSA in Retinal Explant media) and spun at 500g for 10 min at 4 °C to separate the cells from the debris. The supernatant was removed and resuspend the cell pellet in 400 μl of explant culture medium. To generate single-nucleus suspensions from flash-frozen tissue frozen fragments were manually dissociated using Noyes spring scissors in 1 mL TST buffer (73 mM sodium chloride, 5 mM Tris [pH 8.0], 0.5 mM calcium chloride, 10.5 mM magnesium chloride, 0.01% bovine serum albumin, 0.03% Tween-20). Nuclear suspensions were filtered through a 40 μm strainer and diluted with an additional 1 mL TST buffer and 3 mL ST buffer (73 mM sodium chloride, 5 mM Tris [pH 8.0], 0.5 mM calcium chloride, 10.5 mM magnesium chloride) and centrifuged at 500xg at 4°C for 5 min. The nuclear pellet was resuspended in ST buffer and filtered through a 40 μm strainer to generate single-nuclear suspensions for snRNA-seq. To generate single-cell suspensions from fresh tissue, samples were rinsed with PBS -/-, minced with a scalpel, and processed using the Papain Dissociation System (Worthington). The tumors were incubated in 5 mL papain-DNase I solution at 37°C for 15-30 min, filtered through a 40 μm strainer, and centrifuged at 500 x g for 5 min. The cell pellets were then resuspended in 3.1 mL resuspension buffer (2.7 mL Earle’s buffered saline solution, 300 μL albumin-ovomucoid inhibitor, 150 μL DNase I) and layered over 5 mL albumin-ovomucoid inhibitor cushion and centrifuged at 100xg for 6 min. The pellet was resuspended in PBS and filtered through a 40 μm strainer.
Single cell RNA sequencing and library preparation was preformed using the 10X genomics 3’ gene expression kits.

value: cells

tag: disease

value: RMS


title: SJNBL030721_X1_DYE4559

description: Library name: SJNBL030721_X1

source_name: tumor

value: tumor

value: NBL

value: PDX


title: Experiment 2_LV_P_23

description:
Library name: LV_P_23
column T


title: Experiment 2_LV_P_20

description:
Library name: LV_P_20
column S


title: SW1463, 50nM Soto, 24 Hours_2

description: Library name: Soto_24h_2


title: SJRHB010928_X1_DYE2511

description: Library name: SJRHB010928_X1


title: SW1463, 50nM Soto, 24 Hours_1

description: Library name: Soto_24h_1


title: SJRHB013758_C4_DYE1672

description: Library name: SJRHB013758_C4


title: SJRHB010928_C1_DYE0282

description: Library name: SJRHB010928_C1


title: Experiment 2_LV_P_19

description:
Library name: LV_P_19
column R


title: SW1463, DMSO, 10 Days_3

description: Library name: DMSO_10d_3

value: DMSO


title: Experiment 2_LV_P_36

description:
Library name: LV_P_36
column Q


title: SW1463 50nM Soto 6 Days_2

description: Library name: Soto_6d_2


title: Experiment 2_LV_P_25

description:
Library name: LV_P_25
column V


title: Experiment 2_LV_P_35

description:
Library name: LV_P_35
column P


title: SW1463, DMSO, 10 Days_1

description: Library name: DMSO_10d_1


title: SW1463, DMSO, 10 Days_2

description: Library name: DMSO_10d_2


title: Multiome ATAC, tumor 18, RPLN

city: Boston

first: Prathyusha

last: Konda

state: Massachusetts

address: 450 BROOKLINE AVENUE

department: Medical Oncology

institute: Dana-Farber Cancer Institute

description: Library name: Tumor 18 RPLN, ATAC

library_source: genomic single cell

data_processing:
Sequencing data were demultiplexed, aligned, and quantified using the 10x Genomics Cell Ranger pipeline (v3.1.0). Reads were aligned to the GRCh38 reference genome (Gencode annotation), and gene-by-cell UMI count matrices were generated.
Assembly: GRCh38
Supplementary files format and content: fragments and Count matrices

molecule: genomic DNA

source_name: RPLN

extract_protocol:
From fresh frozen tissues, nuclei isolation was performed using low-retention microcentrifuge tubes throughout the procedure to minimize nuclei loss. Briefly, OCT-embedded tissue fragments were rapidly thawed on wet ice to remove excess OCT, mechanically dissociated by pipetting, and homogenized in TST solution. The homogenate was filtered through a 30 µm MACS SmartStrainer and pelleted by centrifugation for 4 minutes at 500 × g at 4°C. The resulting nuclei pellet was resuspended in 150 µL of 10x Genomics Diluted Nuclei Buffer. Trypan blue-stained nuclei were counted manually using INCYTO C-Chip Neubauer Improved Disposable Hemacytometers.
Library construction according to the manufacturer’s protocol (Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 User Guide, Rev D or Chromium Next GEM Single Cell Multiome ATAC + Gene Expression User Guide, Rev F). Libraries were normalized and pooled for sequencing on a NovaSeq SP-100 flow cell (Illumina, Inc., San Diego, CA, USA).

value: RPLN

tag: disease state

value: translocation renal cell carcinoma


title: Multiome ATAC, tumor 18, liver 4

description: Library name: Tumor 18 Liver 4, ATAC


title: Experiment 2_LV_P_34

description:
Library name: LV_P_34
column O


title: SW1463 DMSO 6 Days_3

description: Library name: DMSO_6d_3


title: SW1463 DMSO 6 Days_2

description: Library name: DMSO_6d_2


title: Experiment 2_LV_P_31

description:
Library name: LV_P_31
column M


title: Experiment 2_LV_P_26

description:
Library name: LV_P_26
column L


title: Experiment 2_LV_P_33

description:
Library name: LV_P_33
column N


title: SW1463 DMSO 6 Days_1

description: Library name: DMSO_6d_1


title: SW1463, DMSO, 24 Hours_3

description: Library name: DMSO_24h_3


title: C57BL6-Spleen-NK-6

city: Winnipeg

first: Peter

last: Thompson

state: MB

address: 715 McDermot ave

department: Physiology & Pathophysiology

country: Canada

institute: University of Manitoba

description: Library name: C57BL6-6

data_processing:
Read alignment
Normalize read counts
Assembly: mm10
Supplementary files format and content: Differential_expression_analysis_table.csv

source_name: Spleen

extract_protocol:
Trizol total RNA (ZymoResearch RNA microprep kit)
paired end 150 bp

treatment_protocol: Freshly isolated splenocytes

value: Spleen

tag: strain

value: NK cell

value: wild-type


title: Experiment 2_LV_P_24

description:
Library name: LV_P_24
column U


title: Experiment 2_LV_P_21

description:
Library name: LV_P_21
column J


title: SW1463, DMSO, 24 Hours_2

description: Library name: DMSO_24h_2


title: Experiment 2_LV_P_22

description:
Library name: LV_P_22
column K


title: Experiment 1_LV_T_16

description:
Library name: LV_T_16
column U


title: SW1463, DMSO, 24 Hours_1

description: Library name: DMSO_24h_1


title: Multiome RNA, tumor 18, liver 2

description: Library name: Tumor 18 Liver 2

molecule: nuclear RNA


title: TA bio rep 3, ATAC

city: Dallas

first: Lance

last: Terada

state: TX

address: 5323 Harry Hines Blvd, MC 8448

institute: University of Texas Southwestern

description: Library name: TA3

library_source: genomic

data_processing:
After QC, adaptors were trimmed and aligned into the genome reference. Peaks were called using the FindPeaks command from HOMER (v4.9) with false discovery rate (FDR) threshold set to 0.001. Called peaks were merged from all samples and annotatePeaks.pl command was used to produce a raw count matrix.
Assembly: GRCh38
Supplementary files format and content: narrowPeak files for each sample

extract_protocol:
ATAC-seq was performed on biological triplicates using the Omni-ATAC chromatin preparation protocol (Nature Methods 2017;14:959.
Nextera DNA adaptor indexes were added to the libraries and remaining cycles run for a final concentration of 20 nM per library.

value: Lung carcinoma

value: chimera


title: TA bio rep 2, ATAC

description: Library name: TA2


title: TA bio rep 1, ATAC

description: Library name: TA1


title: KYSE30 cells, Combination, 1Day, rep2

city: Ganzhou

first: zhou

last: dongdong

address: hexie avenue

country: China

institute: Gannan Medical University

description: Library name: OX4_2

data_processing:
The clustering of the index-coded samples was performed on a cBotCluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumia)according to the manufacturer’s instructions
After cluster generation, the librarypreparations were sequenced on an Illumina Novaseq platform and 150 bppaired-end reads were generated.
Raw data (raw reads) of fastq format were firstly processed through fastpsoftware.
Index of the reference genome was built usingHisat2 v2.0.5 and paired-end clean reads were aligned to the referencegenome using Hisat2 v2.0.5
featureCounts v1.5.0-p3 was used to count the reads numbers mapped toeach gene.
Differential expression analysis oftwo conditions/groups (two biological replicates per condition) was performedusing the DESeq2 R package (1.20.0), Genes with an adjusted P-value <=0.05 found by DESeq2 wereassigned as differentially expressed
Assembly: hg38
Supplementary files format and content: fpkm for each sample

growth_protocol: KYSE30 cell were cultured in a medium containing RPMI-1640 and F12 medium  in a 1:1 ratio with 1% glutamine at 37 °C under 5% CO2

extract_protocol:
TRIzol(Thermo Fisher)
AMPure XP system and Agilent Bioanalyzer2100 system

treatment_protocol: KYSE30 cells were treated with 6μg/ml IMMU132, 6μM IACS010759 and combanition for 24 h

value: ESCC

value: Combination


title: KYSE30 cells, Combination, 1Day, rep1

description: Library name: OX4_1


title: KYSE30 cells, Combination, 1Day, rep3

description: Library name: OX4_3


title: control bio rep 3, ATAC

description: Library name: control3

value: control


title: control bio rep 1, ATAC

description: Library name: control1


title: KYSE30 cells, IACS010759, 1Day, rep2

description: Library name: OX3_2


title: snRNA, tumor 11, kidney

description: Library name: Tumor 11

source_name: Kidney

value: Kidney


title: snRNA, tumor 12, kidney

description: Library name: Tumor 12

source_name: Psoas muscle

value: Psoas muscle


title: Experiment 1_LV_T_8

description:
Library name: LV_T_8
column Q

value: Liver


title: KYSE30 cells, IMMU132, 1Day, rep2

description: Library name: OX2_2


title: KYSE30 cells, IMMU132, 1Day, rep1

description: Library name: OX2_1


title: KYSE30 cells, Control, 1Day, rep3

description: Library name: OX1_3

value: Control


title: MIA PaCa-2, stiff matrix, 5 days (3)

city: Shanghai

first: Tiancheng

last: Gong

address: No. 366 Longchuan North Road, Xuhui District, Shanghai

institute: Shanghai Xuhui Central Hospital

description:
Library name: MH3
ML_vs_MH-all_samples.txt MH3

data_processing:
Illumina Casava1.7 software used for basecalling.
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence, then mapped to A. thaliana genome (downloaded from ENSEMBL, version 21) using Bowtie 1.0.0 with parameters -q -p 4 -e 100 -y -a -m 10 --best --strata
Trinity was used for assembly with the parameters --min_kmer_cov 3 --normalize_reads
The number of raw reads unambiguously aligned to ORFs in both RNA-Seq and ribosome profiling data sets from two biological replicates were used to generate gene read counts, which were normalized as reads per million of the total mapped reads (rpm) or reads per kilobase per million of the total mapped reads (rpkm)
Assembly: A fasta file the generate with Trinity, named Trinity.fasta
Supplementary files format and content: tab-delimited text files include TPM values for each Sample

source_name: MIA PaCa-2

extract_protocol:
total RNA was extracted using RNA Extraction Reagent (Qiagen), total RNA was used for the construction of sequencing libraries.
RNA libraries for RNA-seq were prepared using illumina TruseqTM RNA sample prep Kit for illumina following manufactuter's protocols.

value: MIA PaCa-2

value: human pancreatic carcinoma cell line

value: cultured on the Polyacrylamide hydrogels with high (40.40 kPa) stiffness for 5 days


title: 259 IL1b 2 vs 257 Ctrl 2

type: RNA

city: Berlin

last: Sigal

address: Augustenburger Platz 1

department: Dept. Hepatology and Gastroenterology

institute: Charité Universitätsmedizin

hyb_protocol: After precipitation, purification, and quantification, 0.75 g of each labeled cRNA was fragmented and hybridized to multipack microarrays (Agilent design 074809; SurePrint G3 Mouse GE v2 8x60K) according to the supplier’s protocol (Agilent Technologies).

scan_protocol: Scanning of microarrays was performed at 3-m resolution using a G2565CA high-resolution laser microarray scanner (Agilent Technologies). Microarray image data were processed with Image Analysis/Feature Extraction software G2567AA vA.11.5.1.1 (Agilent Technologies) using default settings and the GE2_1105_Oct12 extraction protocol.

data_processing: Data was processed in R using package limma.

label_protocol: Total RNA was amplified and labeled with a dual-color Quick-Amp labeling kit (Agilent Technologies). In brief, mRNA was reverse transcribed and amplified using an oligo-dT-T7 promoter primer and labeled with cyanine 3-CTP or cyanine 5-CTP.

growth_protocol: Primary murine gastric stromal cells at passage 2 were grown in culture for 7 days.

extract_protocol: RNA was extracted with the Nucleospin RNA Kit (Machery Nagel). Quality control and quantification of total RNA was assessed using a 2100 bioanalyzer (Agilent Technologies) and a NanoDrop 1000 UV-Vis spectrophotometer (Kisker)

treatment_protocol: Primary murine gastric stromal cells were treated 3 times with 50ng/ml IL-1b. The last treatment was performed the day before harvesting.

tag: condition

source_name: 257 Ctrl 2

value: Ctrl


title: 257 Ctrl 2 vs 259 IL1b 2


title: KYSE30 cells, Control, 1Day, rep1

description: Library name: OX1_1


title: MIA PaCa-2, stiff matrix, 5 days (2)

description:
Library name: MH2
ML_vs_MH-all_samples.txt MH2


title: 267 IL1b 1 vs 265 Ctrl 1

source_name: 265 Ctrl 1


title: 265 Ctrl 1 vs 267 IL1b 1


title: Il1r1 KO_vs_WT_rep2

extract_protocol: RNA from organoids was extracted with the Nucleospin RNA Kit (Machery Nagel). Quality control and quantification of total RNA was assessed using a 2100 bioanalyzer (Agilent Technologies) and a NanoDrop 1000 UV-Vis spectrophotometer (Kisker)

treatment_protocol: After tamoxifen injection, Col1a2CreErt2:Il1r1 fl/fl and control mice were infected with PMSS1 Helicobacter pylori for 2 months.

value: WT

value: Stomach cells

value: KO


title: WT_vs_Il1r1 KO_rep2


title: Il1r1 KO_vs_WT_rep1


title: KYSE30 cells, IACS010759, 1Day, rep3

description: Library name: OX3_3


title: WT_vs_Il1r1 KO_rep1


title: MIA PaCa-2, stiff matrix, 5 days (1)

description:
Library name: MH1
ML_vs_MH-all_samples.txt MH1


title: MIA PaCa-2, soft matrix, 5 days (3)

description:
Library name: ML3
ML_vs_MH-all_samples.txt ML3

value: cultured on the Polyacrylamide hydrogels with low (2.55 kPa) stiffness for 5 days


title: MIA PaCa-2, soft matrix, 5 days (2)

description:
Library name: ML2
ML_vs_MH-all_samples.txt ML2


title: WT_vs_KO_rep1

treatment_protocol: The conditional mouse model Axin2CreErt2:Bmpr1a fl/fl was previously established and RNA from WT and KO mice was generated from mouse tissue already estalished and described in the publication: Kapalczynska M, Lin M. et al, Nature Communications 2022, doi:10.1038/s41467-022-29176-w


title: WT_vs_KO_rep2


title: KO_vs_WT_rep2


title: Bacteroides thetaiotaomicron RNA-seq extracted from a microfluidic device, replicate 4

city: Wurzburg

first: Elise

last: Bornet

address: Josef-Schneider Strasse 2

institute: Helmholtz Insitute

description: Library name: Bt_4_MF

data_processing:
Sequence reads were trimmed for Nextera adapter and MATQ-Seq primers using bbmap. bbduk.sh -Xmx10g t=20
Trimmed reads were then mapped to the reference genome using bowtie2 version 2.3.4.1. bowtie2 -p 30
features were assigned using featureCounts.
Read counts extraction was performed using feature counts version 2.0.1
Assembly: GCF_000011065.1 for Bacteroides thetaiotaomicron and GCF_904425475.1 for E.coli
Supplementary files format and content: tab delimited text file contains raw counts for each sample

organism: Bacteroides thetaiotaomicron

source_name: Bacteroides thetaiotaomicron VPI-5482

growth_protocol: Cells were grown in M9 mininal media comlemented with amylopectin

extract_protocol:
RNA was extracted following the MATQ-seq protocol
MATQ-Seq and ribosomal RNA depletion with DASH. Cells were lysed using mechanical sonication and 5U lysozyme per well. cDNA librairies were obtained using the MATQ-Seq reverse transcription protocol. Ribosomal cDNA was depleted from the cDNA librairies using DASH.

treatment_protocol: 100% RNAlater

value: Bacteroides thetaiotaomicron VPI-5482

value: GFP expressing

value: fixed in 100% RNAlater


title: MIA PaCa-2, soft matrix, 5 days (1)

description:
Library name: ML1
ML_vs_MH-all_samples.txt ML1


title: Bacteroides thetaiotaomicron RNA-seq extracted from a microfluidic device, replicate 2

description: Library name: Bt_2_MF


title: Nap1l1-WT-rep2

city: Jerusalem

first: Tamar

last: Segal

address: Givat Ram

country: Israel

institute: The Hebrew University of Jerusalem

description: Library name: Nap1l1_WT_Serum_S10_L001_R1_001

data_processing:
Illumina output was analyzed using an in-house Perl script that produced a reads matrix that was aligned using RSEM with Bowtie.
The resulting matrix was analyzed in R (Seurat v4.1.1)
Assembly: mm10
Supplementary files format and content:
gene abundances matrices for each sample seperated to single cells; Each column represents a cell, and the column header indicates the specific barcode from the fastq file.

extract_protocol:
Cells (~1,000 cells/µl) were loaded into a chilled syringe pump and injected into a custom-designed microfluidics device. Flow settings were adjusted to allow individual cells to become encapsulated with single hydrogel beads presenting cell-specific barcoding oligonucleotide primers inside 3-5 nL encapsulation mixture droplets that contain 20 U µL-1 reverse transcriptase (SuperScript III, Thermo Fisher Scientific), First-Strand Buffer (Thermo Fisher Scientific), 0.6% (v/v) IGEPAL CA-630 (Sigma-Aldrich), 1 mM dNTPs (New England Biolabs), 6.67 mM DTT (Thermo Fisher Scientific), 2,700 units mL-1 RNase inhibitor (New England Biolabs), and 0.1 M Tris-HCl (pH 8.0). 2 Primers consisted of a poly-T sequence to trap mRNA molecules, two 7-to-10 base cell-specific barcodes, 5-base unique molecular identifier (UMI) sequence, and a T7 promoter sequence. Droplets were collected, the primers were photo-cleaved by exposing the emulsion to UV (365 nm 10 mW cm-2, BlackRay Xenon lamp) for 10 minutes at room temperature, and reverse transcription of trapped mRNA was performed at 50 ºC for 2 hours. Droplets were then demulsified by adding 1H,1H,2H,2H-perfluoro-1-octanol (Sigma Aldrich) dissolved in HFE-7500 (Novec) at 20% (v/v).
Single-stranded DNA primers and primer dimers were digested using 1 unitµL-1 ExoI (New England Biolabs) and 1 U µL-1 HinFI (Thermo Fisher Scientific). mRNA:cDNA hybrids were purified using Agencout AMPure XP beads (Beckman Coulter Genomics) according to the manufacturer’s instructions. Second-strand cDNA synthesis was performed using NEBNext Ultra II Non-Directional RNA Second Strand Synthesis Module (New England Biolabs) and amplified by in vitro transcription (HiScribe, New England Biolabs). RNA products were fragmented using Ambion RNA Fragmentation Reagents (AM8740, Thermo Fischer Scientific), purified using Agencout AMPure XP beads, and reverse-transcribed using random hexamer primers (SuperScript III, Thermo Fisher Scientific). Single-stranded DNA fragments were linearly amplified via 14 PCR cycles (HiFi HotStart ReadyMix, Kapa Biosystems).

value: mESC

value: wild-type


title: Bacteroides thetaiotaomicron RNA-seq extracted from a microfluidic device, replicate 1

description: Library name: Bt_1_MF


title: Nap1l1-WT-rep3

description: Library name: C20_WT_Serum_S5_L001_R1_001


title: Bacteroides thetaiotaomicron RNA-seq extracted from liquid culture, replicate 4

description: Library name: Bt_4_LC


title: Nap1l1-WT-rep1

description: Library name: C6_WT_Serum_S1_L001_R1_001


title: Bacteroides thetaiotaomicron RNA-seq extracted from liquid culture, replicate 3

description: Library name: Bt_3_LC


title: Bacteroides thetaiotaomicron RNA-seq extracted from liquid culture, replicate 2

description: Library name: Bt_2_LC


title: Nap1l1-alpha-KO-rep1

description: Library name: C6_KO_Serum_S2_L001_R1_001

value: NAP1L1 alpha knock-out


title: MEC1 epicardial cells, Independent replicate sample 3, Input DNA

city: Oxford

first: Andia

middle: Nicole

last: Redpath

address: IDRM IMS-Tetsuya Nakamura Building, Roosevelt Dr, Headington

country: United Kingdom

institute: University of Oxford

description: Library name: EPI_Input_Sample3

data_processing:
NGS data were demultiplexed using Illumina bcl2fastq, and processed with the nf-core/cut&run v2 pipeline using TrimGalore, Bowtie2, Samtools, Picard, deepTools, Bedtools, and SEACR. TrimGalore removed adaptor sequences, polyG+ sequences and low-quality reads, and Bowtie2 aligned reads to the mouse genome (mm10) with default parameters. Filtering was conducted with Samtools and Picard to exclude low-quality alignments (MAPQ ≥ 20 for WT1/IgG, MAPQ ≥ 10 for histone modifications), genomic blacklist regions, and duplicate reads. Peaks were called using SEACR, normalizing against IgG controls and scaling background control by a factor of 0.8. Read normalization was performed using Counts Per Million (CPM) with binsize 1, and consensus peaks were computed from at least two biological replicates.
Assembly: Mouse mm10
Supplementary files format and content: bigWig (.bigWig); BigWig files were created by bedGraphtoBigWig (UCSC) as part of the nf-core/cut&run v2 pipeline.
Library strategy: CUT&Run

source_name: MEC1 Mouse Embryonic Epicardial Cell Line

growth_protocol: MEC1, an epicardium-derived cell line, was cultured on 0.1% bovine gelatine in MEC1 expansion medium (10% EmbryoMax ES Cell Qualified FBS, 1% Penicillin/Streptomycin, EmbryoMAX DMEM high glucose) at 37°C in 5% CO2.

extract_protocol:
MEC1 epicardial cells cultured for 2 days in expansion medium were harvested from T75 flasks using 0.05% Trypsin-EDTA (2-3 minutes at 37°C). Cell viability was estimated at >95%, and cells were divided based on immunogen (1x106 cells/reaction). CUT&RUN was performed using the CUT&RUN Assay Kit (Cell Signaling Technologies), following the manufacturer’s instructions with minor modifications. Input DNA samples (1x105 cells/reaction) were prepared using DNA extraction buffer (containing Proteinase K and RNase A) according to the Cell Signaling protocol. Samples were incubated at 55°C for 1 hour with shaking (500 RPM, Eppendorf ThermoMixer), cooled on ice, then sonicated using a Diagenode Bioruptor Pico set to “Easy Mode” (25 cycles of 30 sec on/off at 4°C).
DNA libraries were prepared using the NEBNext Ultra II DNA Library Prep Kit (New England Biolabs), with modifications guided by the protocol "Library Prep for CUT&RUN with NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® (E7645) V.2" (dx.doi.org/10.17504/protocols.io.bagaibse). Less than 6 ng CUT&RUN DNA was used as input. NEBNext End Prep was performed at 20°C for 30 minutes and 50°C for 60 min. Adaptor concentrations were adjusted to input amounts (e.g., 3 µM NEBNext adaptor for 6 ng CUT&RUN DNA). Ligations were performed at 20°C for 15 min, and USER enzyme was used to cleave the uracil loop (37°C for 15 min). DNA cleanup steps used 1.75X SPRIselect beads for WT1/IgG CUT&RUN and 1.1X for histone modifications (H3K27ac, H3K4me3). To amplify the library, the ligation product was mixed with 2x Ultra II Q5 mix, universal primer and index primers; detailed in the key resources table. PCR amplification was typically 12 - 15 cycles (WT1/IgG: 98°C for 30 sec, 12 - 15 cycles of 98°C for 10 sec, 65°C for 10 sec, final extension at 65°C for 5 min; histone modifications: 13sec annealing/extension). Double size selection (0.8X, 1.2X SPRIselect beads) was used for WT1 and IgG, while single size selection (1.1X beads) was applied for histone modifications. Input DNA samples (50 ng input) followed the standard NEBNext Ultra II DNA Library Prep Kit protocol. Libraries were assessed using a High Sensitivity DNA Kit on a on Bioanalyser instrument (Agilent) and quantified using the KAPA Library Quantification Kit. Next-generation sequencing (NGS) was conducted on equimolar pooled libraries using the NextSeq 500/550 High Output v2.5 kit (75 cycles) (Illumina), with paired-end reads (42 bp x 2) and 6 bp single index reads.


title: MEC1 epicardial cells, Independent replicate sample 4, Input DNA

description: Library name: EPI_Input_Sample4

value: MEC1 Mouse Embryonic Epicardial Cell Line


title: MEC1 epicardial cells, Independent replicate sample 2, Input DNA

description: Library name: EPI_Input_Sample2


title: WT, myelin-treated, differentiation 1, clone E2 (scRNA-seq)

city: New York

first: Anna

last: Podlesny-Drabiniok

state: NY

address: 1425 Madison Avenue

institute: Icahn School of Medicine at Mount Sinai

description: Library name: E2_WT_M

data_processing:
Raw base call files were demultiplexed into FASTQ files using PIPseeker (Fluent Biosciences). Reads were aligned to the human reference genome (GRCh38), followed by barcode assignment and UMI counting to generate feature-barcode matrices. Sensitivity setting 3 was used
Quality control, normalization, clustering, and visualization were performed using Seurat v4.0. Cells were filtered based on the following criteria: <100 detected genes, <500 UMIs, >95% of UMIs from a single gene, or >10% mitochondrial reads. Count data were normalized using SCTransform (v0.4.1) with regression for mitochondrial gene percentage. Principal component analysis (PCA) was used for dimensionality reduction.
Assembly: hg38
Supplementary files format and content: barcode.tsv, features.tsv, matrix.mtx files provided for each sample

growth_protocol: iPSC lines were differentiated to hematopoietic precursor cells (HPCs) using StemDiff kit (StemCell Technologies). 20,833 cells/cm2 HPCs were plated and differentiated for 24 days to microglia (iMGL) in iMGL media (DMEM/F12, 2xB27, 0.5xN2, 2xITSG, 400uM monothioglycerol, 5ug/ml insulin, 1xNEAA, 1xGlutaMax) supplemented with three factors (IL34 (100ng/ml), TGFb (50ng/ml), and MCSF (25ng/ml). iMGLs were fed Monday, Wednesday and Friday with 1ml of iMGL media with three factors. Cells were splitted in 1:2 ratio if too confluent. Day 24 iMGLs were lifted with gentle pipetting and Accutase and plated 62,500 cells/cm2 in 1ml of conditioned media and 1ml of fresh media iMGL media with three factors supplemented additionally with two factors (CX3CL1 and CD200 100ng/ml each) for 3 additional days (day 25, day 26,  day 27). At day 26 of iMGL differentiation, iMGL received 1ml of five factor iMGL media and treatment with myelin or LPS. Cells were collected at day 27.

extract_protocol:
Cells were washed with PBS and dissociated using Accutase (Stemcell Technologies, cat. 07920) for 10 minutes at 37°C. Cells were centrifuged at 400 × g for 5 minutes and resuspended in 1.0% PBS/bovine serum albumin (BSA). The cell suspension was filtered through a 40 μm cell strainer to remove aggregates. Cell number and viability were assessed using trypan blue exclusion and an automated cell counter (Countess, Thermo Fisher Scientific). Only samples with high viability (typically >90%) were used for downstream processing.
Single cells were partitioned into PIP droplets using the PIPseq T20 3’ Single Cell RNA Kit v4.0 (Fluent Biosciences, Watertown, MA, USA) according to the manufacturer’s instructions. Briefly, approximately 40,000 live cells were combined with template beads, gently mixed, vortexed for 3 minutes to generate stable PIP emulsions, and processed following the recommended workflow. Captured mRNA was released upon cell lysis within the droplets, followed by reverse transcription, cDNA amplification, cDNA purification, and library preparation. Libraries were sequenced by Azenta (New Jersey, USA) on an Illumina NovaSeq X platform, generating ~50,000 reads per cell (~2 billion reads per sample).

treatment_protocol: Day 26 iMGL were treated with myelin fragments (20ug/ml, 24) and harvested the following day.

value: iPSC-derived microglia

value: myelin


title: MEC1 epicardial cells, Independent replicate sample 1, Input DNA

description: Library name: EPI_Input_Sample1


title: WT, myelin-treated, differentiation 1, clone C2 (scRNA-seq)

description: Library name: C2_WT_M


title: WT, myelin-treated, differentiation 1, clone F2 (scRNA-seq)

description: Library name: F2_WT_M


title: MEC1 epicardial cells, Independent replicate sample 3, IgG antibody

description: Library name: EPI_IgG_Sample3

tag: cut&run antibody

value: Rabbit (DA1E) mAb IgG XP® Isotype Control


title: MEC1 epicardial cells, Independent replicate sample 2, IgG antibody

description: Library name: EPI_IgG_Sample2


title: MEF2C-KO, myelin-treated, differentiation 1, clone D9 (scRNA-seq)

description: Library name: D9_KO_M


title: MEF2C-KO, myelin-treated, differentiation 1, clone H11 (scRNA-seq)

description: Library name: H11_KO_M


title: MEC1 epicardial cells, Independent replicate sample 1, IgG antibody

description: Library name: EPI_IgG_Sample1


title: MEF2C-KO, myelin-treated, differentiation 1, clone G2 (scRNA-seq)

description: Library name: G2_KO_M


title: MEC1 epicardial cells, Independent replicate sample 3, WT1 antibody

description: Library name: EPI_WT1_Sample3

value: Anti-Wilms Tumor Protein antibody [CAN-R9(IHC)-56-2]


title: MEF2C-KO, myelin-treated, differentiation 1, clone D8 (scRNA-seq)

description: Library name: D8_KO_M


title: MEC1 epicardial cells, Independent replicate sample 2, WT1 antibody

description: Library name: EPI_WT1_Sample2


title: WT, non-treated, differentiation 1, clone H2 (scRNA-seq)

description: Library name: H2_WT_NT

value: non-treated


title: MEC1 epicardial cells, Independent replicate sample 1, WT1 antibody

description: Library name: EPI_WT1_Sample1


title: WT, non-treated, differentiation 1, clone E2 (scRNA-seq)

description: Library name: E2_WT_NT


title: MEC1 epicardial cells, Independent replicate sample 3, H3K27ac antibody

description: Library name: EPI_H3K27ac_Sample3

value: Acetyl-Histone H3 (Lys27) (D5E4)


title: WT, non-treated, differentiation 1, clone C2 (scRNA-seq)

description: Library name: C2_WT_NT

value: WT


title: MEC1 epicardial cells, Independent replicate sample 2, H3K27ac antibody

description: Library name: EPI_H3K27ac_Sample2


title: WT, non-treated, differentiation 1, clone F2 (scRNA-seq)

description: Library name: F2_WT_NT


title: MEC1 epicardial cells, Independent replicate sample 4 H3K4me3 antibody

description: Library name: EPI_H3K4me3_Sample4

value: Tri-Methyl-Histone H3 (Lys4) (C42D8)


title: Nap1l1-full-KO-rep1

description: Library name: C20_KO_Serum_S6_L001_R1_001

value: NAP1L1 knock-out


title: MEF2C-KO, non-treated, differentiation 1, clone G2 (scRNA-seq)

description: Library name: G2_KO_NT


title: MEF2C-KO, non-treated, differentiation 1, clone H11 (scRNA-seq)

description: Library name: H11_KO_NT


title: MEF2C-KO, non-treated, differentiation 1, clone D8 (scRNA-seq)

description: Library name: D8_KO_NT


title: MEC1 epicardial cells, Independent replicate sample 1, H3K4me3 antibody

description: Library name: EPI_H3K4me3_Sample1


title: WT, LPS-treated, differentiation 2, clone H2

description:
Library name: H2_WT_LPS_B2
column name in WTC11_bulkRNAseq_iMGL_raw_counts.tsv: H2_WT_LPS_B2

data_processing:
Raw base call files were converted to FASTQ format and processed using the nf-core/rnaseq pipeline (DOI: 10.5281/zenodo.1400710).
Reads were quality controlled, adapter-trimmed, and aligned to the human reference genome (GRCh38). Gene-level quantification was performed within the nf-core/rnaseq pipeline using standard parameters.
Gene-level count matrices were imported into R (v4.3.3) for downstream analysis. Differential gene expression analysis was performed using the DREAM method implemented in the variancePartition package (v1.23.1), applying linear mixed-effects models.
Assembly: hg38
Supplementary files format and content: tab-separated values (tsv) file containing raw counts for each sample

extract_protocol:
Total RNA was extracted using the RNeasy Plus Mini Kit (Qiagen, 74136) according to the manufacturer’s instructions. RNA quantity was assessed using NanoDrop 8000 (Thermo Fisher Scientific). RNA integrity was evaluated by Azenta using Qubit and BioAnalyzer, with all samples demonstrating RNA integrity number (RIN) > 9.0 prior to library preparation.
RNA-seq libraries were prepared by Azenta (New Jersey, USA) using the TruSeq RNA Sample Preparation Kit v2 (Illumina) according to the manufacturer’s protocol. Libraries were paired-end sequenced on an Illumina HiSeq2500 platform with 150 bp read length, generating approximately 20–30 million mapped fragments per sample.

treatment_protocol: Myelin 20ug/ml, 24h, LPS 50ng/ml 24h

value: LPS


title: E17.5 heart cells, including epicardial lineage traced population expressing tdTomato

description:
Library name: E175_heart_epi_lineage
10x Genomics

data_processing:
Sequencing reads were processed with Cell Ranger software (10x Genomics).
Assembly: Mouse mm10
Assembly: tdTomato
Supplementary files format and content: .tsv and .mtx

source_name: Heart

extract_protocol:
Whole embryonic heart was enzymatically dissociated using the Neonatal Heart Dissociation Kit (Miltenyi Biotec) following the manufacturer’s instructions and as previously described. Sample was processed using the Chromium Single Cell 3' Reagent Kit (v3 chemistry, 10x Genomics)
Library was performed according to the manufacturer's instructions (Chromium Single Cell 3' Reagent Kit, v3 chemistry, 10x Genomics).

value: Heart

tag: developmental stage


title: WT, LPS-treated, differentiation 1, clone H2

description:
Library name: H2_WT_LPS_B1
column name in WTC11_bulkRNAseq_iMGL_raw_counts.tsv: H2_WT_LPS_B1


title: FACS sorted epicardial cells (CD31-tdTomato+PDPN+), Biological replicate sample 1, E12.5

description: Library name: EPI_E125_Sample1

data_processing:
Sequencing data were demultiplexed using the Illumina platform and processed using Samtools, Bowtie2, Picard, Genrich, and deepTools. Nextera adaptor sequences and low-quality reads were trimmed during the bcl2fastq demultiplexing step. Reads were aligned to the mouse genome (mm10) using Bowtie2 with default parameters and a maximum fragment length of 1,000 bp. Peak calling and filtering were performed using Genrich, Samtools, and Picard. Duplicate reads were removed, and remaining reads were filtered for high quality (MAPQ ≥ 30), non-mitochondrial chromosomes, genomic blacklist regions (mm10 exclusion), and properly paired reads. Read coverage was normalized using reads per genome coverage (RPGC) and an effective genome size of 2,308,125,349.To visualize peaks at regions of interest, BigWig files were generated using bamCoverage with a binSize of 10 for loading into IGV.
Assembly: Mouse mm10
Supplementary files format and content: bigWig (.bw); BigWig files were generated using bamCoverage with a binSize of 10.

extract_protocol:
Whole embryonic hearts were enzymatically dissociated using the Neonatal Heart Dissociation Kit (Miltenyi Biotec) following the manufacturer’s instructions and as previously described. Biological replicate samples are indipendent samples generated from pooled hearts. E12.5 or E13.5 hearts (Rosa26tdTomato; Wt1CreERT2/+) from littermate embryos were pooled prior to dissociation (E12.5: 7 hearts/sample; E13.5: 9-13 hearts/sample) to maximise cell numbers for FACS and corresponding FMO controls, and achieve a target sort of 50,000 epicardial cells/sample for the downstream ATAC-seq protocol. Single-cell suspensions in cell staining buffer (Biolegend) were pre-treated for 5 minutes on ice with TruStain FcX antibody, followed by surface staining with BV421 anti-CD31 and APC/Cy7 anti-Podoplanin (PDPN) antibody for 30 minutes on ice, then washed with PBS before being incubated with SYTOX Green Dead Cell Stain for 5 minutes on ice. After PBS washes, cells were resuspended in sorting buffer containing 2% FBS in PBS. BD FACSAria III was used to sort 40,000-50,000 live cells negative for SYTOX Green and CD31, and positive for tdTomato and PDPN, into 0.5% BSA in PBS. Live epicardial cells (CD31-tdTomato+PDPN+) were isolated by FACS were subjected to the ATAC-seq protocol as previously described.
ATAC-seq DNA libraries were generated as previously described, with minor modifications. Reactions were purified using the MinElute Reaction Cleanup Kit (Qiagen) following the manufacturer’s instructions. Pre-amplification of transposed fragments (5 cycles) was performed using NEBNext Ultra II Q5 Master Mix (NEB) and Nextera i7 index adapters (IDT). The number of additional amplification cycles (typically 4) was determined using the qPCR method. Final PCR products were purified using a double-sided bead purification method with SPRIselect beads (0.5X volume followed by 1.3X volume of SPRI beads). Final cleanup was performed with the MinElute Reaction Cleanup Kit (Qiagen). Libraries were assessed for quality using an Agilent TapeStation with a D1000 DNA ScreenTape assay, and quantified with the KAPA Library Quantification Kit. Next-generation sequencing (NGS) was conducted on equimolar pooled libraries using the NextSeq 500/550 High Output v2.5 kit (75 cycles) (Illumina), with paired-end reads (40 bp x 2) and 8 bp single index reads.

value: Epicardial cells


title: WT, myelin-treated, differentiation 2, clone H2

description:
Library name: H2_WT_M_B2
column name in WTC11_bulkRNAseq_iMGL_raw_counts.tsv: H2_WT_M_B2


title: FACS sorted epicardial cells (CD31-tdTomato+PDPN+), Biological replicate sample 2, Technical replicate 2, E13.5

description: Library name: EPI_E135_Sample2_r2


title: WT, myelin-treated, differentiation 1, clone H2

description:
Library name: H2_WT_M_B1
column name in WTC11_bulkRNAseq_iMGL_raw_counts.tsv: H2_WT_M_B1


title: WT, non-treated, differentiation 2, clone H2

description:
Library name: H2_WT_NT_B2
column name in WTC11_bulkRNAseq_iMGL_raw_counts.tsv: H2_WT_NT_B2


title: WT, non-treated, differentiation 1, clone H2

description:
Library name: H2_WT_NT_B1
column name in WTC11_bulkRNAseq_iMGL_raw_counts.tsv: H2_WT_NT_B1


title: FACS sorted epicardial cells (CD31-tdTomato+PDPN+), Biological replicate sample 1, E13.5

description: Library name: EPI_E135_Sample1


title: SPREDs-treated lung 2

city: Saga

first: Ryo

last: Yanagiya

state: Saga

address: 5-1-1 Nabeshima

department: Department of Internal Medicine

country: Japan

institute: Saga University

data_processing:
FASTQ files were mapped to mouse genome (mm10) by HISAT2.
Mapped reads were annotated and counted by StringTie2.
Differentially-expressed genes were extracted by DESeq2.
Annotated pathways were extracted using KEGG pathway analysis of clusterProfiler.
Assembly: mm10
Supplementary files format and content: csv-formatted file containing raw counts per gene

source_name: lung

growth_protocol: Mice were bred in adequate condition.

extract_protocol:
Twenty-four hours after LPS infusion, mice were sacrificed, followed by collection of lung tissues and RNA extraction using Sepasol-RNA I Super G (#09379-55; Nacalai Tesque) .
mRNA was isolated by NEBNext Poly(A) mRNA Magnetic Isolation Module (#E7490S; New England BioLabs).  Library preparation was performed with NEBNext Ultra II Directional RNA Library Kit for Illumina (#E7760S; New England BioLabs) and NEBNext Single Oligos for Illumina (#7350S; New England BioLabs).

treatment_protocol: The ARDS model mouse was established by intratracheal infusion of 3 mg/kg of LPSO111:B4 (Sigma-Aldrich). Four hours after, 30 µg of EVs (hepG2 derived) or SPREDs were intravenously injected.

value: lung

value: SPREDs


title: WT, LPS-treated, differentiation 2, clone E2

description:
Library name: E2_WT_LPS_B2
column name in WTC11_bulkRNAseq_iMGL_raw_counts.tsv: E2_WT_LPS_B2


title: control bio rep 2, ATAC

description: Library name: control2

value: Lung carcinoma

value: control


title: MEC1 epicardial cells, Independent replicate sample 3, H3K4me3 antibody

description: Library name: EPI_H3K4me3_Sample3

value: MEC1 Mouse Embryonic Epicardial Cell Line


title: MEC1 epicardial cells, Independent replicate sample 2, H3K4me3 antibody

description: Library name: EPI_H3K4me3_Sample2


title: SPREDs-treated lung 1


title: WT, LPS-treated, differentiation 1, clone E2

description:
Library name: E2_WT_LPS_B1
column name in WTC11_bulkRNAseq_iMGL_raw_counts.tsv: E2_WT_LPS_B1

value: iPSC-derived microglia


title: EVs-treated lung 2

value: EVs


title: KYSE30 cells, IACS010759, 1Day, rep1

description: Library name: OX3_1

value: ESCC


title: DSA-CAMR, peritubular capillary, ROI015

city: Goyang-si

first: Sung-Eun

last: Choi

state: --- Select One ---

address: 100 Ilsan-ro, Ilsan-donggu

country: South Korea

institute: NHIS Ilsan hospital

description:
Library name: ROI015
GeoMx DSP area of illumination (AOI), Full ROI segment; DSP well DSP-1001660023291-D-E04; AOI surface area 14425.1 um2; nuclei count 116; column name in GeoMx_WTA_Q3norm_counts_gene_x_ROI.tsv: ROI015

data_processing:
GeoMx NGS Pipeline v3.1.1.6
Paired-end FASTQ reads were quality-trimmed, adapter-trimmed, and stitched, then aligned to Readout Tag Sequence (RTS) barcodes; PCR duplicates were removed using unique molecular identifiers (UMIs), yielding deduplicated probe counts in DCC files (one per ROI).
DCC files were uploaded to the GeoMx DSP Data Analysis Suite and interpreted using the Hs_R_NGS_WTA_v1.0.pkc probe configuration file to collapse probe-level counts to gene-level (target-level) counts for 18,677 WTA targets.
Gene-level counts were normalized using the third-quartile (Q3) method across the 48 ROIs.
Assembly: GRCh38.p13
Supplementary files format and content: GeoMx_WTA_Q3norm_counts_gene_x_ROI.tsv: tab-delimited text file. Rows are the 18,677 WTA gene targets; columns are the 48 ROIs (ROI001-ROI048); values are Q3-normalized counts. Column names correspond to the 'library name' field of each sample.
Supplementary files format and content: DCC files (one per ROI): raw deduplicated read counts per RTS_ID (probe barcode). RTS_IDs are interpreted using Hs_R_NGS_WTA_v1.0.pkc, provided as a study-level supplementary file.
Library strategy: Spatial Transcriptomics

source_name: kidney allograft biopsy

extract_protocol:
Formalin-fixed paraffin-embedded kidney biopsy sections (5 um) were mounted on slides, deparaffinized, subjected to target retrieval and proteinase K digestion, and hybridized overnight with the GeoMx Human NGS Whole Transcriptome Atlas RNA probe set (v1.0), in which each target-specific probe carries a UV-photocleavable oligonucleotide barcode. Slides were stained with morphology markers and imaged on the GeoMx DSP. Regions of interest corresponding to glomeruli, peritubular capillaries, and vessels were selected, and barcodes were released by UV illumination and collected into a microplate.
Collected oligonucleotide barcodes were uniquely indexed by PCR using GeoMx Seq Code primers, pooled, and purified with AMPure XP beads. Libraries were sequenced paired-end on an Illumina NovaSeq 6000.

value: kidney allograft biopsy

tag: patient id

value: patient 2

tag: diagnosis

value: chronic antibody-mediated rejection

tag: dsa status

value: negative

tag: anatomical compartment

value: peritubular capillary

tag: roi id


title: WT, myelin-treated, differentiation 2, clone E2

description:
Library name: E2_WT_M_B2
column name in WTC11_bulkRNAseq_iMGL_raw_counts.tsv: E2_WT_M_B2

value: WT

value: myelin


title: EVs-treated lung 1

tag: tissue


title: WT, myelin-treated, differentiation 1, clone E2

description:
Library name: E2_WT_M_B1
column name in WTC11_bulkRNAseq_iMGL_raw_counts.tsv: E2_WT_M_B1

tag: cell type


title: syntheticDNAlibrary, 15-rounds, rep1

city: Kobe

first: Tamai

last: Endoh

address: 7-1-20, Minatojima-minamimachi, Chuo-ku

department: FIRST/FIBER

institute: Konan University

description: Library name: after_15_rounds

library_source: other

data_processing:
CLC Genomics Workbench Version 25.0.1
From sequencing data sequences in the randomized region were extracted by trimming the constant regions of primer annealing sites.
Abundance of identical sequences relative to the total reads was calculated.
Supplementary files format and content: Read counts and abundance of individual sequences were summarized in text file.

molecule: other

organism: synthetic construct

source_name: synthetic construct

extract_protocol:
DNA library consisting of constant primer annealing sites at the 5′ and 3′ regions and a 36-nt randomized region in between (5′-GGAGGCTCTCGGGACGAC(N36)GTCGTCCCGATCACTTGAATGGTCT-3′) was mixed with a 5-fold equimolar amount of biotinylated capture strand (5′-GTCGTCCCGAGAGCCATA-biotin-3′) in a Capture-SELEX buffer containing 20 mM HEPES-NaOH (pH 7.5), 1 M NaCl, 10 mM MgCl2, and 5 mM KCl. The mixture was heated at 90 °C and gradually cooled to 25 °C at a rate of 5 °C/min to make an annealed DNA mixture. Strep-Tactin Superflow High-Capacity Column was initialized with the Capture-SELEX buffer. The annealed DNA solution was added to the Strep-Tactin Superflow High-Capacity Column, returning the flow-through to the column at least twice. The column was washed by flowing the Capture-SELEX buffer (250 µL) ten times. The aptamer candidates were eluted by flowing equol-containing Capture-SELEX buffer (250 µL) three times. Eluted DNA fragments were amplified by PCR using sensen and biotinylated antisense primers. From the PCR product, single-stranded DNA library was produced for the next cycle of selection by removing the biotinylated antisense strand.
After 15 rounds of selection, the amplified DNA fragments were purified using QIAquick PCR Purification Kit after the remaining primers were digested by Exonuclease I. The concentration of the purified DNA fragments was quantified using the qPCR system by comparing with known concentrations of a standard DNA fragment.

value: synthetic construct

value: 15-rounds Capture-SELEX


title: WT, non-treated, differentiation 2, clone E2

description:
Library name: E2_WT_NT_B2
column name in WTC11_bulkRNAseq_iMGL_raw_counts.tsv: E2_WT_NT_B2

value: non-treated

tag: batch


title: WT, non-treated, differentiation 1, clone E2

description:
Library name: E2_WT_NT_B1
column name in WTC11_bulkRNAseq_iMGL_raw_counts.tsv: E2_WT_NT_B1

tag: genotype


title: WT, LPS-treated, differentiation 2, clone C2

description:
Library name: C2_WT_LPS_B2
column name in WTC11_bulkRNAseq_iMGL_raw_counts.tsv: C2_WT_LPS_B2

tag: treatment

value: LPS


title: R-o-18 leaf, Mock, 72 h, biological replicate 3

city: An-seong si

first: Hyunjoon

last: Kim

address: 4726, Seodong-daero, Daedeok-myeon, Anseong-si, Gyeonggi-do, Republic of Korea

institute: Chung-ang University

description:
Library name: Mock_3D_3
Leaf tissue from 10-day-old R-o-18 seedling; Mock treatment; harvested at 72 h; biological replicate 3.

data_processing:
Quality of the RNA-seq reads were evaluated using the FastQC software
The raw reads were trimmed and quality-filtered before alignment, and those with more than 90 % threshold (Q>30) were used for mapping. B. rapa reference genome was obtained from Ensembl and were used using the TopHat2 software with default parameters
Aligned reads were converted to digital counts using HTseq-count and were analyzed using edgeR.
Differentially expressed genes (DEGs) were identified based on a 0.05 p-value and a cutoff of two-fold difference in expression.
Assembly: B. rapa reference genome was obtained from Ensembl
Supplementary files format and content: tab-delimited text files with raw gene counts for individual samples

organism: Brassica rapa

source_name: leaf

growth_protocol: Seeds of Brassica rapa var. trilocularis inbred line R-o-18 were surface-sterilized, plated on half-strength Murashige and Skoog medium, and stratified at 4°C in darkness for 3 days. Seedlings were grown at 22°C under long-day conditions (16 h light/8 h dark) with cool-white fluorescent light.

extract_protocol:
Total RNA was isolated from three biological replicates using the RNeasy Plant Mini Kit (QIAGEN). RNA quantity and integrity were assessed using an Agilent 2100 Bioanalyzer, and samples with RNA integrity number greater than 7 were used for library preparation.
Strand-specific mRNA libraries were constructed using the TruSeq Stranded mRNA LT Sample Prep Kit (Illumina) according to the manufacturer's instructions. Libraries were sequenced in paired-end mode on an Illumina NovaSeq 6000 platform.

treatment_protocol: Ten-day-old seedlings were sprayed with 10 mL of 50 μM methyl jasmonate in 0.1% Tween-20 or mock solution (distilled water containing 0.1% Tween-20). Leaf tissues were harvested at 10 min, 24 h, and 72 h after treatment, immediately frozen in liquid nitrogen, and stored at -80°C.

value: leaf

value: mock control


title: R-o-18 leaf, Mock, 72 h, biological replicate 2

description:
Library name: Mock_3D_2
Leaf tissue from 10-day-old R-o-18 seedling; Mock treatment; harvested at 72 h; biological replicate 2.


title: WT, LPS-treated, differentiation 1, clone C2

description:
Library name: C2_WT_LPS_B1
column name in WTC11_bulkRNAseq_iMGL_raw_counts.tsv: C2_WT_LPS_B1

tag: cell line


title: R-o-18 leaf, Mock, 24 h, biological replicate 3

description:
Library name: Mock_1D_3
Leaf tissue from 10-day-old R-o-18 seedling; Mock treatment; harvested at 24 h; biological replicate 3.


title: R-o-18 leaf, Mock, 72 h, biological replicate 1

description:
Library name: Mock_3D_1
Leaf tissue from 10-day-old R-o-18 seedling; Mock treatment; harvested at 72 h; biological replicate 1.


title: WT, myelin-treated, differentiation 2, clone C2

description:
Library name: C2_WT_M_B2
column name in WTC11_bulkRNAseq_iMGL_raw_counts.tsv: C2_WT_M_B2


title: R-o-18 leaf, Mock, 24 h, biological replicate 2

description:
Library name: Mock_1D_2
Leaf tissue from 10-day-old R-o-18 seedling; Mock treatment; harvested at 24 h; biological replicate 2.


title: R-o-18 leaf, Mock, 24 h, biological replicate 1

description:
Library name: Mock_1D_1
Leaf tissue from 10-day-old R-o-18 seedling; Mock treatment; harvested at 24 h; biological replicate 1.


title: FACS sorted epicardial cells (CD31-tdTomato+PDPN+), Biological replicate sample 2, Technical replicate 1, E13.5

description: Library name: EPI_E135_Sample2_r1

value: Heart

value: Epicardial cells


title: KYSE30 cells, Control, 1Day, rep2

description: Library name: OX1_2

value: Control


title: R-o-18 leaf, Mock, 10 min, biological replicate 3

description:
Library name: Mock_0D_3
Leaf tissue from 10-day-old R-o-18 seedling; Mock treatment; harvested at 10 min; biological replicate 3.

value: 10 min


title: WT, myelin-treated, differentiation 1, clone C2

description:
Library name: C2_WT_M_B1
column name in WTC11_bulkRNAseq_iMGL_raw_counts.tsv: C2_WT_M_B1

value: iPSC-derived microglia


title: WT, non-treated, differentiation 1, clone C2

description:
Library name: C2_WT_NT_B1
column name in WTC11_bulkRNAseq_iMGL_raw_counts.tsv: C2_WT_NT_B1


title: WT, non-treated, differentiation 2, clone C2

description:
Library name: C2_WT_NT_B2
column name in WTC11_bulkRNAseq_iMGL_raw_counts.tsv: C2_WT_NT_B2


title: WT, LPS-treated, differentiation 2, clone F2

description:
Library name: F2_WT_LPS_B2
column name in WTC11_bulkRNAseq_iMGL_raw_counts.tsv: F2_WT_LPS_B2


title: R-o-18 leaf, Mock, 10 min, biological replicate 2

description:
Library name: Mock_0D_2
Leaf tissue from 10-day-old R-o-18 seedling; Mock treatment; harvested at 10 min; biological replicate 2.


title: WT, myelin-treated, differentiation 2, clone F2

description:
Library name: F2_WT_M_B2
column name in WTC11_bulkRNAseq_iMGL_raw_counts.tsv: F2_WT_M_B2


title: WT, LPS-treated, differentiation 1, clone F2

description:
Library name: F2_WT_LPS_B1
column name in WTC11_bulkRNAseq_iMGL_raw_counts.tsv: F2_WT_LPS_B1


title: R-o-18 leaf, Mock, 10 min, biological replicate 1

description:
Library name: Mock_0D_1
Leaf tissue from 10-day-old R-o-18 seedling; Mock treatment; harvested at 10 min; biological replicate 1.


title: R-o-18 leaf, MeJA, 72 h, biological replicate 3

description:
Library name: MeJA_3D_3
Leaf tissue from 10-day-old R-o-18 seedling; MeJA treatment; harvested at 72 h; biological replicate 3.

value: 50 μM methyl jasmonate


title: WT, non-treated, differentiation 2, clone F2

description:
Library name: F2_WT_NT_B2
column name in WTC11_bulkRNAseq_iMGL_raw_counts.tsv: F2_WT_NT_B2


title: R-o-18 leaf, MeJA, 72 h, biological replicate 1

description:
Library name: MeJA_3D_1
Leaf tissue from 10-day-old R-o-18 seedling; MeJA treatment; harvested at 72 h; biological replicate 1.


title: WT, myelin-treated, differentiation 1, clone F2

description:
Library name: F2_WT_M_B1
column name in WTC11_bulkRNAseq_iMGL_raw_counts.tsv: F2_WT_M_B1


title: R-o-18 leaf, MeJA, 24 h, biological replicate 3

description:
Library name: MeJA_1D_3
Leaf tissue from 10-day-old R-o-18 seedling; MeJA treatment; harvested at 24 h; biological replicate 3.


title: Bacteroides thetaiotaomicron RNA-seq extracted from a microfluidic device, replicate 3

description: Library name: Bt_3_MF

value: Bacteroides thetaiotaomicron VPI-5482

value: GFP expressing

value: fixed in 100% RNAlater


title: R-o-18 leaf, MeJA, 72 h, biological replicate 2

description:
Library name: MeJA_3D_2
Leaf tissue from 10-day-old R-o-18 seedling; MeJA treatment; harvested at 72 h; biological replicate 2.

value: WT


title: WT, non-treated, differentiation 1, clone F2

description:
Library name: F2_WT_NT_B1
column name in WTC11_bulkRNAseq_iMGL_raw_counts.tsv: F2_WT_NT_B1


title: Nap1l1-alpha-KO-rep2

description: Library name: Nap1l1_KO_Serum_S11_L001_R1_001

value: mESC

value: NAP1L1 alpha knock-out


title: KO_vs_WT_rep1

value: KO


title: MEF2C-KO, LPS-treated, differentiation 2, clone D9

description:
Library name: D9_KO_LPS_B2
column name in WTC11_bulkRNAseq_iMGL_raw_counts.tsv: D9_KO_LPS_B2


title: MEF2C-KO, LPS-treated, differentiation 1, clone D9

description:
Library name: D9_KO_LPS_B1
column name in WTC11_bulkRNAseq_iMGL_raw_counts.tsv: D9_KO_LPS_B1


title: SK-MEL-28, 2 uM ASO2 (NTC), biol rep 3

city: Seattle

first: Benjamin

last: Logsdon

state: WA

address: 1150 Eastlake Ave E Ste 600/700

institute: Cajal Therapeutics

description:
Library name: AN98-ASO2-RNA-4
column AN98-ASO2-RNA-4 in AN98_deseq2_tmm_norm_counts.tsv

data_processing:
Bcl -> FASTQ demultiplexing performed by Azenta (bcl2fastq).
Transcript-level quantification with Salmon v1.10.0 against the GENCODE human transcriptome (confirm release).
Transcript counts aggregated to gene level with tximport; gene-level counts normalized in DESeq2 v1.38.3 using TMM.
Differential expression testing with DESeq2 (Wald test).
Assembly: GRCh38 (GENCODE release to be confirmed)
Supplementary files format and content: AN98_deseq2_tmm_norm_counts.tsv: tab-delimited gene x sample matrix of DESeq2 TMM-normalized counts. 14,473 Ensembl gene IDs x 12 samples with headers matching *library name values.

growth_protocol: SK-MEL-28 cells (ATCC HTB-72) cultured in EMEM (ATCC 30-2003) + 10% FBS + 1% Penicillin/Streptomycin. Cells were thawed at passage 9, expanded to passage 10, and plated on 2026-02-09 into 24-well plates at 100,000 cells/well (viability 96%) in 1 mL medium and grown ~24 h before transfection.

extract_protocol:
Cell pellets lysed in 250 uL cold Trizol on ice, vortexed 2 x 30 s, incubated 5 min at RT; 50 uL chloroform added, shaken 15 s, incubated 3 min at RT, centrifuged 15 min at 12,000 x g / 4 C. Aqueous phase mixed 1:1 with 250 uL of 70% ethanol and purified on Qiagen RNeasy MinElute Micro columns (UPenn hybrid protocol; RW1 wash, two RPE washes, 80% EtOH wash, 3 min dry spin, elution in 15 uL water). RNA quantified by NanoDrop (A260/A280 1.97-2.04, A260/A230 1.78-2.28, concentrations 205-366 ng/uL). Twelve of 16 samples passed QC and were normalized to 40 ng/uL in 30 uL (1.2 ug total) for shipping to Azenta.
RNA (1,200 ng in 30 uL at 40 ng/uL) submitted to Azenta Life Sciences (project 30-1296162709) for Standard RNA-Seq with polyA selection (rRNA removal = PolyA selection for mRNA species, eukaryotic; 30 M reads/sample; Illumina 2x150 bp paired-end). (Exact library-prep kit chemistry to be confirmed with Azenta; likely NEBNext Ultra II Directional RNA Library Prep Kit.)

treatment_protocol: On 2026-02-10, cells were transfected with one of four ASOs (ASO2 non-targeting control, ASO3 pan-SNCA, ASO43 dIRE-selective from IDT; ASO550 dIRE-selective from Biospring) at 2 uM final in-well concentration; n=4 biological replicates prepared per ASO. ASO stocks were diluted to 200 uM in water; 7.7 uL of 200 uM ASO + 27.3 uL Opti-MEM were combined with 2.1 uL Lipofectamine RNAiMAX + 32.9 uL Opti-MEM (pre-mixed dilute-lipo master mix); after 5 min at RT, 60 uL of the ASO/lipid complex added to 600 uL medium per well (final [ASO] = 2 uM). Cell pellets were collected 3 days post-transfection (2026-02-13) by PBS wash x 2, Trypsin/Accutase (5 min), quench with FBS-containing medium, centrifugation at 350-400 x g / 5 min / 4 C, PBS wash, second spin, and -80 C storage.

value: melanoma (metastatic melanoma-derived)

value: wild type

value: Non-targeting control ASO (ASO0002, IDT); transfected with Lipofectamine RNAiMAX in Opti-MEM; 3-day incubation

tag: dose

value: 2 uM

tag: replicate


title: MEF2C-KO, non-treated, differentiation 2, clone D9

description:
Library name: D9_KO_NT_B2
column name in WTC11_bulkRNAseq_iMGL_raw_counts.tsv: D9_KO_NT_B2

value: non-treated


title: MEF2C-KO, myelin-treated, differentiation 1, clone D9

description:
Library name: D9_KO_M_B1
column name in WTC11_bulkRNAseq_iMGL_raw_counts.tsv: D9_KO_M_B1

value: myelin


title: R-o-18 leaf, MeJA, 24 h, biological replicate 2

description:
Library name: MeJA_1D_2
Leaf tissue from 10-day-old R-o-18 seedling; MeJA treatment; harvested at 24 h; biological replicate 2.

value: leaf


title: R-o-18 leaf, MeJA, 24 h, biological replicate 1

description:
Library name: MeJA_1D_1
Leaf tissue from 10-day-old R-o-18 seedling; MeJA treatment; harvested at 24 h; biological replicate 1.


title: WT, myelin-treated, differentiation 1, clone H2 (scRNA-seq)

description: Library name: H2_WT_M


title: R-o-18 leaf, MeJA, 10 min, biological replicate 2

description:
Library name: MeJA_0D_2
Leaf tissue from 10-day-old R-o-18 seedling; MeJA treatment; harvested at 10 min; biological replicate 2.

value: 10 min


title: MEF2C-KO, myelin-treated, differentiation 2, clone D9

description:
Library name: D9_KO_M_B2
column name in WTC11_bulkRNAseq_iMGL_raw_counts.tsv: D9_KO_M_B2

value: iPSC-derived microglia


title: Bacteroides thetaiotaomicron RNA-seq extracted from liquid culture, replicate 1

description: Library name: Bt_1_LC


title: SNCA-GR mouse M9378, SNc-VTA (midbrain), snRNA-seq 10x 5' v2 (short-read)

description:
Library name: LX3-250506-05
10x Chromium Next GEM Single Cell 5' v2, dual-indexed with SI-TT-A5; libraries sequenced across three Illumina NextSeq 2000 P3 flow-cell runs (RX1-0227, RX1-0228, RX1-0229) and merged before Cell Ranger v6.1.1 processing.

data_processing:
[10X] Bcl -> FASTQ demultiplexing with Illumina DRAGEN BCL Convert v3.10.4 on BaseSpace, one FASTQ set per NextSeq 2000 P3 flow cell (RX1-0227 / RX1-0228 / RX1-0229). Per-sample FASTQs from all three runs were merged before Cell Ranger input.
[10X] cellranger count --include-introns --chemistry fiveprime (Cell Ranger v6.1.1; martian v4.0.6) against a mouse reference (confirm exact build). Outputs: filtered_feature_bc_matrix {barcodes,features,matrix} + .h5, metrics_summary.csv, web_summary.html, analysis/ (clustering, PCA, tSNE, UMAP, diffexp).
[ONT] Real-time basecalling with MinKNOW on PromethION P2 Solo, R10.4.1 chemistry, LSK114 kit; POD5 signals recorded; basecalls written to <libID>_live_basecall.fastq.gz.
[ONT] minimap2 alignment of ONT basecalls with splice-aware parameters (-ax splice -uf -k14) against the same mouse reference used for Cell Ranger. Alignments sorted and indexed with samtools. No gene- or isoform-level quantification is included in this Series.
Assembly: GRCh38 (GenBank assembly accession GCA_000001405.15, primary assembly/no-alt analysis set)
Supplementary files format and content: [10X] For each 10x library: three 10x Market Exchange files (<libID>_barcodes.tsv.gz cell barcodes, <libID>_features.tsv.gz gene features, <libID>_matrix.mtx.gz sparse UMI matrix), the same matrix in HDF5 (<libID>_filtered_feature_bc_matrix.h5), and <libID>_metrics_summary.csv Cell Ranger QC metrics.
Supplementary files format and content: [ONT] <libID>_live_basecall_minimap2_sorted.bam: coordinate-sorted BAM of minimap2 splice-aware alignments of ONT basecalls to the same mouse reference; retains 10x cell-barcode + UMI, basecalled read sequences, and per-base Q-scores. Same BAM is listed as the *raw file for each ONT library -- basecalled FASTQ can be regenerated with `samtools fastq`. Full FASTQs are archived in the source bucket and available on reasonable request.

source_name: Substantia nigra pars compacta / ventral tegmental area (SNc-VTA), midbrain

extract_protocol:
Brains from three SNCA-GR homozygous male mice (JAX #39166; DOB 2025-03-04; sacrificed 2025-04-28 at age 55 days) were flash-frozen and stored at -80 C. On 2025-04-30 (M9376) and 2025-05-01 (M9377, M9378), 50 um coronal sections were cut on a dry-ice-cooled sectioning stage and the substantia nigra / VTA and olfactory bulb were microdissected under a stereo microscope from both hemispheres, then homogenized in 1 mL Homogenization Buffer (NIM pH 8.0 with 0.2 U/uL RNase Inhibitor, 1x protease inhibitor cocktail, 0.1 mM DTT) using a Dounce homogenizer (10 loose + 10 tight strokes). Nuclei were purified by Debris Removal Solution gradient (Miltenyi), washed in 2X BSA/PBS, filtered through a 30 um strainer, and resuspended in 1% BSA blocking buffer. Nuclei counts (Trypan blue, Neubauer hemocytometer) were adjusted to 200-700 nuclei/uL for 10x loading.
[10X] 10x Genomics Chromium Next GEM Single Cell 5' v2: nuclei loaded onto a Chromium Next GEM Chip K at target recovery of 7,500 cells (12,000 loaded). GEM-RT, Dynabead cleanup, and 13-cycle cDNA amplification per manufacturer protocol. Gene expression libraries constructed from 50 ng of amplified cDNA with the 10x Library Construction Kit; 14-cycle indexing PCR with Dual Index Kit TT Set A wells A1-A6 (one index per sample).
[ONT] 10 ng of the same amplified 10x cDNA per sample was re-amplified (7 cycles, LongAmp Hot Start Taq 2X, 66->58 C ramp, 6 min extension) with custom oligos Fwd_3580_partial_read1_defined_for_5'_cDNA (5'-/5phos/ACTTGCCTGTCGCTCTATCTTCCTACACGACGCTCTTCCGATCT-3') and Rev_PR2_partial_TSO_defined_for_5'_cDNA (5'-/5phos/TTTCTGTTGGTGCTGATATTGCAAGCAGTGGTATCAACGCAGAG-3') to add ONT-compatible ends preserving the 10x cell barcode + UMI at the 5' of the read. Products bead-cleaned (1x SPRIselect), end-prepped (NEBNext Ultra II End Repair / dA-tailing), ligated to ONT LSK114 sequencing adapters with Salt-T4 DNA Ligase; cleaned with SPRIselect (0.4x) and eluted in 34 uL Elution Buffer.

value: nuclei

tag: age

value: 55 days

tag: Sex

value: male

value: SNCA-GR homozygous (humanized SNCA gene-replacement knock-in; JAX #39166)

tag: strain

value: SNCA-GR (JAX #39166)


title: R-o-18 leaf, MeJA, 10 min, biological replicate 3

description:
Library name: MeJA_0D_3
Leaf tissue from 10-day-old R-o-18 seedling; MeJA treatment; harvested at 10 min; biological replicate 3.

value: WT

value: 50 μM methyl jasmonate


title: SNCA-GR mouse M9378, olfactory bulb, snRNA-seq 10x 5' v2 (short-read)

description:
Library name: LX3-250506-06
10x Chromium Next GEM Single Cell 5' v2, dual-indexed with SI-TT-A6; libraries sequenced across three Illumina NextSeq 2000 P3 flow-cell runs (RX1-0227, RX1-0228, RX1-0229) and merged before Cell Ranger v6.1.1 processing.

source_name: Olfactory bulb

value: Olfactory bulb


title: R-o-18 leaf, MeJA, 10 min, biological replicate 1

description:
Library name: MeJA_0D_1
Leaf tissue from 10-day-old R-o-18 seedling; MeJA treatment; harvested at 10 min; biological replicate 1.


title: MEF2C-KO, non-treated, differentiation 1, clone D9

description:
Library name: D9_KO_NT_B1
column name in WTC11_bulkRNAseq_iMGL_raw_counts.tsv: D9_KO_NT_B1


title: MEF2C-KO, LPS-treated, differentiation 2, clone H11

description:
Library name: H11_KO_LPS_B2
column name in WTC11_bulkRNAseq_iMGL_raw_counts.tsv: H11_KO_LPS_B2

value: LPS


title: MEF2C-KO, LPS-treated, differentiation 1, clone H11

description:
Library name: H11_KO_LPS_B1
column name in WTC11_bulkRNAseq_iMGL_raw_counts.tsv: H11_KO_LPS_B1


title: SNCA-GR mouse M9377, SNc-VTA (midbrain), snRNA-seq 10x 5' v2 (short-read)

description:
Library name: LX3-250506-03
10x Chromium Next GEM Single Cell 5' v2, dual-indexed with SI-TT-A3; libraries sequenced across three Illumina NextSeq 2000 P3 flow-cell runs (RX1-0227, RX1-0228, RX1-0229) and merged before Cell Ranger v6.1.1 processing.

value: Substantia nigra pars compacta / ventral tegmental area (SNc-VTA), midbrain


title: MEF2C-KO, myelin-treated, differentiation 2, clone H11

description:
Library name: H11_KO_M_B2
column name in WTC11_bulkRNAseq_iMGL_raw_counts.tsv: H11_KO_M_B2


title: SNCA-GR mouse M9377, olfactory bulb, snRNA-seq 10x 5' v2 (short-read)

description:
Library name: LX3-250506-04
10x Chromium Next GEM Single Cell 5' v2, dual-indexed with SI-TT-A4; libraries sequenced across three Illumina NextSeq 2000 P3 flow-cell runs (RX1-0227, RX1-0228, RX1-0229) and merged before Cell Ranger v6.1.1 processing.


title: Primary microglia, DEVDfmk, 120min, rep6

city: Seville

first: Miguel

last: Burguillos

address: Antonio Maura Montaner

country: Spain

institute: IBiS-CSIC

description:
Library name: DEVDfmk_6
primary microglia isolated from whole brain excluding cerebellum

data_processing:
DRAGEN BCL convert software version 4.2.7 was used for basecalling
Sequence reads were trimmed for adaptor sequence/low-quality sequence using FASTQ Generation based on bcl2fastq with default parameters. Reads were mapped to murine genome assembly (GRCm39) using HISAT2  using default parameters but specifying --rna-strandness RF
Differential expressed genes between samples were analysed using edgeR, genes positions were obtained from the GTF file from GENCODE (M36, GRCm39)
Assembly: GRCm39
Supplementary files format and content: Feature count files for each sample from edgeR

source_name: brain

growth_protocol: Postnatal P1–P4 mice were sacrificed, and brains were carefully dissected with meninges and cerebellum removed. Cortices were washed in ice-cold calcium- and magnesium-free Hank's Buffered Salt Solution (HBSS; Biowest). Brains were then minced and resuspended in ice-cold HBSS. After washing, tissues were incubated for 15 minutes at 37ºC in HBSS containing 0.1% trypsin and 0.05% DNase, then resuspended in DMEM (high glucose, GlutaMAX, Gibco) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (P/S). Cells from one brain were seeded into T25 flasks; cells pooled from 4–5 brains were seeded into T75 flasks. Medium was fully replaced 24 hours after seeding. GM-CSF (5 ng/mL) was added 7 days after seeding. Microglial cells were harvested from confluent astrocyte monolayers, 10–12 days after the initial seeding, by tapping the side of the culture flask. These microglial cells found in the medium were plated into 24-well dishes. Experiments were performed 24 h after the final plating.

extract_protocol:
Total RNA was extracted using the AllPrep DNA/RNA Mini Kit (Qiagen, Cat# 80204), according to the manufacturer's instructions. Cells were lysed directly in the culture flasks using the provided lysis buffer containing guanidine thiocyanate. Homogenized lysates were passed through the AllPrep spin columns to separate DNA and RNA. To ensure high purity and remove genomic DNA contamination, on-column DNase treatment was performed on the RNeasy spin columns using the Qiagen RNase-Free DNase set (Cat# 79254). Specifically, 80 µl of DNase I incubation mix (10 µl DNase I stock solution in 70 µl Buffer RDD) was applied directly to the membrane and incubated at room temperature for 15 minutes. After washing, RNA was eluted in RNase-free water and stored at -80°C until use. RNA concentration and purity were assessed using a NanoDrop spectrophotometer and RNA integrity was confirmed using an Agilent Bioanalyzer
Total RNA-Seq libraries were prepared using Illumina Stranded Total Prep, Ligation with Ribo.Zero Plus

treatment_protocol: Cells were treated with vehicle or 20uM DEVDfmk (MedChemExpress; Cat# HY-12466) for 120 minutes

value: brain

value: microglia

value: wild type

value: DEVDfmk (20 uM, 120 min)


title: Primary microglia, vehicle, 120min, rep6

description:
Library name: C_6
primary microglia isolated from whole brain excluding cerebellum

value: vehicle (120 min)


title: MEF2C-KO, myelin-treated, differentiation 1, clone H11

description:
Library name: H11_KO_M_B1
column name in WTC11_bulkRNAseq_iMGL_raw_counts.tsv: H11_KO_M_B1

value: myelin


title: MEF2C-KO, non-treated, differentiation 1, clone H11

description:
Library name: H11_KO_NT_B1
column name in WTC11_bulkRNAseq_iMGL_raw_counts.tsv: H11_KO_NT_B1

value: iPSC-derived microglia

value: non-treated


title: MEF2C-KO, non-treated, differentiation 2, clone H11

description:
Library name: H11_KO_NT_B2
column name in WTC11_bulkRNAseq_iMGL_raw_counts.tsv: H11_KO_NT_B2


title: Primary microglia, vehicle, 120min, rep5

description:
Library name: C_5
primary microglia isolated from whole brain excluding cerebellum


title: Primary microglia, DEVDfmk, 120min, rep5

description:
Library name: DEVDfmk_5
primary microglia isolated from whole brain excluding cerebellum


title: MEF2C-KO, LPS-treated, differentiation 2, clone G2

description:
Library name: G2_KO_LPS_B2
column name in WTC11_bulkRNAseq_iMGL_raw_counts.tsv: G2_KO_LPS_B2


title: MEF2C-KO, myelin-treated, differentiation 2, clone G2

description:
Library name: G2_KO_M_B2
column name in WTC11_bulkRNAseq_iMGL_raw_counts.tsv: G2_KO_M_B2


title: MEF2C-KO, LPS-treated, differentiation 1, clone G2

description:
Library name: G2_KO_LPS_B1
column name in WTC11_bulkRNAseq_iMGL_raw_counts.tsv: G2_KO_LPS_B1


title: MEF2C-KO, non-treated, differentiation 2, clone G2

description:
Library name: G2_KO_NT_B2
column name in WTC11_bulkRNAseq_iMGL_raw_counts.tsv: G2_KO_NT_B2


title: MEF2C-KO, myelin-treated, differentiation 1, clone G2

description:
Library name: G2_KO_M_B1
column name in WTC11_bulkRNAseq_iMGL_raw_counts.tsv: G2_KO_M_B1


title: Primary microglia, DEVDfmk, 120min, rep4

description:
Library name: DEVDfmk_4
primary microglia isolated from whole brain excluding cerebellum


title: MEF2C-KO, non-treated, differentiation 1, clone G2

description:
Library name: G2_KO_NT_B1
column name in WTC11_bulkRNAseq_iMGL_raw_counts.tsv: G2_KO_NT_B1


title: Primary microglia, vehicle, 120min, rep4

description:
Library name: C_4
primary microglia isolated from whole brain excluding cerebellum


title: rd10.F.HAL.last_rd10_F1_F

city: Bethesda

first: Matthew

middle: J

last: Brooks

state: MD

address: 6 Center Dr Bldg 6, Rm 303

department: NEI

institute: NIH

description:
Library name: rd10.F.HAL.last_rd10_F1_F
transcriptome of HAL treated rd10 female retina

data_processing:
Illumina bulk RNAseq reads were quality checked and aligned to Ensembl reference transcriptomes of Mus musculus (version 115) for control and treated samples of rd10 retinas. Analysis pipeline has been described previously (Song, et al. 2025). Briefly, transcriptome alignments were performed with Kallisto and processed with the R package tximport, before differential expression analysis with edgeR and limma.
Differentially expressed genes (DEG) were identified by setting a p-value filter of 0.05 and selecting the top 500 over and under expressing genes with at least 1.2-fold change. DEGs were mapped to KEGG pathways using gProfiler and filtered to identify pathways that had minimum 3 DEG mappings and at least 5% of the pathway impacted by differential genes.
Assembly: GRCm39/ENSv115
Supplementary files format and content: Comma-delimited text files containing transcript level counts, gene level counts, normalized gene level counts-per-million results, or diffierential expressed gene results.

source_name: Retina

growth_protocol: B6.CXB1-Pde6brd10/J (Strain #: 004297; RRID: IMSR_JAX:004279), including both female and male, were obtained from the Jackson Laboratory and intercrossed to generate the pups. Mice were maintained under controlled environment conditions (temperature: 22 °C ± 2 °C, humidity: 30-70%) with a 12-hour light/dark cycle and had ad libitum access to food and water. Bedding, food, and water were replaced on a weekly basis.

extract_protocol:
Total RNA was isolated from homogenized mouse retinas using RNeasy Mini Kit (QIAGEN), following the manufacturer’s instructions. RNA quality was evaluated using the TapeStation system (Agilent), and only samples with high quality (RNA RIN ≥9) were used for the downstream applications.
For library preparation, 100 ng of total RNA was used to generate  strand-specific mRNA sequencing libraries using the TruSeq stranded RNA Kit (Illumina).

treatment_protocol: For intravitreal injections in mice at P10 and P24, pups received intraperitoneal anesthesia with ketamine (50 mg/kg body weight) and xylazine (5 mg/kg body weight). Either  DMSO or Halofantrine (1 μL, 20 μM) was delivered into the vitreous using a Hamilton syringe fitted with a 34-gauge needle. Mice showing evidence of ocular or lens injury following injection were excluded from further analysis.

value: Retina

tag: group

value: F

value: HAL


title: rd10.F.HAL.set2_F1

description:
Library name: rd10.F.HAL.set2_F1
transcriptome of HAL treated rd10 female retina


title: rd10.F.DMSO.final_rd10_F1_F

description:
Library name: rd10.F.DMSO.final_rd10_F1_F
transcriptome of DMSO treated rd10 female retina

value: DMSO


title: MEF2C-KO, myelin-treated, differentiation 1, clone D8

description:
Library name: D8_KO_M_B1
column name in WTC11_bulkRNAseq_iMGL_raw_counts.tsv: D8_KO_M_B1


title: rd10.F.DMSO.set2_F5

description:
Library name: rd10.F.DMSO.set2_F5
transcriptome of DMSO treated rd10 female retina


title: MEF2C-KO, myelin-treated, differentiation 2, clone D8

description:
Library name: D8_KO_M_B2
column name in WTC11_bulkRNAseq_iMGL_raw_counts.tsv: D8_KO_M_B2


title: MEF2C-KO, LPS-treated, differentiation 1, clone D8

description:
Library name: D8_KO_LPS_B1
column name in WTC11_bulkRNAseq_iMGL_raw_counts.tsv: D8_KO_LPS_B1


title: MEF2C-KO, non-treated, differentiation 1, clone D8

description:
Library name: D8_KO_NT_B1
column name in WTC11_bulkRNAseq_iMGL_raw_counts.tsv: D8_KO_NT_B1


title: rd10.F.DMSO.set2_F4

description:
Library name: rd10.F.DMSO.set2_F4
transcriptome of DMSO treated rd10 female retina


title: WT.F.DMSO.set1_F2

description:
Library name: WT.F.DMSO.set1_F2
transcriptome of DMSO treated WT female retina

value: WT


title: WT, AD ReN, differentiation 1, clone E2

description: Library name: E2_WT_AD

growth_protocol: iPSC lines were differentiated to hematopoietic precursor cells (HPCs) using StemDiff kit (StemCell Technologies). 20,833 cells/cm2 HPCs were plated and differentiated for 24 days to microglia (iMGL) in iMGL media (DMEM/F12, 2xB27, 0.5xN2, 2xITSG, 400uM monothioglycerol, 5ug/ml insulin, 1xNEAA, 1xGlutaMax) supplemented with three factors (IL34 (100ng/ml), TGFb (50ng/ml), and MCSF (25ng/ml). iMGLs were fed Monday, Wednesday and Friday with 1ml of iMGL media with three factors. Cells were splitted in 1:2 ratio if too confluent. Day 24 iMGLs were lifted with gentle pipetting and Accutase and plated 62,500 cells/cm2 in 1ml of conditioned media and 1ml of fresh media iMGL media with three factors supplemented additionally with two factors (CX3CL1 and CD200 100ng/ml each) for 3 additional days (day 25, day 26,  day 27). At day 26 of iMGL differentiation, iMGL received 1ml of five factor iMGL media and treatment with myelin or LPS. Cells were collected at day 27.  ReNcell VM human neural progenitor cells (NPCs) were obtained from the laboratories of Drs. Rudolph Tanzi and Doo Kim. Two cell lines were used: (1) control cells expressing GFP and (2) Alzheimer’s disease (AD) cells harboring APP (Swedish and London) and PSEN1 (ΔE9) mutations, expressing both GFP and mCherry.For maintenance, ReN NPCs were cultured on BD Matrigel–coated T75 flasks (Corning, cat. 356231) in expansion medium consisting of DMEM/F12 supplemented with 2% B27 neural supplement, heparin (2 µg/mL), epidermal growth factor (EGF, 20 ng/mL), basic fibroblast growth factor (bFGF, 20 ng/mL), and 1× penicillin/streptomycin. Media were replaced every 72 hours until cultures reached approximately 80% confluency. For 3D neuron/astrocyte differentiation, cells were dissociated with Accutase, counted, and plated at 250,000 cells per well in 96-well plates in expansion medium supplemented with 10% cold Matrigel. Twenty-four hours later, medium was fully replaced with differentiation medium (expansion medium lacking EGF and bFGF). Media were refreshed by 50% every 3–4 days. Cultures were differentiated for four weeks to generate mature neuronal and astrocytic populations. After four weeks of differentiation, mature neuronal/astrocyte cultures were overlaid with mature iPSC-derived microglia (iMGLs). A total of 50,000 iMGLs were added per well. The neuronal/astrocyte medium was supplemented with microglia-supporting three cytokines (IL34, TGFb, MCSF) to maintain iMGL survival. Tricultures were maintained for one additional week prior to scRNAseq

extract_protocol:
Cells were washed with PBS and dissociated using Papain Dissociation System (Worthington Biochemical, cat.LK003153) according to the manufacturer’s instructions for 40 minutes at 37°C according to manufacturer's instructions. Cells were centrifuged at 400 × g for 5 minutes and resuspended in 1.0% PBS/bovine serum albumin (BSA). The cell suspension was filtered through a 40 μm cell strainer to remove aggregates. Cell number and viability were assessed using trypan blue exclusion and an automated cell counter (Countess, Thermo Fisher Scientific). Only samples with high viability (typically >90%) were used for downstream processing.
Single cells were partitioned into PIP droplets using the PIPseq T20 3’ Single Cell RNA Kit v4.0 (Fluent Biosciences, Watertown, MA, USA) according to the manufacturer’s instructions. Briefly, approximately 40,000 live cells were combined with template beads, gently mixed, vortexed for 3 minutes to generate stable PIP emulsions, and processed following the recommended workflow. Captured mRNA was released upon cell lysis within the droplets, followed by reverse transcription, cDNA amplification, cDNA purification, and library preparation. Libraries were sequenced by Azenta (New Jersey, USA) on an Illumina NovaSeq X platform, generating ~50,000 reads per cell (~2 billion reads per sample).

value: triculture

value: AD ReN


title: WT.F.DMSO.set1_F1

description:
Library name: WT.F.DMSO.set1_F1
transcriptome of DMSO treated WT female retina


title: WT.F.DMSO.set1_5_F3

description:
Library name: WT.F.DMSO.set1_5_F3
transcriptome of DMSO treated WT female retina


title: WT, AD ReN, differentiation 1, clone H2

description: Library name: H2_WT_AD