title: D4_ki67-myc+_ROI1_S3_Milademetan

city: Umeå

first: Lixiao

last: Wang

state: Sverige

address: Medicinsk biovetenskap, M21, Försörjningsvägen 1

department: Department of Medical Biosciences

country: Sweden

institute: Umeå University

description:
Library name: DSP-1001660037306-B-E05
Tumor D4, Slide S3, ROI 1, Segment ki67-myc+, Replicate D4_ki67-myc+. Raji xenograft in NSG mouse, NanoString GeoMx DSP WTA v1.0.

data_processing:
FASTQ files were converted to DCC count files via NanoString GeoMx NGS Pipeline. DCC files loaded into R using GeomxTools (Bioconductor). Segment QC: min 1,000 reads, 75% alignment, 50% saturation, 20 nuclei, 1,000 um2 area. Probe QC: ratio >0.1, Grubbs outlier removal. Aggregated to gene-level by geometric mean.
Counts imported into standR (Bioconductor) as SpatialExperiment. Low-expression genes filtered (sample_fraction=0.9, min_count=5). TMM normalization (edgeR). Batch effects assessed by PCA.
DE analysis: limma-voom with duplicateCorrelation (tumor as block). Treatment comparisons per cell population. Gene set analysis: limma::camera for MYC targets, ribosomal, cell cycle, apoptosis pathways. Significance: BH-adjusted P < 0.05 and |log2FC| > 0.5.
R (v4.4.2), GeomxTools, standR, limma, edgeR, SpatialExperiment (Bioconductor).
Assembly: hg38 (GRCh38)
Supplementary files format and content: DCC files: per-AOI probe-level count files from NanoString GeoMx NGS Pipeline, containing raw/trimmed/stitched/aligned read counts and per-probe expression values
Supplementary files format and content: standR_counts.txt: tab-delimited gene-level raw count matrix (genes x 92 AOIs)
Library strategy: Spatial Transcriptomics

source_name: Raji Burkitt lymphoma xenograft

growth_protocol: Raji Burkitt lymphoma cells (ATCC CCL-86, p53 R213Q) were harvested during logarithmic growth, washed, and resuspended in serum-free media. A total of 5x10^6 cells in 200 uL of a 1:1 mixture of Matrigel and medium were injected subcutaneously into the right flank of 6-8-week-old NSG mice (Charles River). Tumors grew to approximately 100-150 mm3 before randomization.

extract_protocol:
FFPE tumor sections were processed on the NanoString GeoMx Digital Spatial Profiler (DSP) at the Genomic Medicine Sweden Center North / Clinical Genomics Umea. Sections were stained with fluorescent antibodies against Ki67 and c-Myc. Within each ROI, cells were segmented into four populations: Ki67+MYC+, Ki67-MYC+, Ki67-MYC-, and Ki67+MYC-. UV-cleavable oligonucleotide tags from each segment (AOI) were collected for library preparation.
Libraries were prepared using the NanoString GeoMx NGS Library Preparation Kit per manufacturer protocol. Human NGS Whole Transcriptome Atlas (WTA v1.0) panel (~18,000 genes). Each AOI received a unique indexing barcode. 92 AOIs across 10 tumors on four slides (S1-S4), each slide containing both conditions.

treatment_protocol: Mice were randomized into milademetan-treated (n=5) and vehicle control (n=5) groups. Milademetan (20 mg/kg) or vehicle was administered daily by oral gavage for 15 days. Tumors were harvested, formalin-fixed, and paraffin-embedded (FFPE).

value: Raji Burkitt lymphoma xenograft

value: Raji (ATCC CCL-86)

value: Milademetan 20mg/kg


title: CTRL_3

status: Public on Jul 07 2026

type: SRA

city: St Louis

first: Carolina

middle: B

last: Lopez

state: MO

address: 425 S Euclid Ave

country: USA

institute: Washington University in St. Louis

description: Library name: CTRL_3

library_source: transcriptomic

data_processing:
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 and 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, with results aggregated into a comprehensive quality control report using MultiQC v1.32. 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.  Sample-sample correlations  for sample-sample heatmap and PCA were calculated on normalized counts  (TMM, trimmed mean of M-values) using Pearson correlation. Differential expression  was done with edgeR v4.0.16 using standard practice including filtering for  low-expressed genes with edgeR::filterByExprwith default values. Functional  enrichment, when available, is performed using gene set enrichment analysis with  gseapy v0.12 using the MSigDB Hallmark gene set.
Assembly: GRCh38
Supplementary files format and content: fastq sequencing files
Supplementary files format and content: csv gene expression tables
Supplementary files format and content: csv gene set enrichment tables

molecule: polyA RNA

organism: Homo sapiens

growth_protocol: A549 human lung epithelial cells (ATCC CCL-185) were maintained in Dulbecco’s Modified Eagle Medium (DMEM; Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 1 mM sodium pyruvate, and 50 μg/mL gentamicin at 37°C in a humidified incubator containing 5% CO2.

extract_protocol:
RNA was extracted by Kingfisher (Thermofisher) with MagMax Mirvana Total RNA isolation kit (Thermofisher, A27828).
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). Second strand synthesis and tagmentation: double stranded-cDNA is generated. Tagmentation creates fragments and incorporates Read 2 sequence. Illumina library amplification: adds unique dual indices (UDI = i5, i7) and P5/P7 sequences.

treatment_protocol: Cells were infected with Sendai Virus (MOI 1.5) for 24 hours

tag: cell line

tag: cell type

value: lung epithelial cells

tag: treatment

value: mock


title: CTRL_2

description: Library name: CTRL_2


title: CTRL_1

description: Library name: CTRL_1


title: LATSi_3

city: Miami

first: xiaomin

last: cai

state: Florida

address: Rosenstiel Medical Science Building 1600 NW, 10th Ave.

institute: University of Miami

description: Library name: I3

data_processing:
Raw FASTQ files were processed in a Linux (Ubuntu 22.04) environment. Reads were aligned to the Mus musculus GRCm38 reference genome using HISAT2. Gene-level read counts were generated using featureCounts with the GENCODE vM25 annotation (gencode.vM25.annotation.gtf).
Assembly: Mus musculus GRCm38
Supplementary files format and content: The processed data file is a gene-level raw count matrix generated by featureCounts.

molecule: total RNA

organism: Mus musculus

source_name: mouse knee joint tissue

extract_protocol:
For the first set, total RNA was extracted from mouse knee joint cartilage tissue using TRIzol reagent (Invitrogen, USA) according to the manufacturer’s instructions. For the second and third sets, total RNA was extracted from mouse knee joint tissues using a modified Trizol/RNeasy. Knee tissues were cryogenically homogenized using stainless steel beads, lysed in TRIzol reagent, treated with Proteinase K, subjected to chloroform extraction, and purified using RNeasy spin columns (Qiagen).
For the first set, Sequencing libraries were then prepared according to the manufacturer’s protocol and sequenced by Innomics Inc. on the DNBSEQ platform using paired-end 100-bp reads.  For the second and third sets, Sequencing libraries were prepared according to the manufacturer’s protocol and sequenced by Admera Health, LLC on an Illumina platform using paired-end 150-bp reads.

tag: tissue

value: mouse knee joint tissue


title: Normal weight control gene expression

city: Madison

first: Aleksandar

middle: K

last: Stanic

state: WI

address: 202 S Park, PAB1

department: Obstetrics and Gynecology

institute: University of Wisconsin-Madison

description:
Library name: 51C-N-GEX
raw_counts_matrix.tsv

data_processing:
CellRanger Count 6.1.2
Alignment to hg38 genome assembly with default parameters.
Filtering based on feature counts, mitochondrial counts, doublet/singlet anotation, and total RNA count was performed in Seurat.
Counts were depth-normalied and log-transformed, and top 2000 variable features determined. Counts were scaled.
CITE-Seq antibody counts were depth-normalized, log-transformed, and counts scaled.
Assembly: hg38
Supplementary files format and content: raw_counts_matrix.tsv
Supplementary files format and content: Wegrzynowicz_PCOS_GEO.Rds
Supplementary files format and content: raw_adt_matrix.tsv
Supplementary files format and content: cell_metadata.tsv
Supplementary files format and content: feature_reference.csv

source_name: ovarian follicular fluid, peripheral blood

extract_protocol:
Cells were isolated from patients via peripheral blood draw and follicular aspiration during transvaginal oocyte retrieval. Cells were extracted using gradient centrifugation and washed with RPMI before freezing in Cryo-stor. Cells were then thawed and single-cell solutions prepared by filter separation. Dead cells were removed with Miltenyi dead cell removal kit according to manufacturer's instructions. RNA was extracted and proceeded to sequencing.
CITE-Seq and hashing protocols described by Stoeckius et al 2017 was used to generate an antibody-derived tag (ADT) library (including hashtag oligos) and a 3' gene expression library using the 10x Genomics library prep kit.


title: LATSi_1

description: Library name: I1


title: KPC Kmt2df/+ Mouse 2500 Rep 3

status: Public on Jul 08 2026

city: Rochester

first: Martin

last: Fernandez-Zapico

state: MN

address: 200 First Street SW

institute: Mayo Clinic

description:
Library name: 11C_2500
Column name 11_2500_htseq.count

data_processing:
RNA-seq FASTq files were processed and trimmed with Trim Galore 0.6.4.
Read quality was evaluated with FastQC and aligned with STAR 2.7.3a and mm10 genome as reference.
Reads were assigned by HTSeq 0.9.1.
BAM files for biological replicates were merged using SAMtools 1.16.
DESeq2 used to generate normalized read counts from merged BAM files (Galaxy Version 2.11.40.6).
Assembly: mm10
Supplementary files format and content: tabular file of DESeq2 normalized counts for each mouse (merged biological replicates)

source_name: pancreas tumor

growth_protocol: Cells were derived from a genetically engineered mouse model (GEMM) pancreas tumors and cultured in DMEM + 10% FBS + 1% Penn/Strep. Cells were maintained at 37°C and 5% CO2.

extract_protocol:
RNA was extracted with TRIzol reagent and then isolated using Qiagen RNeasy kit per manufacturer instructions.
cDNA libraries were prepared using 200 ng of total RNA according to the manufacturer’s instructions for the Illumina TruSeq Stranded mRNA Sample Prep Kit.

value: pancreas tumor

tag: genotype

value: KPC Kmt2df/+

tag: mouse id

value: KPC Kmt2df/+ Mouse 2500

tag: batch


title: Normal weight control feature barcode

description:
Library name: 51C-N-FB
raw_adt_matrix.tsv
feature_reference.csv

library_source: other

data_processing:
CellRanger Count 6.1.2
Alignment to hg38 genome assembly with default parameters.
Filtering based on feature counts, mitochondrial counts, doublet/singlet anotation, and total RNA count was performed in Seurat.
Counts were depth-normalied and log-transformed, and top 2000 variable features determined. Counts were scaled.
CITE-Seq antibody counts were depth-normalized, log-transformed, and counts scaled.
Assembly: hg38
Supplementary files format and content: raw_counts_matrix.tsv
Supplementary files format and content: Wegrzynowicz_PCOS_GEO.Rds
Supplementary files format and content: raw_adt_matrix.tsv
Supplementary files format and content: cell_metadata.tsv
Supplementary files format and content: feature_reference.csv
Library strategy: CITE-seq

molecule: protein

value: ovarian follicular fluid, peripheral blood


title: PKCi_2

description: Library name: H2


title: Obese PCOS gene expression 2

description:
Library name: 50P-OB-GEX
raw_counts_matrix.tsv


title: Obese PCOS feature barcode 2

description:
Library name: 50P-OB-FB
raw_adt_matrix.tsv
feature_reference.csv


title: PKCi_1

description: Library name: H1


title: Overweight control gene expression

description:
Library name: 46C-OW-GEX
raw_counts_matrix.tsv


title: C_3

description: Library name: G3


title: C_2

description: Library name: G2


title: CTRL_ID554_CA1_Neurones_005

city: Genève

first: Marie-Catherine

last: Vozenin

address: Avenue de la Roseraie 53

department: Radio-Oncologie

country: Switzerland

institute: HUG

description:
Library name: DSP-1001660022431-D-B03
FFPE fixed samples GeoMx Nanostring

data_processing:
DSP data were analyzed in R 4.4.0 (2024) according to Nanostring recommendation, using their in-house tools, with the exception of normalization which was performed using the trimmed mean of M values (TMM) algorithm of edgeR. Other packages were used for data manipulation and plotting as well as gene set enrichment analysis.
First, quality control (QC) was performed for the segments using a set of thresholds on chosen descriptive statistics of the segment. Chosen parameters were used as recommended by Nanostring. Then a Grubb test was performed for gene QC, also using parameters recommended by Nanostring. Next, negative probes were used to evaluate the background of quantification and define limit of quantification thresholds for filtering. Segments that contain less than 5% of the genes above the gene detection threshold and genes that are found in less than 5% of the segments were removed from the analysis. After filtering, normalization was performed using the TMM method of the edgeR package. Robust gene detection rate within segments and cell type specificity were confirmed, enabling downstream analysis.
Data was split per region (CA1, CA3, DG, SO and Cortex) and differential gene expression was performed using a linear mixed model (LMM) with the irradiation modality set as the test variable and the slide number as the random intercept.
All scripts used for the analysis and plotting can be found at https://gitlab.unige.ch/lirr/nanostring_flash_mouse_brain.
Assembly: MW0R30722
Supplementary files format and content: dcc
Library strategy: Spatial Transcriptomics

source_name: Brain

extract_protocol:
24h post-treatment completion, whole brains were collected and stored at 4°C in 4% PFA for 48 hours before Formalin-Fixed Paraffin-Embedded (FFPE) before processing
FFPE tissue section of 5-µm were processed according to the provider standard protocol for the GeoMx Digital Spatial Profiler (DSP).Neurons were identified using NeuN  (1:100, Millipore ABN78) revealed with a secondary goat anti-rabbit Alexa 594 (1:1000, Abcam ab150080). Vessels and astrocytes were also stained using an antibodies cocktail composed of CD31 Alexa 488 (Vessels, 1/100, R&D FAB3628G) and GFAP Alexa 647 (Astrocytes, 1/100, Novus NBP2-3318). The nuclear marker Syto83 (Invitrogen 511364) was also used. Whole slides were imaged at x20 magnification and morphologic markers were used to select regions of interest (ROI). Automatic segmentation of ROI based on CD31, NeuN and GFAP markers was performed to define area of illumination (AOIs). AOIs were exposed to 385 nm light (UV), releasing the indexing oligonucleotides to be sequenced. Sequencing libraries were generated by PCR from the photo-released indexing oligos. Pooled libraries were single-sequenced at 27 base pairs and with the single-index workflow  on an Illumina NovaSeq S4 instrument.

treatment_protocol: Irradiation was performed using a prototype 6 MeV electron beam, Oriatron type 6e (eRT6; PMB Alcen), LINAC, available at Lausanne University Hospital and described previously. Dosimetry has been extensively described and published to ensure reproducible and reliable biological studies. Animals were randomized in 5 groups and were sham or whole brain irradiated using a single dose of 10 Gy or 3 fractions of 10 Gy delivered 48 hours apart. A 17-mm graphite applicator was used and CONV was delivered at 0.09 Gy/s whereas FLASH was delivered in 1 pulse of 5.6x106Gy/s.

value: Brain

tag: strain

value: Neurones

value: CTRL


title: Obese control gene expression 2

description:
Library name: 44C-OB-GEX
raw_counts_matrix.tsv


title: C_1

description: Library name: G1


title: Obese control feature barcode 2

description:
Library name: 44C-OB-FB
raw_adt_matrix.tsv
feature_reference.csv


title: Overweight PCOS gene expression

description:
Library name: 35P-OW-GEX
raw_counts_matrix.tsv


title: 72h_2

description: Library name: F2


title: 72h_1

description: Library name: F1


title: Overweight PCOS feature barcode

description:
Library name: 35P-OW-FB
raw_adt_matrix.tsv
feature_reference.csv


title: Obese PCOS gene expression 1

description:
Library name: 30P-OB-GEX
raw_counts_matrix.tsv


title: 7h_3

description: Library name: E3


title: Obese PCOS feature barcode 1

description:
Library name: 30P-OB-FB
raw_adt_matrix.tsv
feature_reference.csv


title: 7h_2

description: Library name: E2


title: Normal weight PCOS gene expression

description:
Library name: 27P-N-GEX
raw_counts_matrix.tsv


title: 7h_1

description: Library name: E1


title: Y/Ti_1

description: Library name: J1


title: LATSi_2

description: Library name: I2


title: 0h_3

description: Library name: D3


title: Normal weight PCOS feature barcode

description:
Library name: 27P-N-FB
raw_adt_matrix.tsv
feature_reference.csv


title: Obese control gene expression 1

description:
Library name: 23C-OB-GEX
raw_counts_matrix.tsv


title: 0h_2

description: Library name: D2


title: Obese control feature barcode 1

description:
Library name: 23C-OB-FB
raw_adt_matrix.tsv
feature_reference.csv


title: 0h_1

description: Library name: D1


title: PKCi_3

description: Library name: H3


title: IFN16_12months_cd34pos_GoT-ChA

city: New York

first: Anna

middle: S.

last: Nam

state: New York

address: Weiss Research Building, Floor 11

department: Pathology and Laboratory Medicine

institute: Weill Cornell Medicine

description:
Library name: IFN16_12months_cd34pos_GoT-ChA
GoT-ChA

library_source: genomic

data_processing:
10x data was processed using Cell Ranger 1 and v6.1.2 using default parameters. RNA reads were aligned to the human referece genome. Amplicon reads was processed following steps: 1) Identification of reads with proper priming, 2) Identification of cell barcodes (CB) that match with the whitelisted CB, 3) Replacement of CB that are not perfectly matched with the whitelist CB, 4) Deduplication of reads, 5) Analyze reads with CB that are also observed in the 10x RNA-seq dataATAC-seq 6) Collapse networks of closely related UMIs within CBs to correct for sequencing error, 7) Matching of CB/UMI pairs found in 10x scRNA-seq data, discarding those matching non-CALR genes and those matching no gene below a supporting read threshold.
CUT&RUN data were down-sampled to have same number of reads across all conditions within the sample replicate to account for difference in sequencing depth. Among the down-sampled reads, low-quality reads were filtered out using Trimmomatic, resulting reads were aligned with hg38 genome with Bowtie2, and PCR duplicates were removed with SAMtools with a previously described workflow. Aligned reads were then used for peak calling with MACS3 with a q-value threshold of 0.01 and reads from IgG antibody as the control. SPI1 over-enrichment in the called peaks was confirmed with Simple Enrichment Analysis (SEA, v5.5.5). Overlapping peaks between replicates of the same condition were kept for further processing. The peaks were processed with multiBamSummary function from deepTools to obtain counts matrix per sample for downstream analysis.
Genotyping data processing was performed according to the previously published instructions and available Gotcha R package. Briefly, initial FASTQ files were filtered based on base quality scores, and the Fastqsplit function was used to split FASTQ files for subsequent parallel computing. Mutations were determined via the BatchMutationCalling function, which identifies reads that are correctly primed, calculates the counts of reads per cell for each specified genotype (i.e., WT, JAK2-V617F).
Assembly: GRCh38
Supplementary files format and content: Processed 10X data is matrix table of gene/peak counts per cell for each sample
Supplementary files format and content: Output matrices processed from genotyping data have two columns: barcodes (BC) and Genotype

molecule: genomic DNA

source_name: Bone marrow

extract_protocol: GoT-IM Extraction Protocol: Cryopreserved bone marrow mononuclear cells approved by the Institutional Review Board of Weill Cornell Medicine were thawed and stained using standard procedures with the surface antibody CD34-PE (clone AC136, dilution 1:50, Miltenyi Biotec) and DAPI (Sigma-Aldrich), according to manufacturer’s protocol. To eliminate experimental batch effects, cells were labelled simultaneously with hashing antibodies with time-point-identifying barcodes using Hashtag Antibodies 1, 2 or 3 (TotalSeq-A, clone LNH-94, Biolegend) for GoT-IM. To link cell identities to expression of cell surface proteins, cells were also incubated with CITE-seq antibodies according to manufacturer protocol (TotalSeq-A, Biolegend). Cells were subsequently sorted for DAPI−, CD34+ cells using BD Influx at the Weill Cornell Medicine flow cytometry core. Genotyping of Transcriptomes (GoT) was performed by adapting the 10x Genomics platform. FACS-sorted CD34+ cells for each time-point from the same individual were pooled, targeting a total of 10,000-15,000 cells.  GoT-ATAC Extraction Protocol: Cryopreserved bone marrow mononuclear cells were thawed and stained using standard procedures with the surface antibody CD34-PE (clone AC136, dilution 1:50, MACS) and DAPI (Sigma-Aldrich), according to manufacturer’s protocol. Cells were subsequently sorted for DAPI−, CD34+ cells using BD Influx at the Weill Cornell Medicine flow cytometry core. Nuclei were isolated from DAPI-, CD34+ cells according to 10x Genomics Demonstrated Low Cell Input Nuclei Isolation protocol. Lysis buffer was prepared following manufacturer’s recommendations and then split into aliquots for each serial sample. Either TotalSeq-A Anti-Nuclei Pore Complex Proteins Hashtag 9 or 10 Antibody (1uL at 1:5 dilution; BioLegend) was added to each aliquot of lysis buffer. Low-input nuclei isolation was otherwise performed following manufacturer’s recommendations. Subsequently, nuclei from each time-point were counted and pooled together at approximately equal proportions. For IFN03, additional nuclei from the IFNa-treated sample were available to be run on a separate lane.  RFX3-OE Extraction Protocol: Cells from frozen umbilical cord blood mononuclear cells were thawed rapidly at 37°C, red blood cell lysis was performed using ACK Lysis Buffer, then centrifuged, and cell pellet was resuspended in cold MACS buffer. Cells were centrifuged at 300g for 5min at 4°C, and resuspended in cold MACs buffer at a concentration of 1x10^7 cells/mL. CD34+ cells were then isolated with the EasySep™ Human CD34 Positive Selection Kit II (StemCell Technologies #17856) according to manufacturer’s protocol. After isolation, cells were counted and plated in 96-well round-bottom plates (Falcon) in StemSpan™ SFEM II media (StemCell Technologies) with StemSpan™ CD34+ Expansion Supplement (StemCell Technologies) and 1% Pen/Strep. After 24 hours in culture, cells were spun down and resuspended in minimal media consisting of StemSpan StemSpan™ SFEM II media with StemSpan™ CD34+ Expansion Supplement, 1% Pen/Strep, 10μM prostaglandin E2 (StemCell Technologies #72192) and 100ng/μl poloxamer 407 (Millipore Sigma P2164030). Cells were split into 2 technical replicates each for untransduced condition, mCherry-control vector transduction, and RFX3-OE vector transduction. Lentivirus or PBS control was added at an MOI of 100, spinoculated at 300g, 32°C for 1 hour, and cells were incubated in same lentivirus-containing media for 24 hours at 37°C before resuspension in fresh media consisting of StemSpan™ SFEM II media with StemSpan™ CD34+ Expansion Supplement and 1% Pen/Strep. After 48 hours of incubation, cells were stained with CD34-APC antibody (1:50). Cells were then FACS isolated for the CD34+ population in the untransduced condition and the CD34+ mCherry+ populations in the RFX3-OE and mCherrycontrol conditions. Three independent transduction experiments were completed with new units of umbilical cord blood cells. Untransduced, mCherry-Control, and RFX3-OE CD34+ cells were stained with Cell Hashing antibodies (HTO TotalSeq-A, BioLegend) for 30 min at 4°C. Cells were then washed with FACs Buffer three times, pooled, and counted for loading. scRNA-sequencing was then performed using the 10x 3’ v3.1 platform according to manufacturer’s protocol. HTO demultiplexing and downstream analyses such as differential gene expression and gene set enrichment were performed as in GoT-IM for CD34+ bone marrow samples. scRNA-seq experiment was completed on one replicate of three independent transduction experiments.  CUT&RUN Extraction Protocol: UT7 cell lines expressing MPL (thrombopoietin receptor) and either the mutant CALR (type 1, L367Tfs*46) or wildtype CALR transgene (93) were seeded in 10cm plates in DMEM supplemented with 10% FBS (Thomas Scientific) and 5 ng/ml GM-CSF (Miltenyi-Biotec). Cells were treated with 0.1μg/ml Recombinant Human IFN-alpha2a (RC217-14) or PBS-1X control for 24hrs. After treatment, cells were washed 1x with PBS and collected for use with the CUTANA™ CUT&RUN assay (EpiCypher) according to the manufacturer’s protocol. 0.5μg of anti-human rabbit monoclonal PU.1 antibody (ab76543) and IgG antibody (ab172730) were used in the assay.  GoT-IM Library Construction Protocol: The standard 10x Genomics Chromium 3′ (v.3.1 chemistry) was implemented according to the manufacturer’s recommendations up to the cDNA amplification step (10X Genomics, Pleasanton, CA). After cDNA amplification and SPRI cleanup, 10x scRNA-seq and ADT/HTO libraries were generated as recommended. A portion of the cDNA was used for somatic genotyping. Briefly, to capture the somatic genotypes of cells, cDNA was amplified with a locus-specific amplification (10 PCR cycles), using the generic forward SI-PCR primer and a following locus-specific reverse primer for the CALR mutations. The amplified locus-specific cDNAs are then cleaned using SPRI purification to remove unincorporated primers. Finally, the targeted amplicon libraries are generated through a PCR performed with a P5 generic forward PCR primer together with an RPI-x primer. The targeted amplicon libraries were spiked into the remainder of the gene expression and immunophenotyping libraries to be sequenced together on a NovaSeq (Illumina, San Diego, CA). The cycle settings were as follows: 28 cycles for Read 1, 90 cycles for Read 2, 8 cycles for i7 sample index and 8 cycles for i5 sample index.  GoT-ATAC Library Construction Protocol: Single-nucleus gene expression (GEX) and chromatin accessibility libraries were constructed from the pooled nuclei according to the Chromium Next GEM Single Cell Multiome User Guide (10X Genomics). Genotyping libraries targeting the CALR mutant transcripts were constructed from the remaining amplified cDNA, similar to the original GoT method. The re-amplified sample was purified and concentrated via 0.7X SPRI cleanup, eluting it into 10uL Buffer EB. To pre-enrich the CALR mutation locus, a gene-specific PCR was performed with 3uL of cleaned re-amplified cDNA and Partial Read 1 and gene-specific primers. CALR locus-specific amplification was then performed with 3uL of cleaned gene-specific amplified cDNA and SI-PCR and loci-specific Primers. A 0.7X SPRI cleanup was performed, and cDNA was eluted into 11uL Buffer EB. Finally, to construct the targeted amplicon library, loci-amplified cDNA was mixed with P5 Generic and RPIx indexing primers and amplified. The constructed library was cleaned via 0.8X SPRI cleanup and eluted into 12uL Buffer EB. At the cDNA amplification stage of the Chromium Next GEM Single Cell Multiome protocol, supernatant from the 0.6X size selection was retained and was used to generate the hashing libraries as per HTO protocol25 with the following modification. For the hashing library construction step, the PCR reaction was prepared with 0.65uL of 10uM SI-PCR primer, 0.65uL of 10uM TruSeq DNA D7xx_s primer, 11.25uL cleaned supernatant and 12.5 uL Kapa HiFI HotStart Ready Mix (Roche, Basel, Switzerland). Hashing, gene expression, and genotyping libraries were pooled and sequenced together on a NovaSeq (Illumina).  Genotyping for JAK2-mutated samples (IFN14, IFN15): Nuclei were processed according to the 10x Genomics Chromium Next GEM Single-Cell ATAC v2 Kit guidelines, with several modifications specific to the GoT-ChA protocol. 1 µl of 22.5 µM GoT–ChA primer mix was added to the barcoding reaction mixture, containing the locus-specific primers GoT–ChA_R1N and GoT–ChA_Rev (primer sequences in Supplementary Table 32). During the post-GEM incubation clean-up, 45.5 µl of elution solution I was used to elute the SPRIselect bead reaction, and 5 µl was used for GoT-ChA library generation. GoT-ChA libraries were generated according to the published protocol.

value: Bone marrow

tag: disease state

value: Essential thrombocythemia

value: IFNa


title: IFN16_12months_cd34pos_sn10x

description:
Library name: IFN16_12months_cd34pos_sn10x
10x genomics


title: BMDMs  treated with PBS rep2

city: Guiyang

first: Yinhui

last: Jiang

address: No. 6, Ankang Avenue, GUI 'an New Area, Guizhou Province

country: China

institute: Guizhou Medical University

description: Library name: PBS rep2

data_processing:
1 Quality control  Raw data (raw reads) of fastq format were firstly processed through fastp software. In this step, clean data (clean reads) were obtained by removing reads containing adapter, reads containing ploy-N and low quality reads from raw data. At the same time, Q20, Q30 and GC content the clean data were calculated. All the downstream analyses were based on the clean data with high quality.
2 Reads mapping to the reference genome  Reference genome and gene model annotation files were downloaded from genome website directly. Index of the reference genome was built using Hisat2 v2.0.5 and paired-end clean reads were aligned to the reference genome using Hisat2 v2.0.5. We selected Hisat2 as the mapping tool for that Hisat2 can generate a database of splice junctions based on the gene model annotation file and thus a better mapping result than other non-splice mapping tools.
3 Novel transcripts prediction  The mapped reads of each sample were assembled by StringTie (v1.3.3b) (Mihaela Pertea.et al. 2015) in a reference-based approach. StringTie uses a novel network flow algorithm as well as an optional de novo assembly step to assemble and quantitate fulllength transcripts representing multiple splice variants for each gene locus.
4 Quantification of gene expression level  featureCounts v1.5.0-p3 was used to count the reads numbers mapped to each gene. And then FPKM of each gene was calculated based on the length of the gene and reads count mapped to this gene. FPKM, expected number of Fragments Per Kilobase of transcript sequence per Millions base pairs sequenced, considers the effect of sequencing depth and gene length for the reads count at the same time, and is currently the most commonly used method for estimating gene expression levels.
Assembly: GRCm39
Supplementary files format and content: table-delimited text file indudes raw counts for each sample

source_name: bone-marrow-derived macrophages

growth_protocol: Briefly, we flushed bone marrow from the femurs and tibiae of adult (6-week-old) male C57BL/6 mice with RPMI 1640 medium. We cultured the cells for 6 days in RPMI 1640 supplemented with 20% fetal bovine serum and 30% L-929 cell-conditioned medium (as a source of macrophage colony-stimulating factor). We collected adherent cells and enumerated them using a Neubauer chamber. We cultured BMDMs (2 × 105 cells per well in 24-well plates) in DMEM supplemented with 10% FBS.

extract_protocol:
We collected the treated cells and extracted total RNA using TRIzol reagent (Invitrogen) according to the manufacturer's instructions.
Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in First Strand Synthesis Reaction Buffer(5X). First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase(RNase H-). Second strand cDNA synthesis was ubsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3’ ends of DNA fragments, Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 370~420 bp in length, the library fragments were purified with AMPure XP system. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system.

treatment_protocol: We incubated BMDMs (2 × 106 cells/ml; 105 cells per well in 24-well plates) with EVs (20 µg per well) or PBS for 6 h.

value: bone-marrow-derived macrophages

value: WT

value: co-culture


title: BMDMs  treated with PBS rep1

description: Library name: PBS rep1


title: IFN16_Baseline_cd34pos_GoT-ChA

description:
Library name: IFN16_Baseline_cd34pos_GoT-ChA
GoT-ChA

value: Control


title: BMDMs  treated with extracellular vesicles (EVs) from A. flavus strain LD-F1_b rep2

description: Library name: EVsLD_F1_b rep2


title: IFN16_Baseline_cd34pos_sn10x

description:
Library name: IFN16_Baseline_cd34pos_sn10x
10x genomics


title: BMDMs  treated with extracellular vesicles (EVs) from A. flavus strain LD-F1_b rep1

description: Library name: EVsLD_F1_b rep1


title: Overweight control feature barcode

description:
Library name: 46C-OW-FB
raw_adt_matrix.tsv
feature_reference.csv


title: DFAT_Rosiglitazone_3

city: Los Angeles

first: Qingwen

last: Xie

state: California

address: 405 Hilgard Avenue

institute: University of California, Los Angeles

description:
Library name: Pig Sample 15
DFAT_DeSeq2_Normalized_counts.txt

data_processing:
Sequencing was performed on Illumina NovaSeq X Plus for PE 2x150 run. Data quality check was done on Illumina SAV. Demultiplexing was performed with Illumina Bcl2fastq v2.19.1.403 software.
Reads were aligned using STAR with mouse reference genome GRCm38 (Ensembl release 107) and pig reference genome sus scrofa 11.1 (Ensemble release 110). Gene counts were normalized and differential gene expression was assessed using DESeq2.
Assembly: GRCm38 for 3T3-L1; Sus scrofa 11.1 for DFAT
Supplementary files format and content: Tab delimited text file inlcuded normalized counts for each sample

organism: Sus scrofa

source_name: Subcutaneous fat

growth_protocol: Mouse embryonic fibroblast cells (3T3-L1, ATCC, CL-173) were cultured in Dulbecco's Modified Eagle's Medium (DMEM) (Gibco, 11995065) supplemented with 10% fetal bovine serum (FBS) (GemCell, 100-500) and 1% antibiotic-antimycotic (Gibco, 15240062) in a 37°C, 5% CO2 incubator. De-differentiated porcine cells (DFAT) were cultured in DMEM with 20% FBS and 100 µg/ml Primocin (InvivoGen, ant-pm-1).

extract_protocol:
RNA quality was assessed using a TapeStation instrument prior to library preparation.
Libraries for RNA-Seq were prepared with KAPA mRNA HyperPrep Kit. The workflow consists of mRNA enrichment and fragmentation, first strand cDNA synthesis using random priming followed by second strand synthesis converting cDNA:RNA hybrid to double-stranded cDNA (dscDNA), and incorporates dUTP into the second cDNA strand to maintain strand origin information. cDNA generation is followed by end repair to generate blunt ends, A-tailing, adaptor ligation and PCR amplification.

treatment_protocol:
Adipogenic differentiation of 3T3-L1 was induced by DMEM containing 0.75 µg/mL insulin (Sigma-Aldrich, I0516), 75 nM dexamethasone (Sigma-Aldrich, D4902), and 37.5 µM isobutyl methylxanthine (IBMX, Sigma-Aldrich, I5879) with 10% FBS and 1% antibiotic-antimycotic. Adipogenic differentiation of DFAT was induced by Advanced DMEM (Gibco, 12491015) supplemented with 100 µg/ml Primocin, 0.5 µM dexamethasone, 12.5 µM IBMX, 2 mM (1×) GlutaMAX (Thermo Fisher, 35050-061), 20 µM biotin (TCI America, B04631G), 10 µM calcium-D-pantothenate (TCI America, P001225G), and 2% FBS.
3T3-L1 and DFAT cells were treated with DMSO (vehicle), dicoumarol, magnolol, dicoumarol togerther with magnolol (D+M), or rosiglitazone respectively during the differentiation.

value: Subcutaneous fat

value: Dedifferentiated fat cell

value: Rosiglitazone


title: E0, P0 mys-1, CUT&Tag, replicate 2

city: Hangzhou

first: Lianfeng

last: Wu

state: Please select a state below

address: 600 Dunyu road

institute: Westlake University

description: Library name: NTY388-2

data_processing:
The Illumina adapters and low-complexity adapters were removed from the reads by Trim Galore! (version 0.6.6), cutadapt (version 4.2). The reads were mapped to the C. elegans genome ce11 by bowtie2 (version 2.4.1) with option “-X 2000”. The unmapped reads were further mapped to the Drosophila genome dm6 by bowtie2 (version 2.4.1) with option “-X 2000”. PCR duplicates were removed by sambamba (version 0.8.2) with option “--overflow-list-size 600000”. Alignments were further filtered by sambamba (version 0.8.2) with option “-F 1804”. NormalizeFactor was calculated as: 10000/(number of alignments mapped to spike-in genome). The average NormalizeFactor of two biological replicates was used for all the following analyses.
Assembly: ce11
Supplementary files format and content: bigWig for each sample
Library strategy: CUT&Tag

organism: Caenorhabditis elegans

source_name: embryo

growth_protocol: C. elegans (N2) were maintained on nematode growth media (NGM) seeded with Escherichia coli OP50 as food source under standard culture conditions at 20°C

extract_protocol:
Approximately 100,000 embryos or L1 larvae were pelleted in ddH2O, snap-frozen in liquid nitrogen Frozen worm pellets were crushed to isolate single nuclei in MACS Buffer (10 mM Tris-HCl pH 7.4, 3 mM MgAc2, 5 mM CaCl2, 2 mM EDTA, 0.6 mM DTT, 1×Protease Inhibitor Cocktail) and were filtered through 30 µm Cell-Tric. After centrifugation (500 × g, 10 min, 4°C), pellets were permeabilized in OMNI Buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% Tween-20, 0.1% NP-40, 0.01% digitonin) for 15 min on ice, then washed twice with PBSI (PBS with 1× protease inhibitor). Nuclei were counted under microscope with Hoechst staining For each sample, 5 × 105 nuclei were combined with 5 × 102 Drosophila S2 cells (as spike-in control). Nuclei-spike-in mixtures were resuspended in MED Buffer #1 (20 mM HEPES, 271 mM NaCl, 2% BSA, 0.5 mM Spermidine, 0.01% Digitonin, 2 mM EDTA, 1×Protease Inhibitor Cocktail) containing 1 µL Bridged Protein A-Tn5 and 2 µg H4K16ac antibody (Abcam ab109463), and incubated overnight at 4°C. Nuclei were washed in MED Buffer #2 (MED Buffer #1 without EDTA), incubated in 10 mM MgCl2, at 550 rpm for 1 h at 37 °C for tagmentation, and then terminated by adding 10 mM EDTA. The nuclei were lysed in 500 mM NaCl, 1% SDS and protease K at 55 °C, 850 rpm for at least 2 h followed by column purification of DNA. Libraries were prepared using NEBNext® High-Fidelity 2X PCR Master Mix and Nextera N5/N7 primers for PCR ampliﬁcation according to recommended cycle number, followed by post-PCR clean-up with SPRIselect Beads and fragments distribution analysis. Biological duplicates per group were prepared for CUT&Tag and sent for sequencing on Illumina NovaSeq 6000.
A total amount of 3 µg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer’s recommendations and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer(5X). First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase(RNase H-). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of  3’ ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 250~300  bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter,Beverly, USA). Then 3 µl USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37°C for 15 min followed by 5 min at 95 °C before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system.

treatment_protocol: Parental generation (P0) hermaphrodites were fed with HT115(DE3) bacteria, with empty vector L4440 or mys-1 RNAi from the L1 larval stage until Day 1 of adulthood. P0 adults were then bleached to isolate embryos (F1) in M9 buffer. The F1 early-stage were collected immediately after bleach (embryo 0 h), 6h after bleach (embryo 6 h) and 24 h after bleach (L1).

tag: developmental stages

value: embyro 0 h

value: embryo

value: mys-1 RNAi in P0

value: batch 1


title: BMDMs  treated with extracellular vesicles (EVs) from A. flavus strain LD-F1 rep2

description: Library name: EVsLD_F1 rep2


title: DFAT_Rosiglitazone_2

description:
Library name: Pig Sample 14
DFAT_DeSeq2_Normalized_counts.txt


title: DFAT_Rosiglitazone_1

description:
Library name: Pig Sample 13
DFAT_DeSeq2_Normalized_counts.txt


title: BMDMs  treated with extracellular vesicles (EVs) from A. flavus strain LD-F1 rep1

description: Library name: EVsLD_F1 rep1


title: E0, P0 mys-1, CUT&Tag, replicate 1

description: Library name: NTY388-1


title: 3T3_L1_Rosiglitazone_3

description:
Library name: Mouse Sample 15
3T3_DeSeq2_Normalized_counts.txt

value: fibroblast


title: 72h_3

description: Library name: F3

value: mouse knee joint tissue


title: DFAT_D+M_3

description:
Library name: Pig Sample 12
DFAT_DeSeq2_Normalized_counts.txt

value: Dicoumarol and magnolol


title: L1, P0 mys-1, replicate 3

description: Library name: L1-M-3

data_processing:
The sequencing reads were de-multiplexed using bcl2fastq and quantified using kallisto 0.46.0, with default parameters to WormBase reference genome (WS274).
Assembly: WS274
Supplementary files format and content: gene-counts

source_name: whole worm

extract_protocol:
Total RNA was extraced from Trizol following manufacturer's instructions
A total amount of 3 µg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer’s recommendations and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer(5X). First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase(RNase H-). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of  3’ ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 250~300  bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter,Beverly, USA). Then 3 µl USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37°C for 15 min followed by 5 min at 95 °C before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system.

treatment_protocol: Parental generation (P0) hermaphrodites were fed with HT115(DE3) bacteria, with empty vector L4440 or mys-1 RNAi from the L1 larval stage until Day 1 of adulthood. P0 adults were then bleached to isolate embryos (F1) in M9 buffer. The F1 early-stage were collected immediately after bleach (embryo 0 h), 6h after bleach (embryo 6 h) and 24 h after bleach (L1). The F1 embryos were also transferred  onto L4440 plates and grown for 70 hours to collect adult. All samples were frozen in Trizol.

value: whole worm

value: batch 2


title: 3T3_L1_Rosiglitazone_2

description:
Library name: Mouse Sample 14
3T3_DeSeq2_Normalized_counts.txt


title: DFAT_D+M_2

description:
Library name: Pig Sample 11
DFAT_DeSeq2_Normalized_counts.txt


title: 3T3_L1_Rosiglitazone_1

description:
Library name: Mouse Sample 13
3T3_DeSeq2_Normalized_counts.txt


title: DFAT_D+M_1

description:
Library name: Pig Sample 10
DFAT_DeSeq2_Normalized_counts.txt


title: 3T3_L1_D+M_3

description:
Library name: Mouse Sample 12
3T3_DeSeq2_Normalized_counts.txt


title: DFAT_Magnolol_3

description:
Library name: Pig Sample 9
DFAT_DeSeq2_Normalized_counts.txt

value: Magnolol


title: L1, P0 control, replicate 3

description: Library name: L1-L-3

value: control RNAi in P0


title: 3T3_L1_D+M_2

description:
Library name: Mouse Sample 11
3T3_DeSeq2_Normalized_counts.txt


title: DFAT_Magnolol_2

description:
Library name: Pig Sample 8
DFAT_DeSeq2_Normalized_counts.txt


title: DFAT_Magnolol_1

description:
Library name: Pig Sample 7
DFAT_DeSeq2_Normalized_counts.txt


title: 3T3_L1_D+M_1

description:
Library name: Mouse Sample 10
3T3_DeSeq2_Normalized_counts.txt


title: E6, P0 control, replicate 4

description: Library name: L4-6

value: embyro 6 h


title: DFAT_Dicoumarol_3

description:
Library name: Pig Sample 6
DFAT_DeSeq2_Normalized_counts.txt

value: Dicoumarol


title: 3T3_L1_Magnolol_3

description:
Library name: Mouse Sample 9
3T3_DeSeq2_Normalized_counts.txt


title: DFAT_Dicoumarol_2

description:
Library name: Pig Sample 5
DFAT_DeSeq2_Normalized_counts.txt


title: 3T3_L1_Magnolol_1

description:
Library name: Mouse Sample 7
3T3_DeSeq2_Normalized_counts.txt


title: 3T3_L1_Magnolol_2

description:
Library name: Mouse Sample 8
3T3_DeSeq2_Normalized_counts.txt


title: 3T3_L1_Dicoumarol_3

description:
Library name: Mouse Sample 6
3T3_DeSeq2_Normalized_counts.txt


title: DFAT_Dicoumarol_1

description:
Library name: Pig Sample 4
DFAT_DeSeq2_Normalized_counts.txt


title: 3T3_L1_Dicoumarol_2

description:
Library name: Mouse Sample 5
3T3_DeSeq2_Normalized_counts.txt


title: DFAT_Vehicle_3

description:
Library name: Pig Sample 3
DFAT_DeSeq2_Normalized_counts.txt

value: DMSO


title: DFAT_Vehicle_2

description:
Library name: Pig Sample 2
DFAT_DeSeq2_Normalized_counts.txt

value: Subcutaneous fat


title: 3T3_L1_Dicoumarol_1

description:
Library name: Mouse Sample 4
3T3_DeSeq2_Normalized_counts.txt


title: 3T3_L1_Vehicle_3

description:
Library name: Mouse Sample 3
3T3_DeSeq2_Normalized_counts.txt


title: DFAT_Vehicle_1

description:
Library name: Pig Sample 1
DFAT_DeSeq2_Normalized_counts.txt

value: Dedifferentiated fat cell


title: E0, P0 control, replicate 1

description: Library name: L1-0


title: mm_atac_gcb_AIDcreBRWD1_DZ.2

city: Chicago

first: Mark

last: Maienschein-Cline

state: IL

address: 900 S Ashland Ave, Rm 1058, M/C 937

department: Research Resources Center

institute: University of Illinois at Chicago

description:
gcb_counts.txt
gcb_peaks.bed

data_processing:
Raw reads were aligned to reference genome mm9 using BWA MEM. Apparent PCR duplicates were removed using Picard MarkDuplicates. Read alignments were first adjusted to account for TAC transposon binding: +4 bp for +strand alignments, -5 bp for -strand alignments. Open chromatin peaks were called using Macs2 with no model set and no background provided; peaks with a score greater than 5 were retained.
Peaks were merged into a uniform set of regulatory elements using bedtools merge. Enrichment levels for each regulatory element were then quantified with featureCounts. This analysis was performed separately for Follicular B cells and Germinal Center B cells.
Assembly: mm9
Supplementary files format and content: .bed files: BED formatted open chromatin peak calls. Per-sample BED files are peak calls for individual samples from Macs2. folb_peaks.bed and gcb_peaks.bed are merged peak calls over Follicular B cell samples or germinal center B cell samples, respectively.
Supplementary files format and content: .txt files: tab-delimited text files of counts per peak

source_name: Germinal Center DZd B cells B220+ GL7+ CD95+ CXCR4+ CD83-

extract_protocol:
Cells were washed with PBS, then lysed with ATAC lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% IGEPAL CA-630). Resulting nuclei were then incubated with tagmentation enzyme (Illumina).
Libraries from purified samples were made with the Nextera Indexing kit (Illumina).

treatment_protocol: Follicular B cell samples were isolated from mouse spleens. Follicular B cells were flow sorted as B220+ CD93+ CD21+ CD23+. Follicular B cells from Cd23-Cre Brwd1fl/fl mice were also sorted as tdTomato+. For Germinal Center B cell samples, mice were immunized intraperitoneally with 10^9 sheep red blood cells in PBS, then boosted with 10^9 sheep red blood cells 5 days later. On day 14, germinal center B cells were isolated from spleens. Germinal Center B cells were sorted as B220+ GL7+ CD95+ tdTomato+. LZ cells were defined as CXCR4- CD83+. DZp cells were defined as CXCR4+ CD83+. DZd B cells were defined as CXCR4+ CD83-.

value: Aicda-Cre Brwd1fl/fl

value: Mice were immunized intraperitoneally with 10^9 sheep red blood cells in PBS, then boosted with 10^9 SRBC 5 days later. On day 14, germinal center B cells were isolated from spleens.


title: 3T3_L1_Vehicle_2

description:
Library name: Mouse Sample 2
3T3_DeSeq2_Normalized_counts.txt


title: mm_atac_gcb_AIDcreBRWD1_GZ.2

description:
gcb_counts.txt
gcb_peaks.bed

source_name: Germinal Center DZp B cells B220+ GL7+ CD95+ CXCR4+ CD83+


title: mm_atac_gcb_AIDcreBRWD1_DZ.1

description:
gcb_counts.txt
gcb_peaks.bed

value: Germinal Center DZd B cells B220+ GL7+ CD95+ CXCR4+ CD83-


title: Nasal epithelial brushing, pediatric ICU control Control_001

type: genomic

city: Little Rock

first: James

middle: Garrett

last: Williams

state: AR

address: 13 Children's Way

department: Pediatrics

institute: University of Arkansas for Medical Sciences

hyb_protocol: Bisulfite-converted, amplified DNA was enzymatically fragmented and hybridized to the Illumina Infinium MethylationEPIC v2.0 BeadChip, followed by single-base extension and staining, per the Illumina Infinium HD Methylation Assay protocol (CCHMC DNA Sequencing and Genotyping Core).

scan_protocol: BeadChips were imaged on the Illumina iScan system.

data_processing:
IDAT files were processed in R (v4.5.0) with the sesame package. openSesame was run with prep='QCDPB' (quality masking, channel/dye-bias correction, noob background correction, and pOOBAH detection-p masking) on platform EPICv2 using a pinned local manifest; beta values were computed with getBetas. Probes flagged by the EPICv2 KYCG mask (cross-reactive and SNP-affected) and probes with detection p-value > 0.05 were removed. Replicate EPICv2 probes were collapsed to one value per CpG by averaging (limma::avereps on the base CpG identifier). M-values were computed as log2(beta/(1-beta)). Cell-type composition was estimated with EpiDISH, and M-values were adjusted for composition (limma::removeBatchEffect) for downstream analyses. The submitted processed matrix contains normalized beta values per CpG per sample.
[processed value definition] Beta value = methylated signal / (methylated + unmethylated signal), ranging from 0 (unmethylated) to 1 (fully methylated), after noob/dye-bias correction and pOOBAH detection-p masking (sesame getBetas). NA indicates a probe that failed detection in that sample.

label: Cy3 and Cy5

source_name: nasal epithelium

label_protocol: Standard Illumina Infinium HD Methylation Assay. Genomic DNA was bisulfite-converted and whole-genome amplified per the manufacturer's protocol.

extract_protocol: Nasal brushes were thawed on ice in 1:100 beta-mercaptoethanol to reduce mucus disulfide bonds, passed through a QIAshredder (Qiagen), and treated with DNase. Genomic DNA was extracted with the AllPrep DNA/RNA Mini Kit (Qiagen) and quantified on a Qubit Flex fluorometer (Thermo Fisher Scientific).

value: nasal epithelium

tag: condition

value: PICU control without lung disease


title: 3T3_L1_Vehicle_1

description:
Library name: Mouse Sample 1
3T3_DeSeq2_Normalized_counts.txt

value: fibroblast


title: exosc4-cyto-exp6_1

city: Bad Nauheim

first: Jan

last: Detleffsen

state: Hessen

address: Ludwigstraße 43

department: Bioinformatics

country: Germany

institute: Max Planck Institute for Heart and Lung Research

description: Library name: Sample 7

data_processing:
Trimmomatic version NA was employed to trim reads after a quality drop below a mean of Q15 in a window of 5 nucleotides and keeping only filtered reads longer than 15 nucleotides (Bolger et al., Trimmomatic: a flexible trimmer for Illumina sequence data).
Reads were aligned versus Ensembl human genome version hg38 (Ensembl release 109) with STAR 2.7.11b (Dobin et al., STAR: ultrafast universal RNA-seq aligner).
Alignments were filtered to remove: duplicates with Picard 3.4.0 (Picard: A set of tools (in Java) for working with next generation sequencing data in the BAM format), multi-mapping, ribosomal, or mitochondrial reads.
Gene counts were established with featureCounts 2.0.4 by aggregating reads overlapping exons on the correct strand excluding those overlapping multiple genes (Liao et al., featureCounts: an efficient general purpose program for assigning sequence reads to genomic features).
The raw count matrix was normalized with DESeq2 version 1.36.0 (Love et al., Moderated estimation of fold change and dispersion for RNA-Seq data with DESeq2).
Assembly: hg38
Supplementary files format and content: The tab-delimited matrix shows DESeq normalized counts.

extract_protocol:
Nuclei were isolated according to the 10x Genomics protocol CG000124 Rev. F. RNA was then extracted from both whole cells and isolated nuclei using the PureLink miRNA Isolation Kit (ThermoFisher, 250753) to recover short and long RNA fractions. Long RNAs were further purified using the miRNeasy Micro Kit (Qiagen, 217084) with on-column DNase digestion using the RNase-Free DNase Set (Qiagen, 79254).
RNA-Seq

treatment_protocol: transfected twice (day 1 and day 3 after seeding)

value: retinal pigment epithelial cells

value: EXOSC4 knockdown


title: exosc4-nucl-exp6_1

description: Library name: Sample 8


title: wt-nucl-exp6_1

description: Library name: Sample 6

value: WT


title: mm_atac_gcb_AIDcreBRWD1_LZ.2

description:
gcb_counts.txt
gcb_peaks.bed

source_name: Germinal Center LZ B cells B220+ GL7+ CD95+ CXCR4- CD83+


title: GBM TAM, Pracinostat, IgG control CUT&Tag, biological replicate 4

city: Stanford

first: Mona

last: Khalaj

address: 265 Campus Drive

institute: Stanford University

description: Library name: CT1208_14

data_processing:
Paired-end reads were aligned using Bowtie2 to UCSC mm10 (--end-to-end --very-sensitive --no-mixed --no-discordant -q --phred33 -I 10 -X 700).
Peaks were called using MACS2 (2.2.9.1) and a consensus peak set was generated using MACS2 summits.
Fixed-width 500 bp regions centered on peak summits were defined to generate a consensus set of non-overlapping bins. Read counts within these regions were used for differential analysis with DESeq2.
Peaks were assigned to genes based on proximity to transcription start sites using HOMER.
Assembly: UCSC mm10
Supplementary files format and content: Tab-delimited peak count matrix containing read counts per peak region (rows) per sample (columns), used for differential acetylation analysis with DESeq2.
Library strategy: CUT&Tag

source_name: brain tumor, orthotopic xenograft

growth_protocol: NSG mice bearing orthotopic T387 GBM xenografts were treated with Pracinostat (30 mg/kg, oral gavage) or DMSO vehicle control four times per week for three weeks beginning 7 days after tumor implantation.

extract_protocol:
At study endpoint, tumors were dissociated and CD11b+ tumor-associated macrophages were FACS sorted. Cells were cross-linked for 2 minutes at room temperature using 0.1% methanol-free formaldehyde (Cell Signaling Technologies). Glycine was added to 75 mM final concentration and incubated 5 minutes to stop cross-linking. Samples were washed in 1x PBS and bound to Concanavalin A magnetic beads (Cell Signaling Technologies).
CUT&Tag was performed using the CUT&Tag-direct protocol (Henikoff laboratory). Bound cells were assayed using rabbit anti-H3K27ac (Cell Signaling Technologies, #8173S) or normal rabbit IgG control (Cell Signaling Technologies, #2729). Libraries were generated using 12-14 rapid PCR cycles and cleaned with SPRIselect beads (Beckman Coulter, B23319) at 1.3:1 ratio. Libraries were quantified on an Agilent Bioanalyzer, pooled at equimolar concentrations, and sequenced on an Illumina NovaSeq X Plus platform using paired-end 150 bp sequencing (Novogene Corporation Inc.), targeting 10 million reads per sample and antibody target.

treatment_protocol: Pracinostat was prepared in 10% DMSO, 40% PEG300, 5% Tween-80, 45% PBS at 3 mg/kg final concentration and administered orally. Vehicle-treated mice received the same solvent mixture without drug.

value: brain tumor, orthotopic xenograft

value: CD11b+ tumor-associated macrophages

tag: cut&tag antibody

value: Normal rabbit IgG (Cell Signaling Technologies, #2729)

value: Pracinostat 30 mg/kg oral 4x/week 3 weeks


title: mm_atac_gcb_AIDcreBRWD1_GZ.1

description:
gcb_counts.txt
gcb_peaks.bed

value: Germinal Center DZp B cells B220+ GL7+ CD95+ CXCR4+ CD83+


title: exosc4-cyto-exp4_1

description: Library name: Sample 5


title: mm_atac_gcb_AIDcreBRWD1_LZ.1

description:
gcb_counts.txt
gcb_peaks.bed

value: Germinal Center LZ B cells B220+ GL7+ CD95+ CXCR4- CD83+


title: mm_atac_folb_CD23creBRWD1.3

description:
folb_counts.txt
folb_peaks.bed

source_name: Follicular B cells B220+ CD93+ CD21+ CD23+ tdTomato+

value: Cd23-Cre Brwd1fl/fl

value: Cells were isolated from spleens


title: GBM TAM, Pracinostat, IgG control CUT&Tag, biological replicate 3

description: Library name: CT1208_13


title: dis3-nucl-exp4_1

description: Library name: Sample 4

value: DIS3 knockdown


title: mm_atac_folb_CD23creBRWD1.1

description:
folb_counts.txt
folb_peaks.bed

value: Follicular B cells B220+ CD93+ CD21+ CD23+ tdTomato+


title: GBM TAM, Pracinostat, IgG control CUT&Tag, biological replicate 2

description: Library name: CT1208_12


title: mm_atac_folb_CD23creBRWD1.2

description:
folb_counts.txt
folb_peaks.bed


title: VenAza_IP_Rep2

city: Boston

first: Qingyu

last: Luo

state: MA

address: 450 Brookline Ave

institute: Dana-Farber Cancer Institute

description: Library name: Sample 8

data_processing:
After sequencing, raw reads are trimmed to remove adapter sequences and mapped to a reference genome. Duplicate reads are removed to avoid amplification bias, and peaks representing RNA-binding protein (RBP) interaction sites are identified by comparing IP samples to size-matched input controls using statistical models.
Assembly: hg38
Supplementary files format and content: bigWig files for peaks

extract_protocol:
Cells or tissues are lysed using eCLIP Lysis Buffer with inhibitors and homogenized via sonication. Proteins are digested, and RNA is purified and quantified. RNase is then used to fragment RNA into ~200 nucleotide segments, which are critical for downstream mapping of RBP-RNA interactions.
The fragmented RNA is immunoprecipitated using antibody-conjugated magnetic beads, and adapters are ligated to RNA ends. After SDS-PAGE separation and membrane transfer, size-selected RNA-protein complexes are excised, digested, and the RNA purified. Reverse transcription converts RNA into cDNA, which is ligated to a second adapter and amplified by PCR to generate sequencing-ready libraries.

value: BPDCN

value: Venetoclax+azacitidine


title: Nasal epithelial brushing, PARDS subject ARDS_029

value: nasal epithelium

value: PARDS


title: VenAza_IP_Rep1

description: Library name: Sample 7


title: DMSO_IP_Rep2

description: Library name: Sample 6

value: DMSO


title: DMSO_IP_Rep1

description: Library name: Sample 5


title: GBM TAM, DMSO vehicle, IgG control CUT&Tag, biological replicate 3

description: Library name: CT1208_10

value: DMSO vehicle


title: GBM TAM, Pracinostat, IgG control CUT&Tag, biological replicate 1

description: Library name: CT1208_11


title: VenAza_Input_Rep2

description: Library name: Sample 4

value: WT


title: VenAza_Input_Rep1

description: Library name: Sample 3


title: FBL2 knockdown 1 background primer set 3

city: Uppsala

first: Rubin

last: Dasgupta

address: Husargatan 3, BMC 582, D9:3

institute: Uppsala University

description: Library name: fbl2_dmso_d562-d563_filt.fastq.gz

data_processing:
After sequencing using PromethIon, basecalling was performed using dorado v1.1.0. The sequence was aligned to the reference sequence using bowtie2 and samtools. The aligned BAM file was used to count the mutations per position using perbase and RNAFramework
Assembly: The sequence was aligned to the reference sequence. There was not genome building or assembly performed
Supplementary files format and content: The processed data is provides as a CSV file containing the nucleotide position, nucleotide identity (A, T, G, or C) as provided in the reference sequence, number of time it was correctly sequenced and mutated to other nucleotide, number of times the identity was not possible and finally number of time the alignment tool classified the position as an insertion or deletion.

molecule: other

extract_protocol:
RNA was purified using the GeneJET RNA Purification Kit (Thermo Scientific, cat. K0732) according to the manufacturer’s instructions. Residual genomic DNA was removed by treatment with TURBO DNase (Invitrogen™, cat. AM1907), followed by ethanol precipitation.
Libraries were prepared using specific primers and Platinum SuperFi II PCR Master Mix (Invitrogen™, cat. 12368010), and dsDNA was purified using the GeneJET Gel Extraction Kit (Thermo Scientific, cat. K0692).

value: human breast cancer


title: DMSO_Input_Rep2

description: Library name: Sample 2


title: LPS+Dexamethasone 3

first: Dr. Liang

last: Lei

state: NY

address: 1130 St. Nicholas Ave

department: Pathology and Cell Biology

institute: Columbia University

description: Library name: DP021

data_processing:
Libraries are sequenced using Element AVITI at Columbia Genome Center. Multiplex samples were in each lane, which yields targeted number of paired-end 75bp reads for each sample. Bases2fastq (version 1.7.0.1196148384) was used to convert BCL to fastq format, coupled with adaptor trimming. Pseudoalignment using kallisto (0.44.0) was performed. Differentially expressed genes were performed using Sleuth or DESeq2, R packages.
Assembly: Ensembl v96, Human:GRCh38.p12
Supplementary files format and content: count

source_name: Microglia

extract_protocol:
RNA was extracted with Rneasy mini kit (Qiagen)
Poly-A pull-down was used to enrich mRNAs from total RNA samples, then proceed with library construction using Illumina TruSeq chemistry. The final PCR step has been modified using the KAPA HiFi HotStart Ready Mix.

value: Microglia


title: DMSO_Input_Rep1

description: Library name: Sample 1


title: ChIPseq, MAU2, release

city: Tokyo

first: Shoin

last: Tei

address: 1-1-1 Yayoi, Bunkyo-ku

department: Institute for Quantitative Biosciences

country: Japan

institute: The University of Tokyo

description: Library name: ChIPseq_MAU2_release

data_processing:
Sequenced reads were aligned to hg38 and mm10 using bowtie (version 1.2.3) with “-n2-m1” parameters. The removal of redundant reads and the normalization of the read number for each sample were performed with parse2wig (version 3.7.2). For peak-calling, we used drompa_peakcall (version 3.7.2) in “PC_SHARP” mode, and for visualization, we used drompa+ PC_SHARP (version 1.17.2) and deeptools (version 3.5.1.)
Assembly: hg38
Supplementary files format and content: BigWig

growth_protocol: HCT116 cells were cultured in McCoy’s 5A medium containing 10% FBS, penicillin, and streptomycin solution (Wako) at 37 C with 5% CO2.

extract_protocol:
Mouse C2C12 cells were used as a spike-in control. HCT-116 and C2C12 cells were crosslinked with 1% formaldehyde for 10 min at room temperature, quenched with 125 mM glycine for 5 min, washed with PBS, pelleted by centrifugation, and stored at –80°C until use. For standard ChIP-seq, thawed cells were resuspended in 1 mL of LB1 Buffer [50 mM HEPES-KOH (pH 7.5), 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100, 1 mM PMSF, 10 mM DTT, 1x cOmplete EDTA-free (Roche)] and incubated on ice for 10 min. After centrifugation, the pellet was resuspended in 1 mL of LB2 Buffer [20 mM Tris-HCl (pH 7.5), 200 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1x cOmplete EDTA-free] and incubated on ice for 10 min. Following another centrifugation, the pellet was resuspended in 1 mL of LB3 Buffer [20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Na-Deoxycholate, 0.1% SDS, 1x cOmplete EDTA-free] and incubated on ice for 10 min. The centrifugation and resuspension steps with LB3 buffer were repeated. Chromatin was sonicated on Sonifier 250D (BRANSON) at 17% of amplitude for six cycles of 12 sec each. Between each cycle, chromatin was collected by centrifugation. After chromatin fragmentation, insoluble debris was removed by centrifugation. HCT-116 and C2C12 chromatin were combined at a 4:1 ratio. To prepare antibody-conjugated beads, 50 μL of Dynabeads Protein A/G (Invitrogen) were washed twice with PBS containing 5 mg/mL BSA and resuspended in the same buffer. After the addition of 4 μg of antibody, the mixture was rotated for 3 hours at 4°C. The beads were then washed twice with BSA/PBS and once with LB3 buffer, followed by resuspension in LB3 buffer. Chromatin mixture was rotated with the antibody-conjugated beads for 14 hours at 4°C. The beads were washed with LB3 Buffer once, twice with LB3 High Buffer [20 mM Tris-HCl (pH 7.5), 500 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Na-Deoxycholate, 0.1% SDS], three times with RIPA wash Buffer [50 mM HEPES-KOH (pH 7.4), 0.25 M LiCl, 1 mM EDTA, 0.5% Na-Deoxycholate, 1% NP-40], and once with TE50 Buffer [50 mM Tris-HCl (pH 7.5), 10 mM EDTA]. Bead-bound DNA was eluted with Elution Buffer [50 mM Tris-HCl (pH 7.5), 10 mM EDTA, 1% SDS] and incubated at 65°C for 6 hours, followed by sequential treatment with RNase A and proteinase K. The samples were purified with QIAquick PCR purification kit (QIAGEN).
The library for sequencing was prepared with NEBNext DNA Library Prep Master Mix Set for Illumina and NEBNext Multiplex Oligos for Illumina (New England Biolabs).

treatment_protocol: Treatment with 0.5 mM indole-3-acetic acid (IAA, SIGMA) for 3h was performed to deplete RAD21. 100 μM 5,6-Dichlorobenzimidazole 1-β-D-Ribofuranoside (DRB; TCI) was added concurrently with IAA for 3 hours, or 1 μM flavopiridol (Santa Cruz) during the final 1 hour of the 3-hour IAA treatment.


title: GBM TAM, DMSO vehicle, IgG control CUT&Tag, biological replicate 1

description: Library name: CT1208_08

value: brain tumor, orthotopic xenograft

value: CD11b+ tumor-associated macrophages

value: Normal rabbit IgG (Cell Signaling Technologies, #2729)


title: ChIPseq, MAU2, DRB

description: Library name: ChIPseq_MAU2_DRB


title: ChIPseq, MAU2, control

description: Library name: ChIPseq_MAU2_control


title: LPS+Dexamethasone 1

description: Library name: DP019


title: ChIPseq, NIPBL, release

description: Library name: ChIPseq_NIPBL_release


title: GBM TAM, Pracinostat, H3K27Ac CUT&Tag, biological replicate 3

description: Library name: CT1208_06

value: H3K27Ac (Cell Signaling Technologies, #8173S)

value: Pracinostat 30 mg/kg oral 4x/week 3 weeks


title: ChIPseq, NIPBL, DRB

description: Library name: ChIPseq_NIPBL_DRB


title: LPS 3

description: Library name: DP018


title: ChIPseq, RAD21, release

description: Library name: ChIPseq_RAD21_release


title: ChIPseq, RAD21, DRB

description: Library name: ChIPseq_RAD21_DRB


title: GBM TAM, Pracinostat, H3K27Ac CUT&Tag, biological replicate 1

description: Library name: CT1208_04


title: ChIPseq, RAD21, control

description: Library name: ChIPseq_RAD21_control


title: LPS 1

description: Library name: DP016


title: ChIPseq, Input, release

description: Library name: ChIPseq_Input_release


title: GBM TAM, DMSO vehicle, H3K27Ac CUT&Tag, biological replicate 3

description: Library name: CT1208_03


title: ChIPseq, Input, DRB

description: Library name: ChIPseq_Input_DRB


title: ChIPseq, Input, control

description: Library name: ChIPseq_Input_control


title: GBM TAM, DMSO vehicle, H3K27Ac CUT&Tag, biological replicate 2

description: Library name: CT1208_02


title: ChIPseq, RPB1, DRB

description: Library name: ChIPseq_RPB1_DRB


title: Control 2

description: Library name: DP014


title: Control 1

description: Library name: DP013


title: GBM TAM, DMSO vehicle, H3K27Ac CUT&Tag, biological replicate 1

description: Library name: CT1208_01


title: ChIPseq, RPB1, control

description: Library name: ChIPseq_RPB1_control


title: ChIPseq, Input, RPB1, release

description: Library name: ChIPseq_Input_RPB1_release


title: CHI224 Active Het

city: Memphis

first: Stanislav

last: Zakharenko

state: Tennessee

address: 262 Danny Thomas Pl, M2426

department: Developmental Neurobiology

institute: St. Jude Children's Research Hospital

description: Library name: CHI224

data_processing:
Adapter sequences were removed by TrimGalore (v0.6.3).
STAR (v2.7.9a) was then used for alignment to the mouse reference genome (GRCm38) in a splice-site aware manner based on Gencode M22 primary assembly annotations.
Gene level quantifications were derived using RSEM (v1.3.3).
Assembly: Mouse mm10
Supplementary files format and content: Processed data contains raw counts per gene for each sample.

source_name: dorsal striatum

extract_protocol:
Cell contents were extracted using the recording electrode following patch-clamp recordings. Contents were directly lysed in buffer from the SMART-Seq mRNA Single Cell LP kit (Takara, 634787).
Libraries were constructed from single cells using the SMART-Seq mRNA Single Cell LP kit (Takara, 634787).

value: dorsal striatum

tag: group

value: Active

value: cholinergic interneuron

value: Het

tag: Sex

value: male

tag: date ephys

tag: dob

tag: age days

tag: electrophysiologist

tag: rt lot

tag: date lp

tag: lp lot

tag: index lot

tag: date seq

tag: seq srm_sample_id


title: ChIPseq, Input, RPB1, DRB

description: Library name: ChIPseq_Input_RPB1_DRB

tag: cell line

tag: treatment


title: Dexamethasone 3

description: Library name: DP003


title: Dexamethasone 2

description: Library name: DP002


title: ChIPseq, MED1, ctrl

description: Library name: ChIPseq_MED1_ctrl


title: ChIPseq, BRD4, aux

description: Library name: ChIPseq_BRD4_aux

value: Auxin


title: ChIPseq, BRD4, ctrl

description: Library name: ChIPseq_BRD4_ctrl


title: CHI220 Active Het

description: Library name: CHI220

tag: tissue

tag: cell type

tag: genotype

tag: batch

tag: mouse id


title: TTseq, aux, rep2

description: Library name: TTseq_aux_rep2

data_processing:
Reads were aligned to human (hg38) and Drosophila (dm6) reference genomes with STAR (version 2.7.1a) using the following parameters: --outFilterScoreMinOverLread 0.2, --outFilterMatchNminOverLread 0.2, and --outFilterMismatchNoverLmax 0.5. Mapped reads were separated into forward- and reverse-strand reads based on SAM flag information (forward: 99 or 147; reverse: 83 or 163) using samtools (version 1.15.1). To calculate scale factors, a Drosophila gene-level count matrix was generated with htseq-count (version 1.99.2) and passed to DESeq2’s estimateSizeFactors function. For differential expression analysis, a read count table was generated for genes, with TT-seq reads counted using drompa3 (version 3.7.2). Differential expression analysis was performed with edgeR (version 4.2.2) with the likelihood ratio test (FDR < 0.05).
Assembly: hg38
Supplementary files format and content: BigWig

extract_protocol:
Drosophila S2 cells were used as a spike-in control. Nascent transcripts were labelled by incubating HCT-116 cells and S2 cells with 1 mM 4-thiouridine (Cayman Chemical) for 10 min and 20 min, respectively. Total RNA was extracted from HCT-116 and S2 cells with TRIzol reagent (Invitrogen) and purified using Direct-zol RNA Miniprep Plus (ZYMO RESEARCH), following the manufacturers’ instructions. For each library, 100 μg of HCT-116 RNA was mixed with 1 μg S2 RNA cells and sheared on M220 Focused-ultrasonicator (Covaris) under the following conditions: peak power, 75 W; duty factor, 25%; 200 cycles per burst for 20 seconds at 20°C. For biotinylation of 4-thiouridine-labeled RNA, sonicated RNA was mixed with Tris-HCl (10 mM, pH 7.4), EDTA (1 mM, pH 8.0), and MTSEA-biotin-XX (20 mg/mL) in a total volume of 250 μL. After phenol-chloroform extraction, biotinylated RNA was isolated using mMACS Streptavidin Kit (Miltenyi Biotec), eluted in 100 mM DTT, and purified using Direct-zol RNA Miniprep (ZYMO RESEARCH).
The library for sequencing was prepared with NEBNext Ultra II Directional RNA Library Prep Kit for Illumina and NEBNext Multiplex Oligos for Illumina (New England BioLabs)..

treatment_protocol: Treatment with 0.5 mM indole-3-acetic acid (IAA, SIGMA) for 3h was performed to deplete RAD21.


title: scRNA-Seq of homo sapiens: Sample (KS9) obtained from Skin (tumor) of patient with Kaposi Sarcoma.

city: St. Louis

first: Lee

last: Ratner

state: Missouri

address: 4451 Clayton Ave, St. Louis

department: Department of Medicine

institute: Washington University in St. Louis

description: Library name: KS9

data_processing:
Cell Ranger count and multi v7.1.0 were used for alignment and gene expression quantification.
Reads were aligned against a custom reference including the human genome (GRCh38/hg38), KSHV genome (Strain GK18, GCF_000838265.1), and HIV-1 genome (GCF_000864765.1).
Feature-barcode matrices were quality controlled, normalized, and scaled using Seurat v4.2.1.
Principal component analysis was performed using highly variable genes followed by clustering (resolution = 0.7).
Dimensionality reduction and visualization were performed using t-SNE and UMAP.
Cell type annotations were assigned using SingleR v2 with the Human Primary Cell Atlas reference dataset and refined manually.
Differential expression analysis was performed using Seurat FindMarkers with the Wilcoxon rank-sum test.
Assembly: GRCh38/hg38 human genome; KSHV genome (Strain GK18, GCF_000838265.1); HIV-1 genome (GCF_000864765.1)
Supplementary files format and content: KS_scRNAseq_seurat.rds: This file contains quality-controlled single-cell gene expression count matrices, normalized expression data, cell-level metadata, clustering assignments, dimensionality reduction embeddings (UMAP and PCA), cell type annotations, and KSHV-related annotations used in the published analyses. The Seurat (.rds) file is intended for use in R/Seurat workflows. The file contains the processed assays and metadata required to reproduce the downstream analyses and visualizations presented in the study.
Supplementary files format and content: KS_scRNAseq_seurat.h5ad: This file contains quality-controlled single-cell gene expression count matrices, normalized expression data, cell-level metadata, clustering assignments, dimensionality reduction embeddings (UMAP and PCA), cell type annotations, and KSHV-related annotations used in the published analyses. The AnnData (.h5ad) file is intended for use in Python/Scanpy workflows. The file contains the processed assays and metadata required to reproduce the downstream analyses and visualizations presented in the study.

source_name: Skin

extract_protocol:
Viably frozen (in Bambanker, serum-free cell freezing media. Sigma) single-cell suspensions were prepared from fresh primary skin biopsy samples using enzymatic digestion and gentle manual tissue dissociation (Whole Skin Dissociation Kit, Miltenyi Biotech) and thawed immediately prior to submission for single cell RNA sequencing. It should be noted that sample preparation without enzymatic digestion dramatically diminished the diversity and abundance of cell populations obtained from skin lesions. An estimated 50,000 cells were submitted for sequencing for each sample, though there was some variation between samples in the viable cells submitted. As for Peripheral blood mononuclear cell samples (PBMC), they were processed separately according to standard PBMC isolation procedures prior to single-cell RNA sequencing.
Single-cell libraries were prepared using the 10x Genomics 5′ or 3′ immune profiling/scRNA-seq protocol by GTAC@MGI. Resulting 10x libraries were sequenced on an Illumina NovaSeq 6000 using an S4 flow cell and 300-cycle sequencing, targeting approximately 100,000 reads per cell.

value: Skin

value: Mixed skin lesion cells

tag: subject status

value: patient with Kaposi Sarcoma


title: TTseq, ctrl, rep2

description: Library name: TTseq_ctrl_rep2

value: DMSO


title: CHI219 Active Het

description: Library name: CHI219

tag: group

tag: Sex

tag: date ephys

tag: dob


title: TTseq, aux, rep1

description: Library name: TTseq_aux_rep1


title: scRNA-Seq of homo sapiens: Sample (KS8) obtained from Skin (tumor) of patient with Kaposi Sarcoma.

description: Library name: KS8


title: CHI218 Active Het

description: Library name: CHI218

tag: age days

tag: electrophysiologist

tag: rt lot

tag: date lp


title: mm_atac_folb.2

description:
folb_counts.txt
folb_peaks.bed

source_name: Follicular B cells B220+ CD93+ CD21+ CD23+

value: WT

value: Cells were isolated from spleens


title: scRNA-Seq of homo sapiens: Sample (KS7B) obtained from Skin (tumor) of patient with Kaposi Sarcoma.

description: Library name: KS7B


title: CHI217 Active Het

description: Library name: CHI217

tag: lp lot

tag: index lot

tag: date seq

tag: seq srm_sample_id


title: Squamocolumnar junction, rep3

type: RNA

city: Newcastle upon Tyne

first: Heiko

last: Peters

address: Central Parkway

department: Institute of Human Genetics

country: United Kingdom

institute: Newcastle University

hyb_protocol: Following fragmentation, 15 ug of cRNA were hybridized in the Hybridisation oven (645) for 16 hr at 45C on Mouse Genome 430 2.0. GeneChips were washed and stained using the Affymetrix Fluidics Station 400.

scan_protocol: GeneChips were scanned using the Hewlett-Packard GeneArray Scanner 3000 7G.

data_processing: The data were analyzed with Microarray Suite version 5.0 (MAS 5.0) using Affymetrix default analysis settings and global scaling as normalization method. The trimmed mean target intensity of each array was arbitrarily set to 100.

label: biotin

source_name: squamocolumnar junction epithelium

label_protocol: Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 5 ug total RNA (Expression Analysis Technical Manual, 2001, Affymetrix).

growth_protocol: Mice were maintained under standard housing conditions in a SPF environment. Standard diet and water was given ad libitum.

extract_protocol: Trizol extraction of total RNA was performed according to the manufacturer's instructions.

tag: gender

value: female

tag: strain

tag: tissue

value: squamocolumnar junction epithelium

tag: stage

value: 3 months

tag: health status

value: normal


title: TTseq, ctrl, rep1

description: Library name: TTseq_ctrl_rep1

tag: cell line

tag: treatment


title: scRNA-Seq of homo sapiens: Sample (KS7A) obtained from Skin (tumor) of patient with Kaposi Sarcoma.

description: Library name: KS7A

tag: cell type

tag: subject status


title: CHI216 Active Het

description: Library name: CHI216

tag: group

tag: genotype

tag: Sex

tag: batch

tag: date ephys

tag: dob

tag: age days

tag: mouse id


title: Squamocolumnar junction, rep2

tag: gender


title: mm_atac_folb.1

description:
folb_counts.txt
folb_peaks.bed

value: Follicular B cells B220+ CD93+ CD21+ CD23+


title: dis3-cyto-exp4_1

description: Library name: Sample 3

value: retinal pigment epithelial cells

value: DIS3 knockdown


title: scRNA-Seq of homo sapiens: Sample (KS6-PBMC) obtained from PBMC (non-tumor) of patient with Kaposi Sarcoma.

description: Library name: KS6-PBMC

source_name: PBMC

value: PBMC


title: wt-nucl-exp4_1

description: Library name: Sample 2


title: Squamocolumnar junction, rep1

tag: strain


title: wt-cyto-exp4_1

description: Library name: Sample 1


title: GBM TAM, DMSO vehicle, IgG control CUT&Tag, biological replicate 2

description: Library name: CT1208_09

tag: tissue

value: brain tumor, orthotopic xenograft

value: CD11b+ tumor-associated macrophages

tag: cut&tag antibody

value: Normal rabbit IgG (Cell Signaling Technologies, #2729)

value: DMSO vehicle


title: LPS+Dexamethasone 2

description: Library name: DP020

value: Microglia


title: CHI215 Unknown Het

description: Library name: CHI215

value: dorsal striatum

value: Unknown

value: cholinergic interneuron

value: Het

value: male

tag: electrophysiologist

tag: rt lot

tag: date lp

tag: lp lot

tag: index lot

tag: date seq

tag: seq srm_sample_id


title: Forestomach, rep3

source_name: forestomach epithelium

value: female

value: forestomach epithelium

tag: stage

tag: health status


title: GBM TAM, Pracinostat, H3K27Ac CUT&Tag, biological replicate 4

description: Library name: CT1208_07

tag: cell type

value: H3K27Ac (Cell Signaling Technologies, #8173S)

tag: treatment

value: Pracinostat 30 mg/kg oral 4x/week 3 weeks


title: scRNA-Seq of homo sapiens: Sample (KS6B-PBMC) obtained from PBMC (non-tumor) of patient with Kaposi Sarcoma.

description: Library name: KS6B-PBMC

tag: subject status

value: patient with Kaposi Sarcoma


title: Macrophages_RNA_SCIENCE_2

first: Evgeny

last: Deforzh

address: 60 Fenwood Rd, Brigham and Women's Hospital Neurology Department, 9th Floor

department: Neurology

institute: Brigham and Women's Hospital

description: Library name: Sample 8

data_processing:
Duplicate reads were collapsed into a fasta file, with duplicate counts per read calculated using fastx_toolkit (version 0.0.14).
Chimeric reads were aligned to a reference fasta file containing transcript sequences (hg38_Homo_sapiens.GRCh38.100.fasta) using BWA-MEM in local alignment mode considered transcript strand only
Pairs of genes from each chimeric read were identified, and the number of reads for each gene pair was quantified with ChiRA
Assembly: GRCh38/hg38

source_name: primary glioma tumors

extract_protocol:
Nuclei were distributed into 96-well plates by limiting dilution. Nuclear membranes were lysed and chromatin was reverse-crosslinked by incubation with Proteinase K for 4 h at 65 °C followed by 15 min 95 °C to deactivate Proteinase K.  Biotinylated RNA complexes were captured using streptavidin magnetic beads and transferred to fresh wells. For 3′ RNA end repair, bead-enriched RNA–RNA crosslinked material was washed in ice-cold T4 polynucleotide kinase (PNK) buffer and treated with 0.5 U T4 PNK (NEB) in an 80-µL reaction at 37 °C for 4 h. Subsequently, fresh ATP (1 mM final) and an additional 0.5 U T4 PNK were added in a total volume of 100 µL and incubated for 1 h at 37 °C to repair 5′ RNA ends, followed by the removal of the buffer. RNA–RNA chimeras were generated by ligation in T4 RNA ligase buffer (NEB) supplemented with 1 mM ATP, 0.5 U/µL RNase inhibitor, and 2.5 U/µL T4 RNA ligase 1 (NEB) at 37 °C for 1 h with continuous agitation. Reverse crosslinking was completed by UV irradiation at 254 nm for 5 min on ice. RNA on Streptavidine beads was washed once with 1x scRT Buffer (Takara, #634444).
RT and subsequent steps were performed using the SMART-Seq® Stranded Kit (Takara, #634444), following the manufacturer’s instructions.

value: primary glioma tumors

tag: genotype

value: WT


title: GBM TAM, Pracinostat, H3K27Ac CUT&Tag, biological replicate 2

description: Library name: CT1208_05

value: brain tumor, orthotopic xenograft


title: ChIPseq, NIPBL, control

description: Library name: ChIPseq_NIPBL_control

tag: cell line

value: DMSO


title: CHI214 Unknown Het

description: Library name: CHI214

tag: group

tag: Sex

tag: batch

tag: date ephys

tag: dob

tag: age days

tag: mouse id


title: Forestomach, rep2

tag: gender

tag: strain

tag: tissue

value: 3 months

value: normal


title: Forestomach, rep1

tag: stage


title: LPS 2

description: Library name: DP017

value: Microglia


title: scRNA-Seq of homo sapiens: Sample (KS6B) obtained from Skin (tumor) of patient with Kaposi Sarcoma.

description: Library name: KS6B

value: Skin

value: Mixed skin lesion cells


title: CHI213 Active Het

description: Library name: CHI213

value: dorsal striatum

value: Active

value: cholinergic interneuron

tag: electrophysiologist

tag: rt lot

tag: date lp

tag: lp lot

tag: index lot

tag: date seq

tag: seq srm_sample_id


title: Corpus, rep3

source_name: glandular stomach epithelium

value: female

value: glandular stomach epithelium

tag: health status


title: Corpus, rep2


title: scRNA-Seq of homo sapiens: Sample (KS6A) obtained from Skin (tumor) of patient with Kaposi Sarcoma.

description: Library name: KS6A

tag: cell type

tag: subject status


title: Oligo Pool 2

first: Jeerthi

last: Kannan

address: 401 Park Dr

department: Nephrology

institute: Boston Children's Hospital

description: Library name: Oligo_Pool2

data_processing:
For oligo sequencing, we preprocessed the raw  data using the NGMerge tool (Gaspar, BMC BIoinformatics, 2018)  to combine paired reads and remove flanking adaptor sequences. We then aligned the merged reads to the designed oligo sequences using the Bowtie2 in the end-to-end mode, with the --very-sensitive option. Next, we identified perfectly aligned reads using the optional tag AS=0. We also extracted mismatch information from the XM tags and INDEL information from the XG tags.
For barcode sequencing (both RNA and DNA), we combined the paired-end reads using the NGMerge tool and used only the 13 bp barcode sequences. We used only perfect matches of the designed barcodes for both DNA and mRNA quantification.
Supplementary files format and content: tab-separated value text files

organism: Escherichia coli

source_name: Stable

growth_protocol: Mouse cardiomyocyte HL-1 cells were cultured in Claycomb medium (Sigma)

extract_protocol:
The two 200-mer oligo pools (Twist Bioscience) were first cloned into pLS-mP vector backbone, before sub-cloning into pGL4.23 (Promega) vector backbone. pLS-mP was digested with SbfI and EcoRI (New England Biolabs) to release the minimal promoter-driven eGFP gene. Oligo pools were PCR amplified using primers that are specific to the 5’ and 3’ flanking sequences and introduce overhangs complementary to the SbfI and EcoRI digested pLS-mP vector backbone. Post gel extraction cleanup (Zymo Research), amplified products were cloned using HiFi DNA Assembly master mix (New England Biolabs). The overhangs complementary to the vector backbone were designed to disrupt the original SbfI and EcoRI sites in the vector post-assembly. The cloning reaction was transformed into electrocompetent Stable cells, and transformants (>5 million/pool)  were pooled, grown overnight and maxi-prepped (Thermo Fisher Scientific) to generate the pre-reporter MPRA plasmid libraries for each of the two oligo pools (Pre-reporter/pLS-mP/Stable). These plasmid libraries were then used to generate the oligo sequencing Illumina libraries and sub-cloning of oligos into pGL4.23 vector backbone. We generated these cloned oligo libraries for potential use in future for lentiviral delivery-based MPRA.
For sub-cloning of oligos into pGL4.23, pGL4.23 DNA was first digested with KpnI and XbaI (New England Biolabs) to release the minimal promoter-driven firefly luciferase gene and generate the vector backbone for cloning of MPRA oligos. The cloned oligos in the two pre-reporter MPRA plasmid libraries in pLS-mP backbone were PCR amplified using primers that are specific to the 5’ and 3’ flanking sequences and introduce overhangs complementary to the KpnI and XbaI digested pGL4.23 vector backbone. Post gel extraction cleanup (Zymo Research), amplified products were cloned using HiFi DNA Assembly master mix (New England Biolabs). The cloning reaction was transformed into electrocompetent Stable and DH10β cells, and transformants (>5 million/pool) were pooled, grown overnight and maxi-prepped (Thermo Fisher Scientific) to generate two versions, V1 and V2, of the pre-reporter MPRA plasmid libraries for each of the two oligo pools (V1: Pre-reporter/pGL4.23/Stable; V2: Pre-reporter/pGL4.23/DH10β). Two different E. coli host cells were used for library propagation to maximize representation and recovery of test elements. These pre-reporter plasmid libraries were digested with SbfI and EcoRI (New England Biolabs), sites for which are present only in the deigned oligos, and ligated with the minimal promoter-driven eGFP reporter cassette from pLS-mP, released by SbfI and EcoRI digestion. Like above, the cloning reaction was transformed into electrocompetent Stable and DH10β cells, and transformants (>5 million/pool) were pooled, grown overnight and maxi-prepped (Thermo Fisher Scientific) to generate two versions, V1 and V2, the post-reporter MPRA plasmid libraries for each of the two oligo pools (V1: Post-reporter/pGL4.23/Stable; V2: Post-reporter/pGL4.23/DH10β).
Quality of the synthesized oligos and their representation was assessed by high throughput sequencing. For each of the two oligo pools, oligos cloned in the pre-reporter MPRA plasmid libraries (Pre-reporter/pLS-mP/Stable) were amplified using custom primers that include P5 and P7 Illumina adapter sequences to generate custom amplicon single-indexed oligo sequencing libraries. These two libraries were pooled and sequenced on Illumina MiSeq platform (2×170 + 8 base index) using custom sequencing primers to cover the entire oligo except for the 5’ and 3’ constant flanking sequences.
The post-reporter MPRA plasmid libraries (V1 and V2) were transfected using FuGENE HD Transfection Reagent (Promega) into HL-1 cells, 24h post seeding of 5 million cells in 10 cm dishes, in 10 parallel replicates per pool to ensure robustness and reproducibility. To preserve library complexity, the number of cells at the time of transfection ensures that at least 50× more cells get transfected as compared to the number of unique oligos after adjusting for ~40% transfection efficiency. Forty-eight hours post- transfection cells were harvested, and total RNA was isolated using QIAshredder (Qiagen) and RNeasy Mini Kit (Qiagen). All RNA isolated was used to isolate mRNA using oligo-dT based Magnetic mRNA Isolation Kit (New England Biolabs), which was then treated with TURBO DNase (Thermo Fisher Scientific) to remove contaminating DNA. 500 ng of mRNA from each replicate was reverse transcribed with SuperScript III First-Strand Synthesis System (Thermo Fisher Scientific) in two parallel reactions using a primer downstream of the barcode, which contained a sample index and P7 Illumina adapter sequence. The two cDNA preparations for each replicate transfection were combined, and used as a template in PCR to generate the cDNA (RNA) -based MPRA barcode sequencing library with the same reverse primer and a forward primer complementary to the 3’ end of eGFP with a P5 Illumina adapter sequence. Similarly, the post-reporter MPRA plasmid libraries (V1 and V2) for each oligo pool were used as templates in PCR to generate the input plasmid (DNA) -based MPRA barcode sequencing libraries in three replicates. In total, we generated 26 (3 replicates for input plasmid DNA and 10 transfection replicates for RNA for each of the two oligo pools) custom amplicon single-indexed barcode libraries for V1, which were pooled and sequenced on the Illumina NextSeq 550 platform (2×26 + 8 base index; high-output kit) using custom sequencing primers. The same was repeated for V2.

value: Stable


title: Corpus, rep1


title: Oligo Pool 1

description: Library name: Oligo_Pool1


title: CHI212 Silent Het

description: Library name: CHI212

tag: group

value: Silent

tag: genotype

value: Het

tag: Sex

value: male

tag: batch

tag: date ephys

tag: dob


title: scRNA-Seq of homo sapiens: Sample (KS4) obtained from Skin (tumor) of patient with Kaposi Sarcoma.

description: Library name: KS4

tag: tissue

value: patient with Kaposi Sarcoma


title: Control 3

description: Library name: DP015

tag: cell line


title: CHI211 Unknown Het

description: Library name: CHI211

value: Unknown

tag: age days

tag: electrophysiologist

tag: mouse id

tag: rt lot


title: A15-U

city: Bethesda

first: Aaron

middle: M

last: Cypess

state: Maryland

address: 10 Center Drive, 5N318A

department: NIDDK - DEOB

institute: National Institutes of Health

description: Library name: sample_68

data_processing:
Data quality check was performed using FastQC (v0.11.8). The adapter trimming was performed using fastq-mcf program (v1.05) and cutadapt (v2.5). Contamination removal was performed using Bowtie2 (v2.5.1). Alignment was performed using STAR (v2.7.3a). The raw read counts were estimated using HTSeq (v0.11.2). Read count data were normalized using DESeq2. Additionally the aligned reads were used for estimating expression of the genes using cufflinks (v2.2.1). The expression values are also reported in FPKM (Fragments per kilobase per million) units for each gene. Additionally, RNA-Seq quality control was performed using RNA-SeQC (v1.1.8), RSeQC (v3.0.1) and MultiQC (v1.7).
Assembly: GRCh38/hg38
Supplementary files format and content: Excel spreadsheet containing DESeq2-normalized count
Supplementary files format and content: Salmon-based raw counts as tab-separated value file

source_name: urinary bladder

extract_protocol:
Total RNA was extracted using RNeasy Kit (Qiagen, Germantown, MD) following the manufacturer’s protocol
Takara SMARTer Stranded Total RNA-Seq Kit v2 – Pico Input Mammalian

value: urinary bladder


title: A15-SQ

description: Library name: sample_67

source_name: subcutaneous

value: subcutaneous


title: scRNA-Seq of homo sapiens: Sample (KS3) obtained from Skin (tumor) of patient with Kaposi Sarcoma.

description: Library name: KS3


title: A15-SCLV

description: Library name: sample_66

source_name: supraclavicular

value: supraclavicular


title: CHI210 Unknown Het

description: Library name: CHI210

tag: date lp

tag: lp lot

tag: index lot

tag: date seq


title: ChIPseq, RPB1, release

description: Library name: ChIPseq_RPB1_release

tag: treatment


title: A15-PV

description: Library name: sample_65

source_name: paravertebral

value: paravertebral


title: scRNA-Seq of homo sapiens: Sample (KS2) obtained from Skin (tumor) of patient with Kaposi Sarcoma.

description: Library name: KS2

value: Skin

tag: cell type

tag: subject status