title: LGS13_E24_1

city: Baltimore

first: Pin

last: Lyu

address: 400 N Broadway

institute: JHU

description: Library name: E24_1

data_processing:
Sequencing results for 13LGS and mouse scRNA-Seq were processed through the 10x CellRanger pipeline (version 3.1.0), using the 13LGS genome GCF_016881025.1_HiC_Itri_2 78, and the output loaded into Seurat (version 4.0.4). Cells from Cellplex-labeled libraries were deconvoluted using the cellranger multi pipeline (version 6.0). ScRNA-Seq Seurat objects were analyzed following the Seurat SCT workflow 67,68. Cell type was determined cluster by cluster based on marker gene expression derived from the Seurat function ‘FindAllMarkers’ using default parameters. Differential gene expression between Cone-like Precursors and Rods was identified using the Seurat function “FindMarkers”. ScRNA-Seq samples were aggregated using the CellRanger aggr pipeline (version 3.1.0).
Assembly: GRCm38
Supplementary files format and content: mtx and txt for single cell counts

organism: Ictidomys tridecemlineatus

source_name: E24 retina

extract_protocol:
The use of animals for these studies was conducted using protocols approved by the Johns Hopkins Animal Care and Use Committee, the UW Oshkosh Animal Care and Use Committee, and the Northwestern University Animal Care and Use Committee, in compliance with ARRIVE guidelines and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, and were performed in accordance with relevant guidelines and regulations.Embryonic 13-lined ground squirrel (13LGS) retinal samples were collected from timed-pregnant captive dams and embryonic day was confirmed by comparison with mouse Theiler stages. Postnatal 13LGS retinal samples were collected from captive-bred litters with day of birth set as Postnatal Day 0. 13LGS were euthanized by decapitation.
Retina and explants were dissociated, methanol-fixed cells rehydrated, and nuclei isolated from flash-frozen samples as previously described 71. GEM generation and library preparation for scRNA-Seq were completed according to the 10x 3’ v3.1 user guide (Rev D) using the Chromium Next GEM Single Cell 3ʹ Kit v3.1 and Dual Index Kit TT Set A and for Cellplex samples according to the 10x Next GEM Single Cell 3’ Reagent Kits v3.1 (Dual Index) with Feature Barcode technology for Cell Multiplexing (Rev A) using the 3ʹ CellPlex Kit Set A and Dual Index Kit NN Set A. Transposition, GEM generation, and library preparation for scATAC-Seq were completed according to the 10x ATAC v1.1 user guide (Rev D) using the Chromium Next GEM Single Cell ATAC Reagent Kits v1.1 and Single Index Kit N, Set A. Quality control and sequencing were performed by the Johns Hopkins Transcriptomics and Deep Sequencing Core.

value: E24 retina


title: LGS13_P1_2

status: Public on Jul 28 2025

type: SRA

country: USA

description: Library name: P1_2

library_source: transcriptomic single cell

molecule: total RNA

source_name: P1 retina

tag: tissue

value: P1 retina


title: LGS13_P4_1

description: Library name: P4_1

source_name: P4 retina

value: P4 retina


title: LGS13_P12_2

description: Library name: P12_2

source_name: P12 retina

value: P12 retina


title: LGS13_P8_1

description: Library name: P8_1

source_name: P8 retina

value: P8 retina


title: LGS13_P17_1

description: Library name: P17_1

source_name: P17 retina

value: P17 retina


title: LGS13_P21_1

description: Library name: P21_1

source_name: P21 retina

value: P21 retina


title: LGS13_P8_2

description: Library name: P8_2


title: LGS13_P12_1

description: Library name: P12_1


title: LGS13_E24_2

description: Library name: E24_2


title: LGS13_E26_2

description: Library name: E26_2

source_name: E26 retina

value: E26 retina


title: LGS13_E26_1

description: Library name: E26_1


title: RNA-Seq, PBMCs, NO2-OA [3µM] + untreated 3

city: Leipzig

first: Jes-Niels

last: Boeckel

address: Johannisallee 30

department: Klinik und Poliklinik für Kardiologie

country: Germany

institute: Universitätsklinikum Leipzig

description:
Library name: Sample 9 - JK_5188
x

library_source: transcriptomic

data_processing:
After demultiplexing with bcl2fastq software (Illumina, v2.20), the fastq files underwent trimming and adapter removal using Trim Galore. Read mapping was performed against the respective reference genome (human hg38/mouse mm39) using Hisat2. Gene expression quantification was achieved by read counting using FeatureCounts (R package). The resulting counts were then converted to Transcripts Per Million (TPM) for normalization in R.
Assembly: human hg38
Supplementary files format and content: .txt-file containing TPM counts

organism: Homo sapiens

source_name: human PBMCs

growth_protocol: PBMCs were isolated from buffy coats of healthy blood donors by density gradient centrifugation. In particular, blood was diluted in PBS (ThermoFisher, #10010023) containing 0.3 mM EDTA and layered onto Ficoll® Paque (Cytiva, #17144003). Tubes were centrifuged for 20 min at RT, 2,030 rpm, brake 4 and PBMCs were collected from the interphase between Ficoll® Paque and plasma. PBMCs were washed multiple times with PBS containing 0.3 mM EDTA and 0.2% BSA and used for further experiments.

extract_protocol:
PBMCs were harvested by centrifugation and washed with ice-cold PBS. RNA was purified from cells using the miRNeasy kit (Qiagen, #217004) according to manufacturer’s instructions with additional DNase (Qiagen, #79254) digestion.
Quality control and quantification of the isolated RNA were performed using Fragment Analyzer 5200 (Agilent) using the High Sensitivity RNA quantification kit and Fragment Analyzer Controller Software (Agilent v3.1.0.12). Random primed library preparation was started with 150 ng of total RNA using the Watchmaker RNA library prep kit with Polaris depletion (Watchmaker Genomics) according to the instructions of the manufacturer. The barcoded libraries were purified and quantified using Qubit Fluorometric Quantification (ThermoFisher Scientific). Size distribution of the library DNA was analyzed using the Fragment Analyzer 5200 (Agilent). Sequencing of 2x150 bp was performed with an Illumina NovaSeq 6000 sequencer (Illumina, San Diego, CA, USA).

treatment_protocol: Human primary PBMCs were seeded in cell culture plates in RPMI-1640 medium (Merck, #R8758) supplemented with 10% fetal bovine serum (FBS; Gibco, #10500064). Nitro-oleic acid (50:50 mix of (E)-9- and 10-nitrooctadec-9-enoic acid; NO2-OA) was added for a final concentration of 1 µM or 3 µM. After 2.5 h pre-treatment, PBMCs were treated for an additional 4.5 h with IL-1β (10 ng/mL; Sigma-Aldrich, #H6291-10UG).

tag: cell type

value: human PBMCs

tag: treatment

value: NO2-OA [3µM]


title: WT iBAT 1

city: Iowa City

first: Renata

middle: O

last: Pereira

state: Iowa

address: 169 Newton Rd

department: Internal Medicine

institute: University of Iowa

description: Library name: S2-12

data_processing:
psuedoalignment and quantification performed using Kallisto (v0.45.0)
Assembly: mm10
Supplementary files format and content: tab-delimited text files with transcripts per million

organism: Mus musculus

source_name: brown adipose tissue

extract_protocol:
Total RNA was extracted from tissues with TRIzol reagent (Invitrogen) and purified with the RNeasy kit (Qiagen Inc).
Illumina TruSeq mRNA Stranded kit

value: brown adipose tissue

tag: genotype

value: WT

tag: Sex

value: male

value: 12 weeks of 60% high fat diet


title: RNA-Seq, PBMCs, Vehicle + untreated 2

description:
Library name: Sample 2 - JK_5165
x

value: Vehicle


title: WT_BAY_rep16

city: Copenhagen

first: Lars

middle: Roed

last: Ingerslev

address: Blegdamsvej 3B

department: NNF Center for Basic Metabolic Research

country: Denmark

institute: Copenhagen University

description:
Library name: SCOP_2023_0255_63
63_S63

data_processing:
The sequencing analysis pipeline nf-core [1] was used, with alignment and quantification selection of STAR and Salmon, and mouse GRCm38 set as the reference genome.
UMI-based deduplication was performed using UMI-tools and sorting and index alignments were completed with SAMtools as part of the pipeline.
Assembly: GRCm38
Supplementary files format and content: Tab-seperated file containing the raw gene counts for each sample. Rows specify genes by ENSEMBL_ID and SYMBOL, and columns specify the sample

molecule: polyA RNA

source_name: Primary hepatocytes

extract_protocol:
RNA was isolated using QIAwave RNAeasy kit (CAT #74104) with on-column DNAse treatment using Qiagen RNase-Free DNase set (CAT #79256) following manufacturer’s protocol.
Libraries were prepared using the Universal Plus mRNA-seq protocol (Tecan) as recommended by the manufacturer. Libraries were quantified with NuQuant using the CLARIOstar Plate Reader (BMG Labtech), quality checked using a TapeStation instrument (Agilent Technologies) and subjected to 52-bp paired-end sequencing on a NovaSeq 6000 (Illumina).

value: Primary hepatocytes

value: Wild Type

value: BAY

tag: mouse id

tag: mouse age

tag: cage id

value: Male

tag: isolation pool

value: D

tag: extraction pool

value: C

tag: rin


title: WT_MK-8722_rep16

description:
Library name: SCOP_2023_0255_62
62_S62


title: KO iBAT 1

description: Library name: S1-17

value: KO


title: PPPE.016

city: Rochester

first: Sylvie

last: Girard

state: Minnesota

address: 200 First Street

institute: Mayo Clinic

data_processing:
Libraries were sequenced using the HiSeq 4000 system (Illumina, USA) at the “Integrated Clinical Genomics Centre in Pediatrics”at the CHUSJ.
Data were processed using the RosalinD platform
Assembly: hg19
Supplementary files format and content: Individual text files with the raw counts for each sample
Supplementary files format and content: The same tab-seperated values file for the normalized counts

source_name: Placenta

extract_protocol:
mRNA was extracted using the RNeasy mini kit (Qiagen, Canada) following manufacturer instructions.
Libraries were created using the TruSeq RNA Library Kit v2 (Illumina, USA) and fragment size was validated on a Bioanalyzer (Agilent 2100, USA).

value: Placenta

tag: condition

value: CTL

value: Whole tissue


title: PPPE.012

value: LOPE


title: RNA-Seq, PBMCs, Vehicle + untreated 3

description:
Library name: Sample 3 - JK_5180
x

value: human PBMCs


title: PPPE.015


title: WT_BAY+MK-8722_rep16

description:
Library name: SCOP_2023_0255_64
64_S64


title: WT iBAT 2

description: Library name: S1-20

value: brown adipose tissue


title: LGS13_E21_2

description: Library name: E21_2

source_name: E21 retina

value: E21 retina


title: WT_BAY_rep15

description:
Library name: SCOP_2023_0255_59
59_S59

value: B


title: E7-AMC-R3_S53

city: College Station

first: Ivan

last: Rusyn

state: TX

address: VIDI(Veterinary Medicine 2), 2, 402 Raymond Stotzer Pkwy

department: Interdisciplinary Faculty of Toxicology

institute: Texas A&M University

description: Library name: E7-AMC-R3_S53

data_processing:
The gene count matrices were generated from single-end reads with 2 lane per sample with 75 base pairs (bp).
Reads were quality trimmed using fastp (version 0.21.0) using the default settings with the exceptions of adapter trimming being excluded and the reads being trimmed to 50 bp (Chen et al., 2018).
The reads from the 2 lanes for each sample were combined to generate a single fastq file.
These files were used as input to the TempO-seq analysis pipeline (House et al., 2017) with the human S1500+ probe manifest file to generate a gene count matrix.
The raw count matrix data were aggregated to the gene level if a gene was associated with more than 1 probe in the TempO-seq library. The Bioconductor's DESeq2 package (Love et al., 2014) was utilized to perform differential expression analysis and generate the complete normalized count matrix for all experimental conditions.
Assembly: S1500+ Gene Set
Supplementary files format and content: normcounts.csv file with normalized count data using DESeq2 R package (Love et al., 2014)
Supplementary files format and content: hash.csv file with meta data of the samples

source_name: AMC

extract_protocol:
Cell lysates were collected by aliquoting and pelleting post-thawing.  This pellet was was flushed out by addition of 15 µl of Enhanced 2× lysis buffer (BioSpyder Technologies, Carlsbad, CA) and incubated for 10 minutes at room temperature to allow for complete cell lysis. The lysates were collected from each insert and stored at −80 °C until further processing.   Cell lysates were utilized for the Templated Oligonucleotide Sequencing Assay (TempO-Seq™, BioSpyder Technologies, Carlsbad, CA) as the method for mRNA quantitation (House et al., 2017).
TempO-seq libraries were prepared using the S1500+ targeted transcriptome panel (Mav et al., 2018) consisting of 2,976 transcripts according to the manufacturer’s instructions.

tag: cell line

value: AMC

value: Media


title: WT_BAY+MK-8722_rep15

description:
Library name: SCOP_2023_0255_60
60_S60

value: Primary hepatocytes


title: PPPE.009

value: EOPE


title: tRNA-Arg-TCA-4-1-5

city: London

first: Patrick

last: O'Donoghue

state: Ontario

address: Middlesex Dr.

department: Biochemistry

country: Canada

institute: University of Western Ontario

description: Library name: TCA4-1-5

data_processing:
Cut Adapt
Cut adapt on the galaxy platform was used to remove short and low quality reads (parameters --quality-cutoff=20       --minimum-length=20 )
trimmed reads were aligned with human Hg38 genome using Hisat2
mapped fragments were counted with featureCounts
Assembly: hg38
Supplementary files format and content: txt files include raw counts with Entrez gene IDs

growth_protocol: Human SH-SY5Y cells were grown in DMEM with 10% Fetal Bovine Serum at 37C in 5% CO2

extract_protocol:
72 hours after transfection, cells were lysed, and RNA was extracted using a commercial RNeasy Mini Kit (Qiagen) as per the manufacturer instructions
Libraries were prepared following QC with Tapestation with Illumina library prep with enrichment kits for poly-A enriched transcripts

treatment_protocol: plasmid DNA encoding either empty WTPan, tRNAArgUCA 3-1, or 4-1 was transfected into SH-SY5Y cells with Lipofectamine 3000

value: neuroblastoma

value: Transfected with WT Pan expressing tRNA-Arg-TCA-4-1 under control of endogenous expression elements


title: tRNA-Arg-TCA-4-1-4

description: Library name: TCA4-1-4


title: PPPE.013


title: WT_MK-8722_rep15

description:
Library name: SCOP_2023_0255_58
58_S58

value: Wild Type

value: Male


title: LGS13_E21_1

description: Library name: E21_1


title: LGS13_E18

description: Library name: E18_1

source_name: E18 retina

value: E18 retina


title: E8-AEC-R3_S61

description: Library name: E8-AEC-R3_S61

source_name: AEC

value: AEC


title: tRNA-Arg-TCA-4-1-1

description: Library name: TCA4-1-1


title: PPPE.008

value: Placenta


title: KO_BAY+MK-8722_rep14

description:
Library name: SCOP_2023_0255_56
56_S56

value: AMPKa1a2 KO

value: D

value: C


title: tRNA-Arg-TCA-4-1-3

description: Library name: TCA4-1-3


title: D8-AEC-R2_S60

description: Library name: D8-AEC-R2_S60


title: C8-AEC-R1_S59

description: Library name: C8-AEC-R1_S59


title: PPPE.010

value: LOPE

value: Whole tissue


title: PPPE.007


title: D7-AMC-R2_S52

description: Library name: D7-AMC-R2_S52


title: C7-AMC-R1_S51

description: Library name: C7-AMC-R1_S51


title: KO_BAY_rep14

description:
Library name: SCOP_2023_0255_55
55_S55

value: BAY


title: tRNA-Arg-TCA-3-1-3

description: Library name: TCA3-1-3

value: Transfected with WT Pan expressing tRNA-Arg-TCA-3-1 under control of endogenous expression elements


title: WT_DMSO_rep15

description:
Library name: SCOP_2023_0255_57
57_S57

value: DMSO

value: B


title: tRNA-Arg-TCA-3-1-4

description: Library name: TCA3-1-4


title: PPPE.003


title: TamR cell line, parental

city: Firenze

first: Matteo

last: Benelli

state: FI

address: viale Morgagni 50

department: Department of Experimental and Clinical Biomedical Sciences "Mario Serio"

country: Italy

institute: University of Florence

description:
Library name: TamR_PDS
Bio-Rad ddSEQ

data_processing:
Extract molecular and cellular tags with ddSeeker (v1.0)
Process unaligned and tagged BAM with Drop-seq tools (v1.13)
Assembly: GRCh38
Supplementary files format and content: Compressed Digital Gene Expression (DGE) matrix files
Supplementary files format and content: Summary output of org.broadinstitute.dropseqrna.barnyard.DigitalExpression

source_name: cell line

extract_protocol:
Cells were encapsulated into nanoliter droplets and single cell transcriptomes barcoded using Bio-Rad ddSEQ Single Cell Isolator 2 (Bio-Rad). Briefly, to obtain a single cell suspension, a 60-90% confluent 25 cm2 plate of MDA-MB-361 was trypsinized, filtered through a 35µm nylon strainer (Falcon) and diluted to 2500 cells/µl in 1XPBS (Gibco) + 0.1 BSA (Santa Cruz Biotechnology) solution. We only proceeded with samples showing > 95% viability, according to the manufacturer’s recommendations. Cell count and viability were evaluated with phase contrast microscope (Leica) and automated cell counter (Invitrogen). Finally, 4.5 µl of the cell suspension were loaded in each well of one ddSEQ M cartridge (Bio-Rad) in order to obtain an average of 300 single cells per chamber.
cDNA library preparation was performed following the Illumina Bio-Rad SureCell WTA 3’ Library kit (Illumina). Quantification and quality analysis of cDNA libraries were carried out on Agilent 2100 Bioanalyzer system (Agilent) using High Sensitivity DNA chips (Agilent). Libraries were sequenced on a Hiseq2500 Rapid Run Mode at Centre for Integrative Biology (CIBIO) Next Generation Sequencing (NGS) Facility, Trento, Italy, following protocol recommendations (Illumina); The bases per reads were: R1 (68) - I (8) - R2 (75); 10% PhiX per lane was included and R1 Custom Seq Primer was spiked in HP10.

value: cell line

value: breast cancer


title: ZR751 cell line, palbociclib resistant

description:
Library name: ZR751_PDR
Bio-Rad ddSEQ


title: tRNA-Arg-TCA-4-1-2

description: Library name: TCA4-1-2

value: neuroblastoma

value: Transfected with WT Pan expressing tRNA-Arg-TCA-4-1 under control of endogenous expression elements


title: PPPE.002

value: EOPE


title: KO_MK-8722_rep14

description:
Library name: SCOP_2023_0255_54
54_S54

value: Primary hepatocytes

value: Male


title: D5-DEC-R2_S36

description: Library name: D5-DEC-R2_S36

source_name: DEC

value: DEC

value: Media


title: tRNA-Arg-TCA-3-1-5

description: Library name: TCA3-1-5


title: tRNA-Arg-TCA-3-1-2

description: Library name: TCA3-1-2


title: KO_DMSO_rep14

description:
Library name: SCOP_2023_0255_53
53_S53

value: AMPKa1a2 KO

value: D


title: ZR751 cell line, parental

description:
Library name: ZR751_PDS
Bio-Rad ddSEQ


title: CAL-27_E6E7_2

city: La Jolla

first: Souvick

last: Roy

state: California

address: 3855 Health Sciences Dr

institute: University of California San Diego

data_processing:
Raw reads filtering was as follows: (1) Removed reads with adaptor contamination.
(2) Removed reads when uncertain nucleotides constitute more than 10 percent of either read (N > 10%).
(3) Removed reads reads when low quality nucleotides (Base Quality less than 5) constitute more than 50 percent of the read.
The filtered reads were aligned to the reference genome using HISAT2.
The aligned read were further used for gene expression quantification by FPKM followed by differential gene expression analysis using DESeq2.
Assembly: Homo sapiens Ensemble_94
Supplementary files format and content: csv files include Gene count for each sample
Supplementary files format and content: csv files include FPKM values for each sample

growth_protocol: CAL-27 EV, E5, E6E7 cells were maintained in DMEM supplemented with 10% FBS and 1% Penicillin/streptomycin.

extract_protocol:
RNA was isolated using RNeasy Plus Mini Kit (Qiagen)
RNA libraries for RNA-seq were prepared using NEBNext Ultra II RNA Library Prep Kit following manufacturer's protocols.

value: Squamous cell carcinoma

value: HPV16 E6E7 positive


title: KO_BAY+MK-8722_rep13

description:
Library name: SCOP_2023_0255_52
52_S52

value: A

value: C


title: CAL-27_E6E7_1


title: C5-DEC-R1_S35

description: Library name: C5-DEC-R1_S35


title: tRNA-Arg-TCA-3-1-1

description: Library name: TCA3-1-1

value: Transfected with WT Pan expressing tRNA-Arg-TCA-3-1 under control of endogenous expression elements


title: TamR cell line, palbociclib resistant

description:
Library name: TamR_PDR
Bio-Rad ddSEQ


title: T47D cell line, parental

description:
Library name: T47D_PDS
Bio-Rad ddSEQ


title: CAL-27_E5_2

value: HPV16 E5 positive


title: WTPAN-5

description: Library name: WT5

value: transfected with empty WT Pan


title: KO_BAY_rep13

description:
Library name: SCOP_2023_0255_51
51_S51

value: BAY


title: T47D cell line, palbociclib resistant

description:
Library name: T47D_PDR
Bio-Rad ddSEQ

value: cell line

value: breast cancer


title: WTPAN-4

description: Library name: WT4

value: neuroblastoma


title: CAL-27_E5_1


title: KO_DMSO_rep13

description:
Library name: SCOP_2023_0255_49
49_S49

value: Primary hepatocytes

value: DMSO


title: MDAMB361 cell line, parental

description:
Library name: MDAMB361_PDS
Bio-Rad ddSEQ


title: KO_MK-8722_rep13

description:
Library name: SCOP_2023_0255_50
50_S50

value: Male


title: CAL-27_EV_2

value: Empty vector


title: MCF7 cell line, parental

description:
Library name: MCF7_PDS
Bio-Rad ddSEQ


title: KO_BAY_rep12

description:
Library name: SCOP_2023_0255_47
47_S47

value: AMPKa1a2 KO


title: WTPAN-1

description: Library name: WT1


title: CAL-27_EV_1


title: KO_BAY+MK-8722_rep12

description:
Library name: SCOP_2023_0255_48
48_S48


title: KO_MK-8722_rep12

description:
Library name: SCOP_2023_0255_46
46_S46


title: Bioprint, trophoblast, scRNA-seq

city: Sydney

first: Hao

last: Chen

state: NSW

address: 745 Harris St

department: Faculty of Science

country: Australia

institute: University of Technology Sydney

description: 10x Genomics

data_processing:
The demultiplexing, barcoded processing, gene counting and aggregation were made using the cellranger (v3.1.0)
Assembly: hg38
Supplementary files format and content: Seurat object of integrated scRNA-seq dataset

extract_protocol:
Seven wells of each of bioprinted and Matrigel-embedded organoids were harvested and pooled, producing approximately 700 organoids in total. Organoids were washed twice with PBS and centrifuged for 5 mins at 300 rcf. Organoids were dissociated to single cells by adding 1 mL Accutase to each pooled sample, followed by incubation on a shaker for 30 mins at 800 rpm. The cell suspension was centrifuged for 5 mins at 300 rcf and washed twice with sterile 0.04% BSA in PBS, centrifuging between each step. Cells were then resuspended in 100 µL 0.04% BSA and passed through a 40 µm filter. Cell viability and count was assessed using trypan blue, ensuring viability was > 80 %.
Library was perfromed according to the manufacturer's instruction (single-cell 3' v2 protocol, 10x Genomics)

value: Placenta

value: First trimester trophoblast

value: Bioprinted


title: KO_DMSO_rep12

description:
Library name: SCOP_2023_0255_45
45_S45


title: SH-SY5Y cells, ARS-3

city: Lucknow

first: Aditya

middle: Bhushan

last: Pant

state: Uttar Pradesh

address: vishvigyan Bhawan MG Marg

department: FEST

country: India

institute: CSIR-INDIAN INSTITUTE OF TOXICOLOGY RESEARCH

data_processing:
Preprocessing for FastQ files were done using fastp tool (version 0.20.0)
Assembly: Release 39 (GRCh38.p13)
Supplementary files format and content: fastp.json files include various metrics before and after filtering reads
Supplementary files format and content: .tsv files contain annotation of identified mRNAs, lncRNAs, and circRNAs
Supplementary files format and content: circRNA counts.tsv include circRNA count matrix

source_name: SH-SY5Y cells

growth_protocol: SH-SY5Y human neuroblastoma cells were maintained in 1:1 mixture of MEM-Ham's F12 with 10% fetal bovine serum and antibiotic/antimycotic solution in the humified atmosphere with 5% CO2 at 37°C.To produce differentiated cells displaying neuronal features, SH-SY5Y cells were treated with RA (10 μM) for 5 days with a reduced level of serum, followed by a 3-day exposure to 100 ng/ml BDNF without any supplement of serum.

extract_protocol:
Total RNA was extracted employing the mirVana miRNA kit. 1μg of total RNA was used for the construction of sequencing libraries.
RNA libraries for RNA-seq were prepared using the Illumina TruSeq Stranded Total RNA kit according to the manufacturer’s protocol.

treatment_protocol: Differentiated SH-SY5Y cells were exposed to 10 μM of sodium arsenite for 24 h.

value: SH-SY5Y cells

value: Neuroblastoma cell line

value: WT

value: Arsenic treated


title: EDR cell line, parental

description:
Library name: EDR_PDS
Bio-Rad ddSEQ


title: EDR cell line, palbociclib resistant

description:
Library name: EDR_PDR
Bio-Rad ddSEQ


title: SH-SY5Y cells, ARS-2


title: WT_BAY+MK-8722_rep11

description:
Library name: SCOP_2023_0255_44
44_S44

value: Wild Type

value: A

value: C


title: WTPAN-2

description: Library name: WT2

value: transfected with empty WT Pan


title: WTPAN-3

description: Library name: WT3

value: neuroblastoma


title: BT474 cell line, parental

description:
Library name: BT474_PDS
Bio-Rad ddSEQ

value: cell line

value: breast cancer


title: BT474 cell line, palbociclib resistant

description:
Library name: BT474_PDR
Bio-Rad ddSEQ


title: MCF7 cell line, palbociclib resistant

description:
Library name: MCF7_PDR
Bio-Rad ddSEQ


title: Matrigel, trophoblast, scRNA-seq

description: 10x Genomics

value: Matrigel-embedded


title: WT_BAY_rep11

description:
Library name: SCOP_2023_0255_43
43_S43

value: Primary hepatocytes

value: BAY

value: Male


title: RH30 IkBa-SR;MyoD CRISPR clone 29, replicate 2

type: RNA

city: Charleston

first: Denis

middle: C

last: Guttridge

state: SC

address: 173 Ashley Ave

department: Pediatrics

institute: Medical University of South Carolina

description: Gene expression data from RH30 SR, clonally selected MyoD CRISPR cells

hyb_protocol: Following fragmentation, 15 ug of cRNA were hybridized for 16 hr at 45 C on GeneChip™ Human Transcriptome Array 2.0. GeneChips were washed and stained in the Affymetrix Fluidics Station 400.

scan_protocol: GeneChips were scanned in a GeneChip Scanner 3000 (Affymetrix Inc., USA)

data_processing: Gene expression levels were estimated from Expression console software from affymetrix (RMA-Gene-Core)

label: biotin

source_name: RH30 cells, SR, MyoD CRISPR

label_protocol: Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 6 ug total RNA (Expression Analysis Technical Manual, 2001, Affymetrix).

growth_protocol: Cells were grown in DMEM 10% fetal bovine serum.

extract_protocol: Total RNA was extracted using Trizol.

treatment_protocol: Cells were infected wuth IkBa-SR, Vector, or IkBa-SR + MyoD sgRNA

value: Rhabdomyosarcoma

value: ablated MyoD expression


title: WT_MK-8722_rep11

description:
Library name: SCOP_2023_0255_42
42_S42


title: SH-SY5Y cells, AC-3

value: SH-SY5Y cells

value: Control


title: WT_DMSO_rep11

description:
Library name: SCOP_2023_0255_41
41_S41

value: DMSO


title: SH-SY5Y cells, AC-2

value: Neuroblastoma cell line

value: WT


title: RH30 IkBa-SR;MyoD CRISPR clone 29, replicate 1


title: iSRE, Cut&Run, H3K27me3, Rep3

first: Laurie

last: Steiner

state: NY

address: 601 Elmwood Ave

institute: University of Rochester

library_source: genomic

data_processing:
Bowtie2 alignment to Hg38 genome
Picard dupliate removal
bamCoverage RPKM normalization
Assembly: Hg38
Supplementary files format and content: bigWig, peak bed file
Library strategy: Cut&Run

molecule: genomic DNA

source_name: erythroblast

growth_protocol: Proliferating SREs (untransduced) or iSREs in phase 2 expansion media were cultured past EXD30 and were collected for Cut&Run.

extract_protocol:
CUT&RUN was performed using the CUTANA ChIC/CUT&RUN Kit (Epicypher) on whole cells.
Libraries were generated using the NEBNext Ultra II DNA Library Prep kit (NEB). For transcription factors (BMI-1 and RING1B), size selection was optimized for small fragments (end prep for 20C for 30 minutes followed by 50C for 60 minutes, ligation AMPure XP cleanup at 1.75X, annealing temperature of 65C for library amplification, and library AMPure XP cleanup using 1st round negative selection at 0.8X followed by a 2nd round positive selection at 1.2X). Libraries were sequenced with illumina HiSeq, paired end 150 bp length reads.

treatment_protocol: for iSREs, PBMCs were transduced Bmi1 overexpression plasmid.

value: erythroblast

value: iSRE


title: KO_BAY+MK-8722_rep10

description:
Library name: SCOP_2023_0255_40
40_S40

value: AMPKa1a2 KO

value: B


title: iSRE, Cut&Run, H3K27me3, Rep2


title: RH30 IkBa-SR;MyoD CRISPR clone 29, replicate 3


title: Lgr5 stem cell, IL25 treated wild type mice, mid 2/3 small intestine, biol rep 3

city: San Francisco

first: Victor

middle: S

last: Cortez

state: CA

address: 513 Parnassus Avenue

department: Medicine

institute: University of California, San Francisco

description:
Library name: Lgr5_IL25_6b
Protocol-Buenrostro et al.

data_processing:
Raw reads were mapped to the mouse mm10 genome assembly using STAR alignment (--outFilterMismatchNoverLmax 0.04 --outFilterMismatchNmax 999 --alignSJDBoverhangMin 1 --outFilterMultimapNmax 1 --alignIntronMin 20 --alignIntronMax 1000000 --alignMatesGapMax 1000000). Bam files were generated by STAR. PCR duplicates removed by Picard and peak calling performed using MACS2 (--keep-dup 1 --bw 500 -n output --nomodel --extsize 400 --slocal 5000 --llocal 100000 -q 0.01). Differential binding peaks were identified with DiffBind (method=DBA_DESEQ2), and peaks annotated using ChIPSeeker. To generate bigwig files for ATACseq datasets, all aligned bam files were merged by condition using samtools merge. Samtools view was then run to convert alignments to counts and bedtools genomecov was run to covert the merged bam files into a bedgraph files. Finally, bedGraphToBigWig (ucsc-tools/363) was used to generate the bigwig files displayed on browser tracks using the IGV browser.
Reads aligned using STAR v2.7
Peak calling via MACS2
Assembly: Mouse mm10
Supplementary files format and content: .BED files

source_name: Small Intestine

extract_protocol:
For ILC2s ATAC: Small intestinal single cell suspensions were prepared from the proximal 1/3, mid 2/3, and distal 3/3 of tissue. Luminal contents were flushed with PBS, Peyer’s patches removed, and tissue cut open. Pieces were incubated with epithelial cell extraction buffer (EEB: Ca2+/Mg2+ free HBBS, 3% FCS, 10 mM HEPES, 10 mM EDTA, 10 mM DTT) at 37℃ with gentle shaking for 15 min. Tubes were vortexed at full speed for 30 sec and supernatants discarded. The tissue was placed with fresh EEB at 37℃ for 15 min with gentle shaking, then vortexed for 30 sec and supernatant discarded. Tissue was placed in rinse buffer (Ca2+/Mg2+ sufficient HBBS, 3% FCS, 10 mM HEPES) and incubated at 37℃ with gentle shaking for 5 min. Tissue was placed in GentleMACS C tubes (Miltenyi Biotec) with digestion buffer (DB: Ca2+/Mg2+ sufficient HBBS, 3% FCS, 10 mM HEPES, 30 ug/ml DNase I, 0.1 Wunsch/ml Liberase TM), minced with scissors, and incubated at 37℃ with gentle shaking for 10-15 min. Single-cell suspensions were obtained using a gentleMACS tissue dissociator (Miltenyi Biotec), running program m_intestine_0, and then pouring through a 100 um filter and washing with FACS buffer (PBS, 3% FCS). For Lgr5+ epitheilal stem cells ATAC: GFP+CD44+CD45- cells were sort purified from intestinal crypts isolated from proximal, mid and distal small intestines of Lgr5DTR-EGFP mice 50-60 days after the last treatment.
Cells were subjected to Assay for Transposase-Accessible Chromatin using Sequencing (ATAC-Seq). 30,000–55,000 cells were sorted into PBS + 10% FCS, washed and lysed in 100 μl detergent (100 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% IGEPAL CA-630) on ice for 5 min to release nuclei. Pelleted nuclei were incubated for 30 min with Tagment DNA Enzyme transposase. The reaction was stopped and DNA purified using the MinElute Reaction Cleanup kit. Eluted DNA was amplified with the NEBNext High-Fidelity 2x PCR Master Mix using Nextera index primers. The indexed libraries were purified using the MinElute PCR Reaction Cleanup before sequencing on an Illumina HiSeq 2500 with 25 bp paired-end reads.

value: Small Intestine

value: Lgr5 epitheilal stem cell

value: IL25 treated


title: KO_BAY_rep10

description:
Library name: SCOP_2023_0255_39
39_S39

value: Primary hepatocytes


title: Lgr5 stem cell, IL25 treated wild type mice, distal 3/3 small intestine, biol rep 3

description:
Library name: Lgr5_IL25_6c
Protocol-Buenrostro et al.


title: iSRE, Cut&Run, H3K27me3, Rep4


title: e6_24h_t

city: Bern

first: Olivier

middle: Thierry

last: Guenat

address: Murtenstrasse 50

department: ARTORG Center for Biomedical Engineering Research

country: Switzerland

institute: University of Bern

data_processing:
Seqeunced reads were trimmed for adapter sequences using fastp 0.19.5
Filtered data was aligned to the reference genome GRCh38 and the GTF file, version 111, from Ensembl using HISAT2, version 2.2.1
FeatureCounts, subread package version 2.0.1., was used to assign the mapped reads to genomic features
Assembly: Homo_sapiens.GRCh38
Supplementary files format and content: tab-delimited raw data with each sample in a column

source_name: umbilical cord vein

growth_protocol: Human umbilical vein endothelial cells (HUVECs, Gibco, USA) were maintained in endothelial growth media (EGM-2, Lonza, Switzerland) added supplements (EGM-2 BulletKit, Lonza, Switzerland) according to the manufacturer’s instructions at 37 ◦C with 5% CO2

extract_protocol:
The channels were washed with DPBS and lysed by adding 1-Thioglycerol and lysis buffer solution. RNA was isolated from two chips according to the manufacturer's protocol (ReliaPrep™ RNA Miniprep Systems, Promega).
Sequencing libraries were made with 50 ng input RNA using a Revelo mRNA-Seq for MagicPrep NGS kit A (Tecan, PN 30186623) according to the Revelo mRNA-Seq for MagicPrep NGS User Guide (Tecan publication number MO1535, v1).

treatment_protocol: HUVECs seeded on the microfluidics device were either connected to the peristaltic pump flow or put on the tilter to induce the unidirectional and bidirectional flow respectively for 4h and 24h.

value: umbilical cord vein

value: Primary HumanUmbilical VeinEndothelial Cells

value: Endothelial cells

value: bidirectional flow-24h


title: e6_24h_p

value: unidirectional flow-24h


title: iSRE, Cut&Run, H3K27me3, Rep1


title: SH-SY5Y cells, ARS-1

value: SH-SY5Y cells

value: Arsenic treated


title: iSRE, Cut&Run, H2AK119Ub, Rep6


title: RH30 SR, replicate 2

description: Gene expression data from RH30 SR cells

source_name: RH30 cells, SR

value: Rhabdomyosarcoma

value: control


title: Lgr5 stem cell, IL25 treated wild type mice, proximal 1/3 small intestine, biol rep 3

description:
Library name: Lgr5_IL25_6a
Protocol-Buenrostro et al.


title: SH-SY5Y cells, AC-1

value: Neuroblastoma cell line

value: WT

value: Control


title: e6_4h_t

value: bidirectional flow-4h


title: KO_BAY+MK-8722_rep9

description:
Library name: SCOP_2023_0255_36
36_S36

value: AMPKa1a2 KO

value: Male

value: D

value: B


title: Lgr5 stem cell, IL25 treated wild type mice, mid 2/3 small intestine, biol rep 2

description:
Library name: Lgr5_IL25_5b
Protocol-Buenrostro et al.

value: Small Intestine

value: Lgr5 epitheilal stem cell

value: IL25 treated


title: iSRE, Cut&Run, H2AK119Ub, Rep5

value: erythroblast

value: iSRE


title: RH30 SR, replicate 1

description: Gene expression data from RH30 SR cells


title: Lgr5 stem cell, IL25 treated wild type mice, distal 3/3 small intestine, biol rep 2

description:
Library name: Lgr5_IL25_5c
Protocol-Buenrostro et al.


title: KO_BAY_rep9

description:
Library name: SCOP_2023_0255_35
35_S35

value: Primary hepatocytes

value: BAY


title: iSRE, Cut&Run, H2AK119Ub, Rep4


title: e6_4h_p

value: umbilical cord vein

value: Primary HumanUmbilical VeinEndothelial Cells

value: Endothelial cells

value: unidirectional flow-4h


title: Lgr5 stem cell, IL25 treated wild type mice, proximal 1/3 small intestine, biol rep 2

description:
Library name: Lgr5_IL25_5a
Protocol-Buenrostro et al.


title: RH30 SR, replicate 3

description: Gene expression data from RH30 SR cells

value: Rhabdomyosarcoma

value: control


title: e4_24h_p

value: unidirectional flow-24h


title: KO_DMSO_rep10

description:
Library name: SCOP_2023_0255_37
37_S37

value: AMPKa1a2 KO

value: DMSO

value: Male


title: Lgr5 stem cell, IL25 treated wild type mice, distal 3/3 small intestine, biol rep 1

description:
Library name: Lgr5_IL25_4c
Protocol-Buenrostro et al.

value: Small Intestine


title: iSRE, Cut&Run, H2AK119Ub, Rep2


title: KO_MK-8722_rep9

description:
Library name: SCOP_2023_0255_34
34_S34

value: D

value: B


title: Lgr5 stem cell, IL25 treated wild type mice, mid 2/3 small intestine, biol rep 1

description:
Library name: Lgr5_IL25_4b
Protocol-Buenrostro et al.

value: Lgr5 epitheilal stem cell

value: IL25 treated


title: WT_BAY+MK-8722_rep8

description:
Library name: SCOP_2023_0255_32
32_S32

value: Primary hepatocytes

value: Wild Type

value: A


title: KO_DMSO_rep9

description:
Library name: SCOP_2023_0255_33
33_S33


title: e4_4h_p

value: umbilical cord vein

value: Primary HumanUmbilical VeinEndothelial Cells


title: Lgr5 stem cell, IL25 treated wild type mice, proximal 1/3 small intestine, biol rep 1

description:
Library name: Lgr5_IL25_4a
Protocol-Buenrostro et al.


title: iSRE, Cut&Run, Ring1b, Rep6

value: erythroblast

value: iSRE


title: WT_BAY_rep8

description:
Library name: SCOP_2023_0255_31
31_S31

value: BAY


title: e4_24h_t

value: Endothelial cells

value: bidirectional flow-24h


title: e3_24h_t


title: iSRE, Cut&Run, H2AK119Ub, Rep1


title: e4_4h_t

value: bidirectional flow-4h


title: WT_MK-8722_rep8

description:
Library name: SCOP_2023_0255_30
30_S30

value: Male


title: Lgr5 stem cell, PBS treated wild type mice, distal 3/3 small intestine, biol rep 3

description:
Library name: Lgr5_PBS_3c
Protocol-Buenrostro et al.

value: Small Intestine

value: PBS treated


title: iSRE, Cut&Run, H2AK119Ub, Rep3


title: e3_24h_p

value: unidirectional flow-24h


title: Lgr5 stem cell, PBS treated wild type mice, mid 2/3 small intestine, biol rep 3

description:
Library name: Lgr5_PBS_3b
Protocol-Buenrostro et al.

value: Lgr5 epitheilal stem cell


title: Lgr5 stem cell, PBS treated wild type mice, proximal 1/3 small intestine, biol rep 3

description:
Library name: Lgr5_PBS_3a
Protocol-Buenrostro et al.


title: WT_DMSO_rep8

description:
Library name: SCOP_2023_0255_29
29_S29

value: DMSO

value: B


title: e3_4h_t


title: iSRE, Cut&Run, Ring1b, Rep3


title: iSRE, Cut&Run, Ring1b, Rep4


title: Lgr5 stem cell, PBS treated wild type mice, distal 3/3 small intestine, biol rep 2

description:
Library name: Lgr5_PBS_2c
Protocol-Buenrostro et al.


title: e3_4h_p

value: unidirectional flow-4h


title: WT_BAY_rep7

description:
Library name: SCOP_2023_0255_27
27_S27

value: Primary hepatocytes

value: Wild Type

value: D


title: iSRE, Cut&Run, Ring1b, Rep2

value: erythroblast


title: Lgr5 stem cell, PBS treated wild type mice, mid 2/3 small intestine, biol rep 2

description:
Library name: Lgr5_PBS_2b
Protocol-Buenrostro et al.


title: Sample_03 Allo15MCAR-NKT

city: Los Angeles

first: Lili

last: Yang

address: 610 Charles E. Young Dr. East

department: Microbiology Immunology & Molecular Genetics

institute: University of California, Los Angeles

description: 10x Genomics

data_processing:
The raw sequencing data were processed and mapped to the human reference (GRCh38) dataset using the Cell Ranger. The processed data were filtered for further downstream analysis.
Assembly: hg38
Supplementary files format and content: matrix.mtx.gz: Matrix Market Format, with rows representing genes and columns representing cells.
Supplementary files format and content: barcode.tsv.gz: Tab-separated values, where each row represents a unique identifier for a cell.
Supplementary files format and content: features.tsv.gz: Tab-separated values, where each row represents a gene.

source_name: Peritoneal fluid

extract_protocol:
Peritoneal cavity cells (the tumor site) harvested from experimental animals at week 4 were subjected to FACS sorting for isolating OVCAR8-FG cells (GFP+; Sample_01: vehicle treated, Sample_02: MCAR-T treated, and Sample_03: Allo15MCAR-NKT treated). Cells isolated from 10 experimental mice were combined for each group.
Cells were quantified using a Cell Countess II automated cell counter (Invitrogen/Thermo Fisher Scientific). A total of 10,000 cells from each experimental group were loaded on the Chromium platform (10X Genomics), and libraries were constructed using the Chromium Next GEM Single Cell 3' Kit v3.1 and the Chromium Next GEM Chip G Single Cell Kit (10X Genomics), according to the manufacturer’s instructions. Library quality was assessed using the D1000 ScreenTape on a 4200 TapeStation System (Agilent Technologies). Libraries were sequenced on an Illumina NovaSeq using the NovaSeq S4 Reagent Kit (100 cycles; Illumina).
Library quality was assessed using the D1000 ScreenTape on a 4200 TapeStation System (Agilent Technologies). Libraries were sequenced on an Illumina NovaSeq using the NovaSeq S4 Reagent Kit (100 cycles; Illumina).

value: Peritoneal fluid

value: Tumor cells


title: iSRE, Cut&Run, Ring1b, Rep1

value: iSRE


title: Lgr5 stem cell, PBS treated wild type mice, proximal 1/3 small intestine, biol rep 2

description:
Library name: Lgr5_PBS_2a
Protocol-Buenrostro et al.


title: Sample_02 MCAR-T

description: 10x Genomics


title: WT_DMSO_rep7

description:
Library name: SCOP_2023_0255_25
25_S25


title: iSRE, Cut&Run, Bmi1, Rep6


title: iSRE, Cut&Run, Bmi1, Rep5


title: Sample_01 NT

description: 10x Genomics


title: Lgr5 stem cell, PBS treated wild type mice, distal 3/3 small intestine, biol rep 1

description:
Library name: Lgr5_PBS_1c
Protocol-Buenrostro et al.


title: RNA-Seq, PBMCs, Vehicle + untreated 1

description:
Library name: Sample 1 - JK_5150
x

source_name: human PBMCs

value: human PBMCs

value: Vehicle


title: E5-DEC-R3_S37

description: Library name: E5-DEC-R3_S37

value: DEC

value: Media


title: WT_MK-8722_rep7

description:
Library name: SCOP_2023_0255_26
26_S26

value: Male


title: WT_BAY+MK-8722_rep7

description:
Library name: SCOP_2023_0255_28
28_S28


title: Allo15MCAR-NKT

description: 10x Genomics

data_processing:
The raw sequencing data were processed and mapped to the human TCR reference genome (hg38) using the Cell Ranger VDJ software. The frequencies of the α or β chain recombination were plotted to identify the unique TCR repertoires in each cell population.
Assembly: hg38
Supplementary files format and content: Sample_filtered_contig_annotations.csv, high-level annotations of each high-confidence contigs from cell-associated barcodes of each sample
Supplementary files format and content: Sample_clonotypes.csv, high-level descriptions of each clonotype of each sample
Library strategy: TCR-seq

source_name: In vitro culture

extract_protocol:
scTCR-seq was utilized to study the TCR sequencing of Allo/15BCAR-NKT cells. Five different cell types were delivered to the UCLA TCGB Core for library construction and scTCR-seq.
Cells were quantified using a Cell Countess II automated cell counter (Invitrogen/Thermo Fisher Scientific). A total of 10,000 cells from each experimental group were loaded on the Chromium platform (10X Genomics), and libraries were constructed using the Chromium Next GEM Single Cell 3' Kit v3.1 and the Chromium Next GEM Chip G Single Cell Kit (10X Genomics), according to the manufacturer’s instructions. Library quality was assessed using the D1000 ScreenTape on a 4200 TapeStation System (Agilent Technologies). Libraries were sequenced on an Illumina NovaSeq using the NovaSeq S4 Reagent Kit (100 cycles; Illumina).
Library quality was assessed using the D1000 ScreenTape on a 4200 TapeStation System (Agilent Technologies). Libraries were sequenced on an Illumina NovaSeq using the NovaSeq S4 Reagent Kit (100 cycles; Illumina).

value: In vitro culture

value: NKT cell


title: KO_BAY+MK-8722_rep6

description:
Library name: SCOP_2023_0255_24
24_S24

source_name: Primary hepatocytes

value: AMPKa1a2 KO

value: C


title: Lgr5 stem cell, PBS treated wild type mice, proximal 1/3 small intestine, biol rep 1

description:
Library name: Lgr5_PBS_1a
Protocol-Buenrostro et al.

value: Small Intestine

value: Lgr5 epitheilal stem cell

value: PBS treated


title: Lgr5 stem cell PBS treated wild type mice, mid 2/3 small intestine, biol rep 1

description:
Library name: Lgr5_PBS_1b
Protocol-Buenrostro et al.


title: KO_BAY_rep6

description:
Library name: SCOP_2023_0255_23
23_S23

value: Primary hepatocytes

value: BAY

value: B


title: iSRE, Cut&Run, Bmi1, Rep3

value: erythroblast


title: iSRE, Cut&Run, Bmi1, Rep4

value: iSRE


title: AlloMCAR-NKT

description: 10x Genomics


title: KO_MK-8722_rep6

description:
Library name: SCOP_2023_0255_22
22_S22


title: iSRE, Cut&Run, Bmi1, Rep2


title: ILC2s, IL25 treated wild type mice, distal 3/3 small intestine, biol rep 3

description:
Library name: ILC2_IL25_6c
Protocol-Buenrostro et al.

value: Wild type, C57BL/6J

value: IL25 treated


title: PBMC-Tc

description: 10x Genomics

value: T cell


title: KO_DMSO_rep6

description:
Library name: SCOP_2023_0255_21
21_S21

value: DMSO


title: iSRE, Cut&Run, Bmi1, Rep1


title: SRE cells replicate 3

data_processing:
Reads were mapped to the human genome using STAR
Reads in genes were quantified using kallisto.
Differential expression between different sample types was identified using DESeq2.
Assembly: hg19
Supplementary files format and content: TPM transcript per million values were obtained from Kallisto and tximport
Supplementary files format and content: Counts per gene were obtained from kallisto

source_name: SRE

growth_protocol: SREs and iSREs from 3 donors were collected at day 13 and after day 30 of expansion culture, respectively, and sorted as live CD36+ CD235amid cells.

extract_protocol:
RNA was harvested using the Qiagen RNeasy kit
Preparation and sequencing of poly-A mRNA sequencing libraries were performed by the Yale Center for Genome Analysis (YCGA). RNA integrity was estimated using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA).  Poly-A mRNA was purified from approximately 200 ng of total RNA and the stranded mRNA sequencing libraries were constructed using KAPA mRNA HyperPrep Kit for Illumina sequencing (Roche, Basel, Switzerland)

value: SRE

value: Control


title: WT_BAY+MK-8722_rep5

description:
Library name: SCOP_2023_0255_20
20_S20

value: Wild Type

value: Male

value: D


title: ILC2s, IL25 treated wild type mice, proximal 1/3 small intestine, biol rep 3

description:
Library name: ILC2_IL25_6a
Protocol-Buenrostro et al.

value: Small Intestine


title: WT_BAY_rep5

description:
Library name: SCOP_2023_0255_19
19_S19


title: ILC2s, IL25 treated wild type mice, distal 3/3 small intestine, biol rep 2

description:
Library name: ILC2_IL25_5c
Protocol-Buenrostro et al.


title: SRE, Cut&Run,  H3K27me3, Rep3

value: untransduced


title: SRE, Cut&Run,  H3K27me3, Rep4


title: ILC2s, IL25 treated wild type mice, mid 2/3 small intestine, biol rep 3

description:
Library name: ILC2_IL25_6b
Protocol-Buenrostro et al.


title: WT_MK-8722_rep5

description:
Library name: SCOP_2023_0255_18
18_S18


title: WT_DMSO_rep5

description:
Library name: SCOP_2023_0255_17
17_S17


title: KO_MK-8722_rep10

description:
Library name: SCOP_2023_0255_38
38_S38

value: AMPKa1a2 KO


title: ILC2s, IL25 treated wild type mice, mid 2/3 small intestine, biol rep 2

description:
Library name: ILC2_IL25_5b
Protocol-Buenrostro et al.


title: SRE, Cut&Run,  H3K27me3, Rep2


title: KO_BAY+MK-8722_rep4

description:
Library name: SCOP_2023_0255_16
16_S16

value: Primary hepatocytes

value: C

value: A


title: iSRE cells replicate 2

source_name: iSRE


title: ILC2s, IL25 treated wild type mice, proximal 1/3 small intestine, biol rep 2

description:
Library name: ILC2_IL25_5a
Protocol-Buenrostro et al.


title: SRE, Cut&Run,  H3K27me3, Rep1

value: erythroblast


title: ILC2s, IL25 treated wild type mice, distal 3/3 small intestine, biol rep 1

description:
Library name: ILC2_IL25_4c
Protocol-Buenrostro et al.


title: SRE cells replicate 1


title: iSRE cells replicate 3

value: iSRE


title: KO_BAY_rep4

description:
Library name: SCOP_2023_0255_15
15_S15

value: BAY


title: SRE, Cut&Run,  H2AK119Ub, Rep4


title: ILC2s, IL25 treated wild type mice, mid 2/3 small intestine, biol rep 1

description:
Library name: ILC2_IL25_4b
Protocol-Buenrostro et al.

value: Wild type, C57BL/6J

value: IL25 treated


title: WT_DMSO_rep16

description:
Library name: SCOP_2023_0255_61
61_S61

value: DMSO


title: SRE, Cut&Run,  H2AK119Ub, Rep3

value: SRE


title: ILC2s, IL25 treated wild type mice, proximal 1/3 small intestine, biol rep 1

description:
Library name: ILC2_IL25_4a
Protocol-Buenrostro et al.


title: KO_DMSO_rep4

description:
Library name: SCOP_2023_0255_13
13_S13


title: SRE, Cut&Run,  H2AK119Ub, Rep2


title: C20, mono-culture, Vehicle-treated, replicate 3

city: Freiburg

first: Michael

last: Schulz

address: Breisacher Straße 64

department: Institute for Neuropathology

institute: University Hospital Freiburg

description:
36_ZEINERetAl
col AM
ZeinerEtAl_allSamples.csv

data_processing:
FastQC Analysis
quality score filtering, poly-A trimming, artifact removal, removal of ‘N’ containing reads and clearing of rRNA contamination using a pipeline provided by the HUSAR platform, DKFZ.
Transcriptomes were mapped to the human genome using the genecode annotations (release v. 32; genome assembly version GRCh38.p13) and TopHat2 (v. 2.0.14)
The number of reads per gene was determined using HTSeq count.
Paired differential analysis was performed using   ‘DESeq2’  (version 1.18.1)
Assembly: Transcriptomes were mapped to the human genome using the genecode annotations (release v. 32; genome assembly version GRCh38.p13) and TopHat2 (v. 2.0.14)
Supplementary files format and content: .csv; processed data file contains annotated ENSEMBL IDs and gene names as well as readcounts which were annotated from HTSeq counts

source_name: C20 microglia cell line

growth_protocol: Cells were cultured in Dulbecco’s modified eagle medium (DMEM) containing 100 IU/ml penicillin and 100 mg/ml Streptomycin and 10% fetal calf serum (fcs)

extract_protocol:
Samples were washed with PBS and resuspended in TRIzol, and stored at -80 °C until further usage. RNA was isolated according to manufacturers instructions.
RNA-sequencing libraries were generated with the SMART-Seq preparation kit (CloneTech) and fragmented with the Nextera XT kit (Illumina).

treatment_protocol: Transwell co-cultivation of the human MG cell lines C20 or HMC3 with the glioma cell line LNT-229 was performed using 6- or respective 24-well cell culture inserts (1 μm pore). 6-well inserts were used for RNA analyses. MG were seeded into the lower chamber (6- well: 500.000/well, 24-well: 120.000/well) and glioma cells into the inserts (6-well insert: 400.000/well, 24-well insert: 30.000/well) separately in DMEM with 10% fcs 24 h prior to co-cultivation. To provide a MG monoculture control condition, C20 or HMC3 MG were seeded into the inserts and lower chambers. Cultivation was performed in serum-free DMEM with a total volume of 3 ml/500 μl or 2 ml/200 μl added to the well and insert (6-/24-well plate, respectively). Cultured cells were treated with the mTOR inhibitors 100 nM rapamycin and 100 nM torin2 as well as 400 μM temozolomide or corresponding vehicle controls for 24 h. Afterwards, C20 or HMC3 cells in the lower chamber were harvested for further RNA-Seq analysis.

value: C20 microglia cell line

value: microglia

tag: culture condition

value: mono-culture with C20

value: vehicle


title: SRE cells replicate 2

value: Control


title: iSRE cells replicate 1


title: ILC2s, PBS treated wild type mice, distal 3/3 small intestine, biol rep 3

description:
Library name: ILC2_PBS_3c
Protocol-Buenrostro et al.

value: Small Intestine

value: PBS treated


title: SRE, Cut&Run,  H2AK119Ub, Rep1

value: untransduced


title: WT_BAY_rep3

description:
Library name: SCOP_2023_0255_11
11_S11

value: Primary hepatocytes

value: Wild Type

value: Male

value: C


title: C20, mono-culture, Rapamycin-treated, replicate 3

description:
34_ZEINERetAl
col AG
ZeinerEtAl_allSamples.csv

value: rapamycin


title: WT_MK-8722_rep3

description:
Library name: SCOP_2023_0255_10
10_S10

value: A


title: ILC2s, PBS treated wild type mice, proximal 1/3 small intestine, biol rep 3

description:
Library name: ILC2_PBS_3a
Protocol-Buenrostro et al.

value: Wild type, C57BL/6J


title: C20, co-culture, Vehicle-treated, replicate 3

description:
33_ZEINERetAl
col AD
ZeinerEtAl_allSamples.csv

value: co-culture with LNT-229 glioma cell line


title: WT_DMSO_rep3

description:
Library name: SCOP_2023_0255_9
9_S9

value: DMSO


title: ILC2s, PBS treated wild type mice, mid 2/3 small intestine, biol rep 3

description:
Library name: ILC2_PBS_3b
Protocol-Buenrostro et al.


title: C20, co-culture,Temozolomide-treated, replicate 3

description:
32_ZEINERetAl
col AA
ZeinerEtAl_allSamples.csv

value: C20 microglia cell line

value: temozolomide


title: SRE, Cut&Run,  Ring1b, Rep4

value: erythroblast

value: SRE


title: ILC2s, PBS treated wild type mice, distal 3/3 small intestine, biol rep 2

description:
Library name: ILC2_PBS_2c
Protocol-Buenrostro et al.


title: SRE, Cut&Run,  Ring1b, Rep2


title: KO_MK-8722_rep4

description:
Library name: SCOP_2023_0255_14
14_S14

value: AMPKa1a2 KO


title: C20, co-culture, Rapamycin-treated, replicate 3

description:
31_ZEINERetAl
col X
ZeinerEtAl_allSamples.csv

value: microglia


title: ILC2s, PBS treated wild type mice, mid 2/3 small intestine, biol rep 2

description:
Library name: ILC2_PBS_2b
Protocol-Buenrostro et al.

value: Small Intestine

value: PBS treated


title: C20, mono-culture, Temozolomide-treated, replicate 3

description:
35_ZEINERetAl
col AJ
ZeinerEtAl_allSamples.csv

value: mono-culture with C20


title: WT_BAY+MK-8722_rep3

description:
Library name: SCOP_2023_0255_12
12_S12

value: Primary hepatocytes

value: Wild Type


title: C20, mono-culture, Vehicle-treated, replicate 2

description:
30_ZEINERetAl
col AL
ZeinerEtAl_allSamples.csv

value: vehicle


title: KO_BAY_rep2

description:
Library name: SCOP_2023_0255_7
7_S7

value: BAY

value: Male

value: C


title: ILC2s, PBS treated wild type mice, distal 3/3 small intestine, biol rep 1

description:
Library name: ILC2_PBS_1c
Protocol-Buenrostro et al.

value: Wild type, C57BL/6J


title: ILC2s, PBS treated wild type mice, proximal 1/3 small intestine, biol rep 2

description:
Library name: ILC2_PBS_2a
Protocol-Buenrostro et al.


title: C20, mono-culture, Temozolomide-treated, replicate 2

description:
29_ZEINERetAl
col AI
ZeinerEtAl_allSamples.csv


title: SRE, Cut&Run,  Ring1b, Rep3

value: untransduced


title: SRE, Cut&Run, Bmi1, Rep3


title: ILC2s, PBS treated wild type mice, proximal 1/3 small intestine, biol rep 1

description:
Library name: ILC2_PBS_1a
Protocol-Buenrostro et al.


title: C20, mono-culture, Rapamycin-treated, replicate 2

description:
28_ZEINERetAl
col AF
ZeinerEtAl_allSamples.csv

value: C20 microglia cell line

value: rapamycin


title: SRE, Cut&Run, Bmi1, Rep2

value: erythroblast


title: KO_DMSO_rep2

description:
Library name: SCOP_2023_0255_5
5_S5

value: DMSO

value: A


title: SRE, Cut&Run, Bmi1, Rep1

value: SRE


title: ILC2s, PBS treated wild type mice, mid 2/3 small intestine, biol rep 1

description:
Library name: ILC2_PBS_1b
Protocol-Buenrostro et al.

value: Small Intestine


title: prox gut c03

city: Rennes

first: Fatima

last: SMAGULOVA

address: 9 av. Prof. Léon Bernard

country: France

institute: Irset - U1085

description: Library name: FMSV438

data_processing:
Image analysis and base calling were performed using RTA 2.7.7 and bcl2fastq 2.20.0.422.
Reads were mapped onto rn7 assembly of rat genome using Hisat2 (v2.1.0).
For each biological replicate, a transcriptome was generated using featureCounts and Gene annotation file ncbiRefSeq.gtf.gz from UCSC
Differentially expressed genes were determined by DESeq2 (Version 2.11.40.7). All analyses were performed using Galaxy platform, Nucleic Acids Research, Volume 50, Issue W1, 5 July 2022, Pages W345–W351
Assembly: rn7
Supplementary files format and content: tab-delimited tex file include raw Counts for each sample

organism: Rattus norvegicus

source_name: proximal colon

growth_protocol: Methods. Sprague-Dawley rats were exposed to nicotine during gestation, and the proximal colon of male progeny was analyzed at 11 weeks of age. Transcriptomics analysis of the proximal colon was carried out by paired-end RNA sequencing.

extract_protocol:
The extraction was performed using RNeasy Plus Mini Kit 250 (QIAGEN™). Approximately 30 mg of each tissue was used. Proximal colon samples were lysed and homogenized using TissueLyser (QIAGEN™). Once homogenized, the RNA extraction protocol was followed. The additional DNA extraction step was performed using RNase-Free DNase Set (Qiagen, 79254).
The libraries were prepared using the NEBNEXT® Ultra II Directional RNA Library kit for Illumina® (NEB E7760S) using 1µg of total RNA according to the manufacturer’s instructions. RNA-Seq (2x100bp)

treatment_protocol: Specific pathogen-free male and female Sprague Dawley rats were purchased from Envigo (USA). Females were purchased at 8 weeks of age and were allowed 1 week to acclimate. At 9 weeks of age, females were randomly assigned to one of 4 treatment groups. Two groups of females were mated, and two groups of females remained virgins. Mating included placing a single female overnight with a male and examination on the following morning for the presence of sperm via vaginal swabs. If sperm was present, the dam was weighed and placed in a separate cage, and this period was marked as gestational day (GD) 0. Using a sterile technique, a 28-day osmotic mini-pump (2ML4-ALZET, Durect Corp., Cupertino, CA, USA) filled with either sterile saline (control; CON) or a nicotine tartrate (6 mg/kg; NIC), dissolved in sterile saline was placed subcutaneously between the scapulae, as described before (Boychuk and Hayward 2011)(Chang, Karatayev, and Leibowitz 2013). On GD19, or 13 days following pump surgery, dams were allowed to give birth. The proximal colon of male progeny was analyzed at 11 weeks of age.

value: proximal colon

tag: strain

value: Sprague-Dawley

value: male

tag: age

value: 11 weeks

tag: group

value: control


title: C20, co-culture, Rapamycin-treated, replicate 2

description:
25_ZEINERetAl
col W
ZeinerEtAl_allSamples.csv

value: microglia

value: co-culture with LNT-229 glioma cell line


title: prox gut c02

description: Library name: FMSV437

tag: tissue

value: vehicle


title: KO_BAY+MK-8722_rep2

description:
Library name: SCOP_2023_0255_8
8_S8

data_processing:
The sequencing analysis pipeline nf-core [1] was used, with alignment and quantification selection of STAR and Salmon, and mouse GRCm38 set as the reference genome.
UMI-based deduplication was performed using UMI-tools and sorting and index alignments were completed with SAMtools as part of the pipeline.
Assembly: GRCm38
Supplementary files format and content: Tab-seperated file containing the raw gene counts for each sample. Rows specify genes by ENSEMBL_ID and SYMBOL, and columns specify the sample

extract_protocol:
RNA was isolated using QIAwave RNAeasy kit (CAT #74104) with on-column DNAse treatment using Qiagen RNase-Free DNase set (CAT #79256) following manufacturer’s protocol.
Libraries were prepared using the Universal Plus mRNA-seq protocol (Tecan) as recommended by the manufacturer. Libraries were quantified with NuQuant using the CLARIOstar Plate Reader (BMG Labtech), quality checked using a TapeStation instrument (Agilent Technologies) and subjected to 52-bp paired-end sequencing on a NovaSeq 6000 (Illumina).

value: Primary hepatocytes

value: AMPKa1a2 KO

value: Male

value: C


title: WT_BAY_rep1

description:
Library name: SCOP_2023_0255_3
3_S3

value: Wild Type

value: BAY


title: SRE, Cut&Run,  Ring1b, Rep1

value: erythroblast

tag: cell type

value: untransduced


title: KO_MK-8722_rep2

description:
Library name: SCOP_2023_0255_6
6_S6

tag: treatment

value: A


title: WT_MK-8722_rep1

description:
Library name: SCOP_2023_0255_2
2_S2

tag: genotype


title: C20, mono-culture, Vehicle-treated, replicate 1

description:
24_ZEINERetAl
col AK
ZeinerEtAl_allSamples.csv

value: C20 microglia cell line

value: mono-culture with C20


title: MDAMB361 cell line, palbociclib resistant

description:
Library name: MDAMB361_PDR
Bio-Rad ddSEQ

value: cell line

value: breast cancer


title: prox gut c01

description: Library name: FMSV436

value: proximal colon

value: Sprague-Dawley

tag: Sex

value: male

value: 11 weeks

value: control


title: 44035_OB_18_5_TST_A23

city: Nijmegen

first: Joost

last: Martens

state: Nederland

address: Geert Grooteplein 28

department: RIMLS

country: Netherlands

institute: Radboud University

description: Library name: 44035_Exp185_OB_18_5_TST_A23

data_processing:
RNA-seq data was analyzed using the seq2science  pipeline which can be found at: https://github.com/vanheeringen-lab/seq2science (van der Sande M, Frölich S, Schäfers T, Smits JGA, Snabel RR, Rinzema S, van Heeringen SJ. 2023. Seq2science: an end-to-end workflow for functional genomics analysis. PeerJ 11:e16380 https://doi.org/10.7717/peerj.16380)
Assembly: GRCh38
Supplementary files format and content: .tsv file with read counts per gene: GRCh38-counts.tsv

source_name: blood

growth_protocol: Primary cells

extract_protocol:
At day 6, cells were harvested by scraping, centrifuged, and resuspended in 350 μl of Lysis Buffer RA1 from the NucleoSpin RNA kit (Macherey-Nagel) and saved at -80°C until RNA isolation. RNA was isolated using the NucleoSpin RNA kit following the manufacturer’s instructions.
Total RNA was used for the preparation of the RNA sequencing libraries using the KAPA RNA HyperPrep Kit with RiboErase (KAPA Biosystems). Oligo hybridization and rRNA depletion, rRNA depletion cleanup, DNase digestion, DNase digestion cleanup, and RNA elution were performed according to protocol. Fragmentation and priming were performed at 94°C for 6 min. First strand synthesis, second strand synthesis and A-tailing was performed according to the protocol. For the adaptor ligation, a 1.5 µM stock was used (NextFlex DNA barcodes, Bio Scientific). First and second post-ligation cleanup was performed according to protocol. A total of 11 PCR cycles were performed for library amplification. The library amplification cleanup was done using a 0.8x followed by a 1.0x bead-based cleanup. Library size was determined using the High Sensitivity DNA bioanalyzer kit, and the library concentration was measured using the dsDNA High Sensitivity Assay (Denovix). Paired-end sequencing of reads of 50 bp were generated using an Illumina NextSeq 500.

treatment_protocol: Adherent PBMCs of 5 healthy individuals were trained with 5 μg/mL BCG, 50 µM testosterone (TST, dissolved in ethanol), both or control (EtOH) according to in vitro trained immunity protocols.

value: blood

value: monocyte

value: TST


title: prox gut N03

description: Library name: FMSV435

value: treatment

value: nicotine 6mg/kg/bw


title: 44037_OB_20_5_BCGTST_A25

description: Library name: 44037_Exp185_OB_20_5_BCGTST_A25

value: BCG+TST


title: SRE, Cut&Run, Bmi1, Rep4

value: erythroblast

value: SRE

value: untransduced


title: C20, co-culture, Vehicle-treated, replicate 2

description:
27_ZEINERetAl
col AC
ZeinerEtAl_allSamples.csv

value: C20 microglia cell line

value: microglia

value: co-culture with LNT-229 glioma cell line

value: vehicle


title: WT_BAY+MK-8722_rep1

description:
Library name: SCOP_2023_0255_4
4_S4

value: Primary hepatocytes

value: Wild Type

tag: mouse id

value: Male

value: A


title: 44034_OB_17_5_EtOH_A22

description: Library name: 44034_Exp185_OB_17_5_EtOH_A22

value: blood

value: EtOH


title: C20, mono-culture, Temozolomide-treated, replicate 1

description:
23_ZEINERetAl
col AH
ZeinerEtAl_allSamples.csv

value: mono-culture with C20

value: temozolomide


title: WT_DMSO_rep1

description:
Library name: SCOP_2023_0255_1
1_S1

value: DMSO

tag: mouse age


title: 44036_OB_19_5_BCGEtOH_A24

description: Library name: 44036_Exp185_OB_19_5_BCGEtOH_A24

value: monocyte

value: BCG+EtOH


title: C20, co-culture, Temozolomide-treated, replicate 2

description:
26_ZEINERetAl
col Z
ZeinerEtAl_allSamples.csv


title: 44033_OB_16_4_BCGTST_A21

description: Library name: 44033_Exp185_OB_16_4_BCGTST_A21

value: BCG+TST


title: SLIT2-knockout, H6

city: Xiamen

first: zuoxing

last: wu

address: Xiang’an South Road

country: China

institute: Xiamen university

description:
Library name: H6
ko3

data_processing:
CLC Genomics Workbench y 11 .0.1
Assembly: GRCm39.107
Supplementary files format and content: tab-delimited text file includes raw counts for each Sample

source_name: sympathetic ganglion

extract_protocol:
Total RNA was extracted from cultured cells or tissues using Trizol (Invitrogen, 15596026), as directed by the manufacturer.
RNA was harvested using Rneasy mini plus kit (Qiagen)1.5 ug of total RNA was used for the construction of sequencing libraries

value: sympathetic ganglion

value: neuron


title: C20, mono-culture, Rapamycin-treated, replicate 1

description:
22_ZEINERetAl
col AE
ZeinerEtAl_allSamples.csv

value: C20 microglia cell line

value: microglia

value: rapamycin


title: prox gut N02

description: Library name: FMSV434

value: proximal colon

value: Sprague-Dawley

value: male

value: 11 weeks

value: treatment

value: nicotine 6mg/kg/bw


title: SLIT2-knockout, H5

description:
Library name: H5
ko2


title: 44032_OB_15_4_BCGEtOH_A20

description: Library name: 44032_Exp185_OB_15_4_BCGEtOH_A20

value: blood


title: 44031_OB_14_4_TST_A19

description: Library name: 44031_Exp185_OB_14_4_TST_A19

value: TST


title: C20, co-culture, Temozolomide-treated, replicate 1

description:
20_ZEINERetAl
col Y
ZeinerEtAl_allSamples.csv

value: co-culture with LNT-229 glioma cell line

value: temozolomide


title: SLIT2-knockout, H4

description:
Library name: H4
ko1


title: prox gut N02

description: Library name: FMSV522

data_processing:
Image analysis and base calling were performed using RTA 2.7.7 and bcl2fastq None.
The reads were mapped to the reference genome rn7 using BWA v.0.7.18. The numbers of mapped reads were normalized by a scale factor to adjust the total number of reads.
From the aligned reads, DNA methylated peaks were identified using 4 biological replicates and the input (from  GSE253596) by MACS2 (2.2.9.1) with  the following parameters:  shift-size window of 73 bp, --nomodel, and q-value threshold <0.05.
To compare the control datasets of the nicotine exposed and control samples, differential peaks were identified using counting reads at each peak using bedtools MultiCovBed (Version 2.30.0). Statistical significance was calculated using Deseq2  with filtering peaks with low counts.
Each normalised bam file was converted to BedGraph tracks by using the Genome Coverage 2.30.0. All analyses were performed using the Galaxy platform, Nucleic Acids Research, Volume 50, Issue W1, 5 July 2022, Pages W345–W351.
Assembly: rn7
Supplementary files format and content: bedgraph

growth_protocol: Methods. Sprague-Dawley rats were exposed to nicotine during gestation, and the proximal colon of male progeny was analyzed at 11 weeks of age.

extract_protocol:
DNA was extracted from 4 biological replicates using DNA Easy Blood and Tissue Kit (Qiagen, 69506) according to instructions provided by the manufacturer. DNA concentration was measured by the fluorescent method using a fluorescent reader (Promega). 10 µgs of DNA was used for MeDIP using methylated DNA Enrichment Kit (NEB, #E2600S). DNA was diluted and sonicated in Qsonica sonicator using the following conditions: 20 s pulse on, 20 sec pulses off, and a total sonication time of 8 min. After that, sonicated DNA was incubated with MBD2-Fc -protein A Magnetic beads. The beads were washed 4 times with 1XBind/Wash Buffer. The enriched methylated DNA was eluted from beads with MilliQ water, and the concentration of methylated DNA was again determined by fluorescence.
6 ng of methylated DNA or input were taken for library preparation. Sequencing libraries were prepared using the NEBNext Ultra DNA Library Prep Kit for Illumina (E7645S; NEB). We performed 15 cycles for the library amplification.

treatment_protocol: Specific pathogen-free male and female Sprague Dawley rats were purchased from Envigo (USA). Females were purchased at 8 weeks old and allowed 1 week to acclimate. At 9 weeks of age, females were randomly assigned to one of 4 treatment groups. Two groups of females were mated, and two groups of females remained virgins. Mating included placing a single female overnight with a male and examination on the following morning for the presence of sperm via vaginal swabs. If sperm was present, the dam was weighed and placed in a separate cage, and this period was marked as gestational day (GD) 0. Using a sterile technique, a 28-day osmotic mini-pump (2ML4-ALZET, Durect Corp., Cupertino, CA, USA) filled with either sterile saline (control; CON) or a nicotine tartrate (6 mg/kg; NIC), dissolved in sterile saline was placed subcutaneously between the scapulae, as described before (Boychuk and Hayward 2011)(Chang, Karatayev, and Leibowitz 2013). On GD19, or 13 days following pump surgery, dams were allowed to give birth. The proximal colon of male progeny was analyzed at 11 weeks of age.


title: prox gut N01

description: Library name: FMSV433


title: SLIT2f/f, H3

description:
Library name: H3
wt3


title: 44030_OB_13_4_EtOH_A18

description: Library name: 44030_Exp185_OB_13_4_EtOH_A18

value: monocyte

value: EtOH


title: 44029_OB_12_3_BCGTST_A17

description: Library name: 44029_Exp185_OB_12_3_BCGTST_A17

value: BCG+TST


title: C20, co-culture, Rapamycin-treated, replicate 1

description:
19_ZEINERetAl
col V
ZeinerEtAl_allSamples.csv


title: prox gut N01

description: Library name: FMSV521


title: 44028_OB_11_3_BCGEtOH_A16

description: Library name: 44028_Exp185_OB_11_3_BCGEtOH_A16

value: BCG+EtOH


title: SLIT2f/f, H1

description:
Library name: H1
wt1

value: sympathetic ganglion

value: neuron


title: prox gut c02

description: Library name: FMSV518

value: control

value: vehcle


title: prox gut c01

description: Library name: FMSV517

value: proximal colon

value: Sprague-Dawley


title: SLIT2f/f, H2

description:
Library name: H2
wt2


title: HMC3, mono-culture, Temozolomide-treated, replicate 3

description:
17_ZEINERetAl
col R
ZeinerEtAl_allSamples.csv

source_name: HMC3 microglia cell line

value: HMC3 microglia cell line

value: microglia

value: mono-culture with HMC3


title: LSC_Ruvbl2_2

city: Chongqing

first: Zhe

address: 1 Yixueyuan Road, Yuzhong District

institute: Chongqing Medical University

data_processing:
The quality control of each sample was accomplished using FASTQC V0.11.5.
Clean reads were aligned to mouse reference genome (mm10) using Bowtie 2 version 2.2.9. MarkDuplicates was used to mark PCR duplicate and unmapped reads. Only unique mapped reads were used in further analysis.
Peak calling and annotation were performed using MACS2 and ChIPseeker package, respectively.
Assembly: mm10
Supplementary files format and content: bigWig
Supplementary files format and content: narrowPeak
Library strategy: CUT&Tag

source_name: Bone marrow

extract_protocol:
The CUT&Tag assay was performed using NovoNGS CUT&Tag 3.0 High-Sensitivity Kit (Novoprotein) according to the manufacturer’s protocol. 2,000-5,000 HSCs were harvested and resuspended in 45 µL wash buffer. Then 6 µL ConA beads were added to the sample and incubated at 37°C for 10 min. Then the supernatant was removed by magnet adsorption and the beads were resuspended in 50 µL antibody buffer (20 mM Hepes, pH 7.5, 150 mM Nacl, 0.5 mM Spermidine, 0.0125% Digitonin, 2 mM EDTA, 0.1% BSA, 1× PIC).
Then, 0.5 µL antibody was added to the sample and incubated at 4°C overnight. The supernatant was removed by magnet adsorption and the beads were resuspended in 50 µL secondary antibody buffer, containing 0.5 µL goat anti-rabbit IgG antibody. After incubation at 37°C for 60 min, the beads were resuspended in 50 µL ChiTag Buffer, containing 0.5 µL ChiTag transposome. After incubation at 37°C for 60 min, the beads were resuspended in 50 µL tagmentation buffer. Then, 1 µL 10% SDS was added to the sample and incubated at 55°C for 10 min, and DNA was extracted using 2×DNA clean beads (Vazyme) and amplified with i5 and i7 primers (Vazyme) by 17-20 PCR cycles. The libraries were purified by 1.2×DNA clean beads (Vazyme).

treatment_protocol: HSCs (CD48-CD150+LSK) from Nup214fl/+ mice and LSCs (Lin-Sca-1-c-Kit+CD16/32+CD34+) from MLL-AF9-driven Nup214fl/+ mice were sorted, followed by Ruvbl2-CUT&Tag-seq

value: Bone marrow

value: Hematopoietic stem cell

tag: cut&tag antibody

value: Ruvbl2 (67851-1-Ig,Proteintech)


title: iSRE, Cut&Run, Ring1b, Rep5

value: erythroblast

value: iSRE


title: 44027_OB_10_3_TST_A15

description: Library name: 44027_Exp185_OB_10_3_TST_A15

value: blood

value: monocyte

value: TST


title: HMC3, mono-culture, Rapamycin-treated, replicate 3

description:
16_ZEINERetAl
col O
ZeinerEtAl_allSamples.csv

value: rapamycin


title: C20, co-culture, Vehicle-treated, replicate 1

description:
21_ZEINERetAl
col AB
ZeinerEtAl_allSamples.csv

value: C20 microglia cell line

value: co-culture with LNT-229 glioma cell line

value: vehicle


title: Scan 38

city: Memphis

first: Jasmine

last: Plummer

state: TN

address: 262 Danny Thomas Pl

department: Developmental Neurobiology

institute: St. Jude Children's Research Hospital

hyb_protocol: The Imaging based platforms used in this study are the CosMX SMI (Nanostring) and the Xenium Analyzer (10X Genomics) with all specifications required for this study outlined in the CosMX SMI was used for both RNA and protein assays. All sections were also stained after data acquisition on their respective imaging platform. The specifics for each assay is outlined in the proceeding sections and found in STSOP. All methods were used across the three institutions: site #1 (UoA), site #2 (St. Jude Children’s Research Hospital), and site #3 (Weill-Cornell Medicine).  CosMx RNA Slide Preparation: Samples were processed following the guidelines outlined in the CosMx slide preparation manual MAN-10159-02 for prostate cancer processed in Adelaide and MAN-10184-01-1 for all other tissues. In summary, 5-μm sections were placed onto the 15 mm x 22 mm scannable area of a Superfrost Plus Micro Slide (VWR, 48311-703) and dried in an oven at 37°C for 2 hours then kept at room temperature with desiccant overnight before storage at 4°C. The slides were then incubated overnight at 60°C for 17–18 hours and then brought to room temperature for 3–5 minutes. This was followed by two xylene washes for 5 minutes each, and two washes in 100% ethanol for 2 minutes each. For normal tissues, CitroSolv workflow was used in place of xylene. The slides were then dried in an oven at 60°C for 5 minutes. The slides were immersed in pre-heated 1X Target Retrieval solution (CosMx FFPE slide preparation kit, RNA) in a pressure cooker (or steamer) at 100°C for 15 minutes, timing started once the retrieval solution reached 100°C. The slides were immediately transferred to water for 15 seconds, then washed in 100% ethanol for 3 minutes, and air-dried at room temperature for 30 minutes to 1 hour. Incubation frames were attached to each slide, and a pre-warmed digestion buffer containing 3 ug/mL Proteinase K (CosMx FFPE slide preparation kit, RNA) and 1X PBS (AM9922, AM9625 was applied and incubated in a hybridization chamber for 30 minutes. The slides were then rinsed twice in water, and fiducials were applied at 0.001%, followed by a 5-minute incubation at room temperature, shielded from light. The slides underwent a 1X PBS wash for 1 minute, were fixed in 10% NBF for 1 minute, immediately followed by two washes in NBF stop buffer for 5 minutes each, and then a 5-minute wash in 1X PBS. A 100 mM NHS-acetate solution was applied to the tissue for 15 minutes at room temperature, followed by two washes in 2X SSC for 5 minutes each. The CosMx Human Universal Cell Characterization RNA Panel (CMX-H-USCP-1KP-R) targeting 950 human genes and a 50-target add-on panel set, were denatured at 95°C for 2 minutes and then cooled on ice for 1 minute. A probe mix containing RNase inhibitor, buffer R, and nuclease free water was applied to the slide and incubated for 17 hours in the hybridization chamber at 37°C. Post-incubation, the slides were washed twice in a final concentration of 50% deionized formamide and 2X SSC mix for 25 minutes each, followed by two washes in 2X SSC for 2 minutes each. DAPI stock was diluted to 1:40 and applied to the slides for 15 minutes at room temperature, protected from light. The slides were then washed in 1X PBS for 5 minutes and stained for 1 hour with a cocktail of CD298/B2M, PanCK, CD45, and CD68 antibodies (Item # 121500020, 121500021, 121500022). Colon, HCC TMA, pancreas, and prostate cancer samples were stained with 4 uL of the CD298/B2M antibody, following the guidelines in MAN-10184-01-1. In contrast, appendix, ileum, and prostate cancer samples received a staining of 8 uL of the CD298/B2M antibody, as specified in MAN-10159-01-1.The slides were washed three times in 1X PBS for 5 minutes each and then stored in 2X SSC. The pre-bleaching profile followed configuration C, while the cell segmentation profile adhered to configuration A, except for the HCC TMA, which used configuration E. These procedures were consistently applied across all sites: St. Jude Children's Research Hospital, Weill Cornell Medical Center, and the University of Adelaide, with FOVs remaining uniform across all three locations.  CosMx Protein Slide Preparation: Samples were processed according to the guidelines provided in the CosMx SMI slide preparation manual MAN-10185-01-1 (NanoString). In summary, 5-μm FFPE sections were placed onto the 15 mm x 22 mm scannable area of a Superfrost Plus Micro Slide and dried in an oven at 37°C for 12 hours, sealed with desiccant and dried for a further 12 hours at room temperature before storage at 4°C. The slides were baked at 65°C for 2 hours and then equilibrated to room temperature for 3–5 minutes, and then washed twice in xylene (or CitriSolv) for 5 minutes each at room temperature. The slides were then washed twice in 100% ethanol for 10 minutes each, twice in 95% ethanol for 5 minutes each, and once in 70% ethanol for 5 minutes, followed by two washes in 1X PBS for 5 minutes each. The slides were immersed in pre-heated 1X Target Retrieval solution and heated in a pressure cooker at 100°C with the valve closed for 15 minutes, then allowed to equilibrate to room temperature in the same solution. Afterward, the slides were washed three times in 1X PBS for 5 minutes each, and incubation frames were attached. Tissue sections were then covered with Buffer W and incubated at room temperature for 1 hour in the dark. The NanoString 64-plex human protein antibody panel was combined with CD298/B2M and PanCK/CD45 segmentation markers in Buffer W. Samples from the appendix, ileum, and pancreas were also incubated with the CD68 segmentation marker. The primary antibody mix was incubated at 4°C for 17 hours, followed by three washes with 1X TBS-T buffer for 10 minutes each and once with 1X PBS for 2 minutes. Fiducials, prepared at the recommended concentration of 0.00005%, were applied to the slides at room temperature for 5 minutes, protected from light. Slides were then washed once with 1X PBS for 5 minutes and fixed with 4% PFA for 15 minutes. This was followed by three washes in 1X PBS for 5 minutes each. Sections were stained with nuclei stain for 10 minutes, washed twice with 1X PBS for 5 minutes, and incubated with 100 mM NHS acetate for 5 minutes, before a final wash in 1X PBS for 5 minutes. The selected pre-bleaching profile was configuration C, and the cell segmentation profile was configuration A. These procedures were uniformly followed at St. Jude Children's Research Hospital, Weill Cornell Medical Center, and the UoA, ensuring consistency in the FOVs across all three locations. Breast cancer, normal appendix, and normal ileum samples processed as part of the NanoString’s Technology Access Program followed the methods previously described (He et al,. Nat Biotechnol, 2022).  Xenium Slide Preparation: Sections for Xenium were prepared according to the Xenium In Situ for FFPE-Tissue Preparation Guide (CG000578 Rev C, 10X Genomics) protocol. Briefly, 5-μm sections were cut, floated in an RNAse-free water bath, and carefully placed onto the 10.45 mm x 22.45 mm sample area of Xenium slides (PN-3000941, 10X Genomics). The slides were then placed on a drying rack at room temperature for 30 minutes to remove excess water. This was followed by a 3-hour incubation at 42 ̊C on a Xenium Thermocycler Adapter plate, positioned atop the 96-well block of a C1000 Touch Thermal Cycler (BioRad) with the lid open. Subsequently, the slides were stored overnight at room temperature with a desiccant to facilitate further drying. The slides were then processed following the Xenium In Situ for FFPE Deparaffinization and Decrosslinking protocol (CG000580 Rev C, 10X Genomics). The slides were placed again on a Xenium Thermocycler Adapter plate atop the 96-well block of a thermocycler with the lid open and incubated at 60 ̊C for 2 hours. They were then equilibrated to room temperature for 7 minutes, deparaffinized, and rehydrated by immersion twice in xylene for 10 minutes each, twice in 100% ethanol for 3 minutes each, twice in 96% ethanol for 3 minutes each, once in 70% ethanol for 3 minutes, and once in water for 20 seconds. Subsequently, the Xenium slides were assembled into Xenium cassettes and hydrated with 1X PBS. The tissue sections were reverse crosslinked using a decrosslinking buffer containing tissue enhancer, urea, and perm enzyme B, at 80 ̊C for 30 minutes, followed by three washes with PBS-T. Slides were then immediately processed following the Xenium In Situ Gene Expression user guide (CG000582 Rev E, 10X Genomics) for the remaining slide preparation steps. Briefly, the pre-designed gene expression probe set, Xenium Human Multi-Tissue and Cancer Panel (10X-1000626, 10X Genomics) targeting 377 human genes, was utilized for prostate cancer, ileum, appendix, pancreas, and colon samples. For breast cancer samples, the Xenium Human Breast Panel (10X-1000463, 10X Genomics) targeting 380 human genes was used. Panels were denatured at 95 ̊C for 2 minutes, crash-cooled on ice for 1 minute, and then equilibrated to room temperature before being added to the probe hybridization buffer and TE buffer to make the probe hybridization mix. The slides were incubated with the hybridization mix at 50 ̊C for 17 hours. Slides were washed three times with PBS-T for 1 minute each then incubated with a post-hybridisation wash buffer for 30 minutes at 37 ̊C. Slides were washed three times with PBS-T for 1 minute each and incubated with ligation mix for 2 hours at 37 ̊C. Three 1-minute PBS-T washes were followed by a 2-hour incubation at 30 ̊C with an amplification mix. Slides were then washed three times with TE buffer then three washes with 1X PBS for 1 minute each followed by incubation with reducing agent B for 10 minutes, washed once in 70% ethanol for 1 minute, twice in 100% ethanol then treated with autofluorescence solution for 10 minutes in the dark. Slides were washed three times with 100% ethanol for 2 minutes each then dried for 5 minutes at 37 ̊C before rehydrating with 1X PBS then PBS-T for 2 minutes. Slides were stained with a nuclei staining buffer for 1 minute at room temperature followed by three washes with PBS-T for 1 minute each in the dark. The slides were then assembled into Xenium Cassettes and loaded into the Xenium Analyzer for imaging and decoding. These procedures were uniformly followed at St. Jude Children's Research Hospital, Weill Cornell Medical Center, 10x Genomics, and the UoA, ensuring consistency in theFOVs across all four locations. Breast cancer samples processed as part of the 10x Genomics’ Catalyst Program followed the methods previously described (19); these experiments were carried out at 10X Genomics headquarters (Pleasanton, CA, USA). Sections from the prostate cancer samples were also subjected to Xenium Multimodal segmentation through an early access program and processed at the 10X Genomics headquarters (Pleasanton, CA). In brief, the procedure for in situ gene expression with cell segmentation staining was conducted following the Demonstrated Protocol for Xenium In Situ for FFPE - Tissue Preparation Guide (CG000578 Rev C, 10X Genomics), the Xenium In Situ Gene Expression User Guide (CG000582 Rev F, 10X Genomics), and the Xenium In Situ Gene Expression Cell Segmentation User Guide (CG000749 Rev A, 10X Genomics). The combined protocol utilized the Xenium Human Multi-Tissue and Cancer Panel for in situ gene expression and the Xenium Cell Segmentation Staining Reagents add-on (PN-1000661, 10X Genomics) for segmentation staining. This add-on kit contains a single cocktail of Xenium Multi-Tissue Stain Mix (PN-2000991, 10X Genomics), which contains a cocktail of antibodies labeling the membranes (anti-ATP1A1/CD45/E-cadherin), antibodies labeling the cell interior (anti-alphaSMA/Vimentin) and a universal interior label against Ribosomal RNA (18S rRNA). Stain was then enhanced by the addition of Xenium Staining Enhancer reagent, followed by treatment of the sections with Xenium Autofluorescence Mix (PN-2000753, 10X Genomics) to diminish unwanted autofluorescence and enhance the signal-to-noise ratio in the tissue sections. Subsequently, Xenium Nuclei Staining Buffer (PN-2000762, 10x Genomics) was used to help the identification of tissues or regions of interest during the instrument's overview scan.  Xenium RNA to CosMx Protein slide preparation: Following completion of the Xenium RNA run, slides were removed from the instrument and stored in the Xenium cassette with PBS-T at 4C. After storage for up to 3 days, slides were washed three times in PBS-T for 1 minute each as recommended in Technical Note: Post-Xenium In Situ Applications: Immunofluorescence, H&E, and Visium CytAssist Spatial Gene Expression (CG000709 Rev A, 10X Genomics), removed from the Xenium cassette and placed into a wash jar of 1X PBS for 5 minutes as described in CosMx SMI Manual Slide Preparation for Protein Assays (MAN-10185-01-1, NanoString), incubation frames were attached to the Xenium slide and tissue sections were covered with Buffer W and incubated at room temperature for 1 hour in the dark. Afterwards, MAN-10185-01-1 and MAN-10161-03-2 were followed as previously described in CosMx Protein slide preparation and CosMx SMI setup and data acquisition.

scan_protocol: CosMx SMI Setup and Data Acquisition: The setup and scan acquisition for the CosMx SMI instrument were carried out as detailed in the CosMx SMI Instrument User Manual   (MAN-10161-02-1 or MAN-10161-03-2, Software Version 1.1, NanoString). A new acquisition process was initiated through the CosMx SMI Control Center web interface. Prior to insertion into the CosMx Flow Cell Assembly Tool, the slides were carefully dried in the areas surrounding the imaging region. The process involved lowering the tailgate, placing the slide, and then securing it by raising the tailgate. After removing the adhesive backing, a new flow cell coverslip was precisely aligned above the slide's imaging area. The Assembly Tool's lid was closed securely to affix the coverslip to the slide, thus creating a functional CosMx flow cell. Following the assembly, 2X SSC was gently introduced through one of the flow cell ports to hydrate the tissue section. Subsequent steps included entering the flow cell configuration data into the Control Center interface, which comprised the flow cell's barcode and the slide's ID number. The prostate sections underwent scanning with Configuration C for the pre-bleaching profile and Configuration A for the cell segmentation profile. Further details regarding the probe panel, cell segmentation, and supplemental markers were entered into the flow cell configuration data for each section. Assembled flow cells/slides, along with Buffer Bottles 1–4, were then loaded into the instrument. Before positioning Bottle 4, catalase and pyranose oxidase enzymes were added. The Control Center configuration was verified and following a period of equilibration to room temperature and the addition of an RNase inhibitor to a designated well in the CosMx imaging tray, the tray was placed in the instrument. A pre-run check and a tissue find scan for each tissue section were performed by the instrument. After the completion of tissue find scans, rectangular scan areas were delineated around each tissue section for the preview scan. The preview scan images facilitated the selection of a contiguous region of FOVs using the grid FOV placement tool, thoroughly covering the tissue areas on each slide. FOV selections for each slide were confirmed before initiating the cycling process. CosMx scan data were automatically uploaded to NanoString’s cloud-based AtoMx Spatial Analysis Platform during the run, as detailed in the CosMx Data Analysis manual (MAN-10162-02, Software Version 1.1, NanoString). Briefly, upon the completion of the run and the full upload of scan data to AtoMx, a study was created for each CosMx scan. Within AtoMx, pipelines were executed for each study, facilitating the export of data in various formats, such as TileDB arrays and Seurat objects, for in-house analysis.     Xenium Analyzer Setup and Data Acquisition: Xenium slides, assembled within Xenium cassettes, were imaged using the Xenium Analyzer, in accordance with the guidelines specified in the Xenium Analyzer User Guide (CG000584 Rev D and Rev E for Multi-modal Segmentation-stained slides, 10X Genomics). The Xenium Decoding Consumables Kit, (PN-1000487, 10X Genomics) was used for instrument loading. Briefly, Xenium slide ID numbers and information on pre-designed gene expression probes—were input into the Analyzer. The necessary consumables and reagents were loaded into the instrument: reagent module B, thawed at 4 ̊C overnight and equilibrated to room temp for 30 minutes prior to loading into the instrument, reagent module A, stored at 4 ̊C until loading in the instrument, instrument wash buffer (100% Milli-Q water), sample wash buffer A (1X PBS, 0.05% Tween-20), sample wash buffer B (100% Milli-Q water), and probe removal buffer (50% DMSO, 50 mM KCl, 0.1% Tween-20) were mixed prior to loading into the instrument with buffer caps along with, pipette tip rack, extraction tip, objective wetting consumable. After loading, the sample was scanned to generate images of the fluorescently labeled nuclei in each section which was used for FOV selection prior scanning. For the prostate cancer sample, the entire section was included in the full scan as 4 regions were selected for the appendix. After the run, the instrument was cleared of consumables, and the Xenium slides were carefully removed. Fresh PBS-T was applied to each slide/cassette, which were then   covered and stored in the dark at 4°C until post-run H&E staining. All procedures were standardized across three sites. The data were then acquired using the Xenium In Situ software suite (v1.9, 10X Genomics), which is a set of software applications for analyzing and visualizing in situ gene expression data produced by the Xenium Analyzer. For prostate samples stained with Multi-Modal Cell Segmentation reagents (10X Genomics Headquarters), both in situ gene expression and segmentation data were simultaneously collected and processed using v2.0 of the Xenium In Situ software suite.

data_processing:
To ensure a fair comparison, cells without detected transcripts were excluded from the analysis following the Xenium Onboard Analysis or the AtoMx processing workflow. Subsequent analyses, including cell type annotation, were conducted using raw transcript counts
Processed data: Transcript counts derived from intensity values within the boundaries of segmented cells.

source_name: Normal pancreas

label_protocol: CosMx and Xenium barcoded probes for RNA and protein assays.

extract_protocol: To confirm the RNA quality of all the samples included in this study three consecutive 10-μm scrolls for the isolation and purification of total RNA using the Norgen Biotek FFPE RNA Purification Kit (Product ID 25300). RNA material (5 ng) was analyzed using a 5200 Fragment Analyzer with the High Sensitivity RNA Assay kit (DNF-472, Agilent Technologies). DV200 values across all samples ranged between 50%–70%. Single Nucleus RNA sequencing was performed following the snPATHO-Seq protocol (Wang et al. BioRxiv, 2023). Briefly, two 30- to 50-μm-thick scrolls from each sample, adjacent to sections for CosMx, Xenium, and H&E staining, were cut and placed into 1.5-mL Eppendorf tubes and stored at 4°C inside Ziplock bags with desiccants to control moisture. Scrolls were stored for up to 1 week. For the preparation of nuclei suspensions, scrolls sections underwent a triple wash in 1 mL of xylene (214736, Millipore Sigma) for 10 minutes each to dewax, followed by rehydration through sequential ethanol (E7023, Millipore Sigma) immersions of decreasing concentrations (100% twice, then 70%, 50%, and 30% once each), each for a duration of 1 minute. Of note, the initial xylene wash was performed at 55°C for 10 minutes to enhance paraffin removal. Subsequent to rehydration, a single wash was performed using 1 mL of wash RPMI 1640 media (11875093, Gibco). After removing the RPMI1640 wash, 50–100 uL of digestion mix [RPMI1640 supplemented with 1 mg/mL of Liberase TH (5401151001, Millipore-Sigma), along with 1 U/uL RNAse Inhibitor (RiboLock RNase Inhibitor, EO0382, Thermo Fisher Scientific)] was added to the tissue scrolls breaking them to smaller pieces with a 1.5-mL pellet pestle before topping up to 1 mL with the same digestion media. Digestion was carried out in a Thermomixer at 800 rpm. After digestion, 400 uL of Ez Lysis Buffer (NUC-101, Sigma-Aldrich) was added, mixed by inversion five times, and centrifuged at 850xRCF for 5 minutes at 4°C. The pellet, consisting of released nuclei and undigested tissue, was resuspended in 250 uL of Ez Lysis buffer with 2% BSA (130-091-376,  MACS BSA Stock Solution, Miltenyi) and 1 U/uL RNAse Inhibitor, followed by homogenization using a 1.5-mL pellet pestle. This was supplemented with an additional 750 uL of the same lysis mix, with further disaggregation achieved through pipetting. The sample was then filtered through a 70-μm strainer (pluriStrainer Mini 70um, 43-10070-50, pluriSelect) and centrifuged, with the flow-through washed again with 800 uL of the lysis mix. Nuclei pellet was washed three times in 1x PBS (70011044, Gibco) with 1% BSA, followed by two washes in 0.5x PBS (70011044, Gibco) with 0.02% BSA. Nuclei was finally resuspended in a volume of 500-1000 uL of the same buffer, dependent on pellet size, and filtered through a 40-μm strainer (pluriStrainer Mini 40um, 43-10040-50, pluriSelect. Nuclei were counted using a Luna-FX7 using Acridine Orange/Propidium Iodide Stain (Logos). For immediate analysis, the suspension was kept on wet ice until processing using the Chromium X controller and the Chromium Fixed RNA Profiling Reagent Kits for multiplexed samples (Chromium Fixed RNA Kit, Human Transcriptome, PN1000475; Chromium Next GEM Chip Q Single Cell Kit, PN-1000418; Dual Index Kit TS Set A, PN-1000251, 10x Genomics) and the User guide CG000527, Rev E. For storage, the pre- or post-hybridized nuclei suspensions were supplemented with 10% Glycerol and 0.1X Enhancer (PN-2000482, 10x Genomics), rested on ice for 10 minutes, and then cryopreserved at −80°C. For the hybridization step, between 500,000 and 1.5 million nuclei were used; hybridization was conducted for 20 hours at 42°C across all samples. BC1-4 were used in all samples used in this study. Post-hybridization washes were carried out in a pool-wash setting as per User guide, with an additional wash included. Approximately 10,000 nuclei/sample were aimed for capture. An extra cycle was introduced for the Pre-Amplification PCR step. Indexing PCR conditions were tailored to attain the target library concentration of 50–200 nM. Libraries were checked for quality control and quantified using a Fragment Analyzer 5200 (Agilent) and a Qbit Flex Fluorometer (Thermo Fisher Scientific), respectively. The indexed libraries were sequenced using the Illumina NovaSeq 6000 system. The read configuration was Read 1: 28, i7: 10, i5: 10, and Read 2: 90. Libraries were sequenced to aim for 20,000–30,000 read pairs/nuclei.

treatment_protocol: Normal and cencerous samples used for RNA and/or protein profiling (Xenium and CosMx platforms) as well as for tissue staining (Hematoxylin and Eosin, H&E) and single nuclei RNA-seq (snPATHO-Seq, using Chromium platform) were fixed in 10% neutral buffered formalin (NBF) and parafin embedeed (FFPE). To confirm the RNA quality of all the samples included in this study three consecutive 10-μm scrolls for the isolation and purification of total RNA (DV200 was used as measure of RNA intgrity post BioAnalyzer, TapeStation or Fragment Analyzer run). for snRNA-Seq from FFPE (snPATHO-Seq).

tag: tissue type

value: Normal pancreas

tag: sample source

value: St. Jude Children's Research Hospital

tag: sample processed

value: Weill Cornell Medical Center

tag: sample type

value: FFPE

tag: technology

value: Xenium


title: 44026_OB_9_3_EtOH_A14

description: Library name: 44026_Exp185_OB_9_3_EtOH_A14

value: EtOH


title: LSC_Ruvbl2_1

value: Bone marrow

value: Hematopoietic stem cell


title: Scan 37


title: HSC_Ruvbl2_2

value: Ruvbl2 (67851-1-Ig,Proteintech)


title: 44025_OB_8_2_BCGTST_A13

description: Library name: 44025_Exp185_OB_8_2_BCGTST_A13

value: BCG+TST


title: Scan 36


title: HMC3, co-culture, Temozolomide-treated, replicate 3

description:
14_ZEINERetAl
col I
ZeinerEtAl_allSamples.csv

tag: cell line

value: HMC3 microglia cell line

value: microglia

tag: culture condition

value: temozolomide


title: HSC_Ruvbl2_1


title: 44024_OB_7_2_BCGEtOH_A12

description: Library name: 44024_Exp185_OB_7_2_BCGEtOH_A12

value: blood

value: monocyte

value: BCG+EtOH


title: HMC3, mono-culture, Vehicle-treated, replicate 3

description:
18_ZEINERetAl
col U
ZeinerEtAl_allSamples.csv

value: mono-culture with HMC3

value: vehicle