title: Hashtag derived Oligo (HTO)

city: Hannover

first: Jaba

last: Gamrekelashvili

address: Carl-Neuberg-Str. 1

department: Nephrology

country: Germany

institute: MHH

description:
Library name: Undetermined
Hashtag-derived oligonucleotide (HTO)

data_processing:
The 10x Genomics CellRanger analysis pipeline set (v7.1.0) was used with default parameters. Briefly, BCL files were demultiplexed into FASTQ files by cellranger mkfastq using the respective sample sheet with utilized 10X barcodes. Fastq files of each sample from both sequencing runs were combined. The cellranger count pipeline was used to align read data to the reference genome provided by 10X Genomics (Mus musculus reference dataset refdata-gex-mm10-2020-A), counting aligned reads per gene, and calculating clustering and summary statistics. Subsequently, outputs from cellranger count of all samples were aggregated, normalized to the same sequencing depth, and then the feature-barcode matrices was recomputed by cellranger aggr.
Simultaneously, feature barcoding data was processed during the cellranger count step with a feature reference build according to the specification of TotalSeq™-A antibodies.
The output from cellranger count was demultiplexed to retrieve values for each previously pooled sample. Demultiplexing with hashtag oligos was performed using a Seurat (v.4.0.0) based workflow developed in a collaboration of Dresden-concept Genome Center (TU Dresden) and Research Core Unit Genomics (Hannover Medical School) (https://github.com/ktrns/scrnaseq). Briefly, the workflow (conducted via R version 4.0.2) log-normalized and clustered the HTO counts, classified cells based on normalised HTO data, removed HTO doublet or negative cells, and generated demultiplexed data.
Assembly: gex-mm10-2020-A
Supplementary files format and content: tab-delimited text files includes raw counts for each pooled sample
Supplementary files format and content: tab-delimited text files includes RPKM counts for each pooled sample

molecule: protein

source_name: Mononucler phagocytes and the progenitors

extract_protocol:
Ctrl, N2ΔCx3cr1 and RbpjΔCx3cr1 spleens (n=2 per genotype) were isolated and digested on ice for 30 min. in DMEM medium supplemented with 500 U/ml Collagenase II (Worthington) and 2% FCS. Erythrocytes were removed using red blood cell lysis buffer (BioLegend). Following thorough washing, filtering and counting, single cell suspensions of equal cell numbers (one tube per spleen) were prepared. For sample multiplexing, a cell hashing-based approach was performed using oligo-tagged antibodies (TotalSeqTM-A Hashtag derived Oligos, HTO, #A1-#A6, BioLegend). Blocking was done with anti-mouse CD16/32 (TruStain fcX) and anti-mouse CD16.2, followed by HTO tagging (#A1-#A6) and anti-F4/80-BV650 staining. After thorough washing, cells were pooled and stained with Lineage-PE antibodies (CD3, CD19, B220, Ly6G, Ter119, NK1.1, CD90.2, CD49b, CD117, FcεRIα) and anti-mouse CD11b FITC, CD11c APC and Ly6C PE-Cy7. Following the exclusion of dead (DAPI+) and CD11b/CD11c double-negative cells, Lineage-negative cells were sorted using FACSAria Fusion (BD Biosciences). Collected cells were loaded on one 10x Genomics lane for downstream scRNA-seq processing and analysis. Bone marrow cells from Ctrl, N2ΔCx3cr1 and RbpjΔCx3cr1 mice (n=2 per genotype) were counted and equal numbers were taken from each mouse in separate tubes (one tube per mouse). A cell hashing-based experimental approach via oligo-tagged antibodies (TotalSeqTM –A Hashtag derived Oligos (HTO), #A1-#A6, BioLegend) was used for sample multiplexing (Table S4). After initial blocking (anti-mouse CD16/32 BV510 and anti-mouse CD16.2 antibodies) cells were incubated with individual HTOs (#A1-#A6) and with anti-mouse CD115 APC. After an extensive washing step, cells were pooled, and stained with a cocktail of anti-mouse Lineage-PE (CD90.2, CD3, CD19, B220, Ly6G, Ter119, NK1.1 Ly6A/E), anti-PE beads (Mojosort BioLegend) and Lineage-PE+ cells were depleted using QuadroMACS and LS columns (Miltenyi Biotec). Remaining Lineage-negative cells were stained with anti-mouse CD11b BV650, CD34 PE-Dazzle 594, Ly6C PE-Cy7 and CD117 APC-Cy7 and were sorted using FACSAria Fusion (BD Biosciences). Doublets and DAPI+ cells were excluded from sorted populations. Gates were set to collect four subpopulations of myeloid cells: #1 'Progenitors' (Lineage-negativeCD115+CD11blo/-CD34+CD117+), #2 'Pre-monocytes' (Lineage-negativeCD115+CD11blo/-CD34lo/-CD117lo/-Ly6Chi), #3 'Ly6Chi monocytes' (Lineage-negativeCD115+CD11b+CD34negCD117lo/-Ly6Chi), #4 'Ly6Clo cells' (Lineage-negativeCD115+CD117negLy6Clo/-). These subpopulations, #1 - #4, were mixed with the ratio: 1 : 1 : 1.5 : 2.75 to have representative subpopulations and loaded on one 10x Genomics lane for downstream processing and scRNA-seq.
Library preparation for single cell mRNA-Seq analysis was performed according to the Chromium NextGEM Single Cell 3ʹ Reagent Kits v3.1 CellSurfaceProtein User Guide (Manual Part Number CG000206 Rev D; 10x Genomics). According to the protocol a given excess of cells was loaded to the 10x controller in order to reach a target number of 25,000 cells per sample. Fragment length distribution of generated libraries was monitored using ‘Bioanalyzer High Sensitivity DNA Assay’ (5067-4626; Agilent Technologies). Quantification of libraries was performed by use of the ‘Qubit® dsDNA HS Assay Kit’ (Q32854; ThermoFisher Scientific).  Sequencing run: Generated mRNA expression libraries were pooled accordingly, denatured with NaOH, and were finally diluted to 1.8pM or 2pM according to the ‘Denature and Dilute Libraries Guide’ (Document # 15048776 v02; Illumina). 1.3 ml of denatured pool (including 1% PhiX) was sequenced on an Illumina NextSeq550 sequencer using one high output flowcell for 75 cycles and 400 million clusters (#20024906; Illumina). HTO libraries was loaded with a molar proportion corresponding to around 5% of sequencing run capacity. Sequencing was performed according to the following settings: 28bp as sequence read 1; 56bp as sequence read 2; 8bp as index read 1; no index read 2. In total, two such sequencing runs were performed.

tag: library type

value: HTO

tag: hto


title: MCF10A, UT

status: Public on Oct 30 2025

type: SRA

city: Boulder

first: Brianna

last: Fernandez

state: CO

address: 3415 Colorado Avenue

department: Biochemistry

country: USA

institute: University of Colorado Boulder

description:
Library name: Sample 1
10x Genomics

library_source: transcriptomic single cell

data_processing:
scRNA-seq data were processed and analyzed using the Seurat (v5.1.0) package in R (v4.4.1). Raw sequencing reads were pre-processed using CellRanger (v7.1.0) and aligned to the Human (GRCh38) 2020-A reference genome. Gene-cell count matrices were generated, and low-quality cells were filtered out based on the number of detected genes, unique molecular identifiers (UMIs), and mitochondrial gene percentage. Cells with fewer than 3,500 genes were excluded to remove low-quality cells. Cells with greater than 15% mitochondrial gene expression were also removed. Data were log-normalized using Seurat’s NormalizeData() function with a scale factor of 10,000.
Assembly: Human (GRCh38) 2020-A
Supplementary files format and content: matrix files for counts, features, and barcodes

molecule: polyA RNA

organism: Homo sapiens

extract_protocol:
Single-cell suspensions were prepared following the 10x Genomics “Cell Preparation for Single Cell Protocols” guide with a viability of >95%. Single-cell encapsulation, barcoding, cDNA amplification, library preparation, and sequencing were performed by the University of Colorado Anschutz Genomics Shared Resource facility. Cells were encapsulated into droplets using the Chromium Controller, where reverse transcription occurred within individual gel beads-in-emulsion (GEMs).
Single-cell RNA sequencing (scRNA-seq) libraries were generated using the 10x Genomics Chromium platform with the 3' mRNA capture kit. Single-cell suspensions were prepared following the 10x Genomics “Cell Preparation for Single Cell Protocols” guide with a viability of >95%. Single-cell encapsulation, barcoding, cDNA amplification, library preparation, and sequencing were performed by the University of Colorado Anschutz Genomics Shared Resource facility. Cells were encapsulated into droplets using the Chromium Controller, where reverse transcription occurred within individual gel beads-in-emulsion (GEMs). Following barcoding and cDNA amplification, sequencing libraries were constructed according to the manufacturer’s protocol. Libraries were sequenced on an Illumina NovaSeqX to a depth of 81,810 reads per cell for untreated, 74,214 reads per cell for 2.5µM, 81,254 reads per cell for 10µM, and 76,973 reads per cell for 25µM.

treatment_protocol: MCF10A cells were split 1:10 (from a confluent plate) into a plastic 10cm culture plate before being treated with 2.5, 10, or 25μM etoposide the following day for 24 h. Cells were then washed twice with PBS before being returned to full-growth media. Full-growth media was refreshed every 3d during the drug recovery period.

tag: cell line

tag: cell type

value: normal human breast epithelial cell

tag: treatment

value: Untreated


title: IL-10 pos splenic B cells, control mouse 1, D3 aBCR IL-5 IFNg IL-2

city: Rennes

first: Simon

last: Leonard

address: 2 avenue du Prof Léon Bernard

country: France

institute: Université Rennes

library_source: transcriptomic

data_processing:
Sequence reads were trimmed for low quality reads, adaptor sequence, contaminant DNA or PCR duplicates using Trimmomatic program.
Trimmed reads were mapped to reference genome GrCM38/mm10 with HISAT2, splice-aware aligner.
Transcript was assembled by StringTie with aligned reads.
Read count extraction and normalization were performed using -e option of StringTie.
DEG analysis was conducted using DESeq2 tool. Samples were grouped using Hierarchical Clustering Analysis or Multidimensional Scaling Analysis. Gene-set enrichment analysis was performed based on gene ontoloy on significant DEG lists.
Assembly: GrCM38/mm10
Supplementary files format and content: tab-delimited text file including read counts, FPKM and TPM values of all the samples

molecule: total RNA

organism: Mus musculus

source_name: CD25+ IL10+ splenic B cells

growth_protocol: B cells were harvested from mice spleen using "B Cell Isolation Kit, mouse" (Miltenyi #130-090-892) and cultured at 500.000 cells/mL in RPMI supplemented with 10% Fetal Bovine Serum (FBS) and sodium pyruvate, non-essential amino acids, HEPES, beta-mercaptoéthanol and anitbiotics in humidified atmosphere with 5% CO2 at 37°C.

extract_protocol:
Sorted cells were frozen direclty in lysis buffer. RNA extraction was performed using "NucleoSpin RNA Plus XS" kit from Macherey-Nagel.
RNA library for RNA-seq were prepared using Watchmaker mRNA Library Prep Kit.

treatment_protocol: Splenic B cells were stimulated with anti-BCR (8 µg/mL, Invitrogen #16-5092-85), IL-5 (50 U/mL, Stemcell #78049.1), IFNg (1 ng/mL, Miltenyi #130-105-790) and IL-2 (50 U/mL, Miltenyi #130-120-662) for 3 days. IL-10pos CD25pos B cells and IL-10neg CD25pos B cells were sorted by FACS at D3.

tag: Sex

value: Female

tag: age

value: 25 weeks

value: CD25+ IL10+ splenic B cells

tag: strain

tag: genotype

value: Il2rb fl/fl, CD19 +/+

value: in vitro stimulation:anti-BCR + IL-5 + IFNg + IL-2

tag: time

value: Day 3


title: Senescent MRC5 fibroblast; siMAVS; Replicate 4

status: Public on Oct 29 2025

city: Rochester

first: Dominik

last: Saul

state: Minnesota

address: 200 First Street SW Rochester

institute: Mayo Clinic

description: Library name: SV29

data_processing:
After investigating the quality of the raw data, sequence reads were trimmed to remove possible adapter sequences and nucleotides with poor quality using Trimmomatic v.0.36. The trimmed reads were mapped to the reference genome available on ENSEMBL using the STAR aligner v.2.5.2b. BAM files were generated as a result of this step. Unique gene hit counts were calculated by using feature Counts from the Subread package v.1.5.2. Only unique reads that fell within exon regions were counted. After extraction of gene hit counts, the gene hit counts table was used for downstream differential expression analysis. Using DESeq2, a comparison of gene expression between the groups of samples was performed. The Wald test was used to generate P values and Log2 fold changes. Genes with adjusted P values < 0.05 and absolute log2 fold changes >1 were called as differentially expressed genes for each comparison. Gene ontology analysis was performed on the statistically significant set of genes by implementing the software GeneSCF. The GO list was used to cluster the set of genes based on their biological process and determine their statistical significance.
Assembly: Data were aligned and quantified using the 10X Genomics Cell Ranger Software Suite (v6.1.1) against the murine reference genome (mm10) and human genome (hg38).
Supplementary files format and content: tab-delimited text file including raw counts for each sample

source_name: Fibroblast

growth_protocol: For cell culture experiments, MRC5 fibroblasts were grown in DMEM supplemented with 10% FBS, 100 units/ml penicillin, 100 ug/ml streptomycin and 2mM glutamine. Cells were maintained at 37C with 5% CO2.

extract_protocol:
RNA was extracted using Rneasy mini kit (Qiagen).
RNA samples were quantified using Qubit 2.0 Fluorometer (Life Technologies, Carlsbad, CA, USA) and RNA integrity was checked using Agilent TapeStation 4200 (Agilent Technologies, Palo Alto, CA, USA). RNA sequencing libraries were prepared using the NEBNext Ultra II RNA Library Prep Kit for Illumina and the NEBNext Poly(A) mRNA Magnetic Isolation Module following the manufacturer's instructions (NEB, Ipswich, MA, USA). Briefly, mRNAs were initially enriched with Oligod(T) beads. Enriched mRNAs were fragmented for 15 minutes at 94 °C. First-strand and second-strand cDNA were subsequently synthesized. cDNA fragments were end-repaired and adenylated at 3'ends, and universal adapters were ligated to cDNA fragments, followed by index addition and library enrichment by PCR with limited cycles. The sequencing library was validated on the Agilent TapeStation (Agilent Technologies, Palo Alto, CA, USA), and quantified by using Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA) as well as by quantitative PCR (KAPA Biosystems, Wilmington, MA, USA). The sequencing libraries were clustered on a flowcell. After clustering, the flowcell was loaded on the Illumina NovaSeq X Plus instrument according to the manufacturer’s instructions. The samples were sequenced using a 2x150bp Paired-End (PE) configuration, targeting approximately 30 M reads/sample. The control software conducted image analysis and base calling. Raw sequence data (.bcl files) generated by the sequencer were converted into fastq files and de-multiplexed using Illumina's bcl2fastq 2.20 software. One mismatch was allowed for index sequence identification.

treatment_protocol: For senescence induction in vitro, cells were exposed to 20Gy X-ray irradiation and collected at Days 10-20 post-irradiation for analysis. For mouse experiments, 10-12 week old Bax fl/fl Bak KO male mice were were injected via tail vein with 1.5 x 1011 GC/mouse of either AAV8-TBG-Cre (for hepatocyte-specific deletion) or with control virus (AAV8-GFP). Two weeks post-injection, mice were placed on FFC diet containing high fat (40% calories) and high cholesterol (0.2%) (#AIN-76A Western Diet; TestDiet), with fructose (18.9 g/L) and glucose (23.1 g/L) added to the drinking water (total sugar 42 g/L) for induction of MASH. Animals were kept on this diet for 16 weeks.

tag: tissue

value: Fibroblast

value: Human lung fibroblast

value: MAVS knockdown

value: 20Gy X-ray irradiation


title: Senescent MRC5 fibroblast; siMAVS; Replicate 3

description: Library name: SV28


title: Senescent MRC5 fibroblast; siMAVS; Replicate 2

description: Library name: SV27


title: Senescent MRC5 fibroblast; siScramble; Replicate 4

description: Library name: SV25

value: WT


title: Senescent MRC5 fibroblast; siScramble; Replicate 3

description: Library name: SV24


title: Senescent MRC5 fibroblast; siScramble; Replicate 1

description: Library name: SV22


title: Bone marrow pooled

description:
Library name: N4704m
HTO-A-Mouse-155801-0301 (N4704ms1) and HTO-A-Mouse-155803-0302 (N4704ms2) correspond to Bone marrow of Control mice No1 and No2; HTO-A-Mouse-155805-0303 (N4704ms3) and HTO-A-Mouse-155807-0304 (N4704ms4) correspond to Bone marrow of N2ΔCx3cr1 mice No1 and No2; HTO-A-Mouse-155809-0305 (N4704ms5) and HTO-A-Mouse-155811-0306 (N4704ms6) correspond to Bone marrow of RbpjΔCx3cr1 mice No1 and No2.

source_name: Bone marrow

value: Bone marrow

value: Mononucler phagocytes and the progenitors

value: male

value: mRNA


title: Proliferating MRC5 fibroblast; siScramble; Replicate 2

description: Library name: SV20

value: Proliferating


title: Proliferating MRC5 fibroblast; siScramble; Replicate 3

description: Library name: SV21


title: Papain ILC2-2

city: Shanghai

first: Dongdi

last: Wang

address: 227 South Chongqing Road, Shanghai, China

country: China

institute: Shanghai Jiaotong University School of Medicine

data_processing:
Illumina Casava1.7 software used for basecalling.
The raw reads were aligned to the mouse reference genome (version mm10) by using Hisat2 RNASeq alignment software.
Quantitated relative mRNA expression levels (FPKM) were calculated based on exon regions using Cufflinks and the mm10 reference genome annotations.
Assembly: mm10
Supplementary files format and content: comma-separated values files include readcounts  for each Sample and every gene
Supplementary files format and content: comma-separated values files include FPKM values  for each Sample and every gene

source_name: lung

extract_protocol:
Total RNA from sortedlung ILC2s mRNA profiles of PBS treated mice and Papain treated mice was extracted by Trizol reagent.  RNA quality was examined with an Agilent Bioanalyzer 2100 (Agilent). The concentration of RNA was from 7ng/ul to 14ng/ul. The SMART-Seq HT Kit (Takara Bio USA, Inc) was used to generate high-quality, full-length cDNA.
Libraries were generated using TruePrep DNA Library Prep Kit V2 for Illumina (Vazyme) following manufacturer’s protocol.

value: lung

value: Papain


title: Senescent MRC5 fibroblast; siScramble; Replicate 2

description: Library name: SV23


title: Proliferating MRC5 fibroblast; siScramble; Replicate 1

description: Library name: SV19


title: Senescent MRC5 fibroblast, DDX58 CRISPR/Cas9; Replicate 2

description: Library name: SV17

value: DDX58 KO


title: Senescent MRC5 fibroblast, DDX58 CRISPR/Cas9; Replicate 3

description: Library name: SV18


title: Senescent MRC5 fibroblast, DDX58 CRISPR/Cas9; Replicate 1

description: Library name: SV16


title: Senescent MRC5 fibroblast, pLenti CRISPR/Cas9; Replicate 3

description: Library name: SV12


title: Senescent MRC5 fibroblast, IFIH1 CRISPR/Cas9; Replicate 3

description: Library name: SV15

value: IFIH1 KO


title: Senescent MRC5 fibroblast, IFIH1 CRISPR/Cas9; Replicate 2

description: Library name: SV14


title: Senescent MRC5 fibroblast, IFIH1 CRISPR/Cas9; Replicate 1

description: Library name: SV13


title: Senescent MRC5 fibroblast, pLenti CRISPR/Cas9; Replicate 2

description: Library name: SV11


title: Proliferating MRC5 fibroblast, DDX58 CRISPR/Cas9; Replicate 1

description: Library name: SV07


title: Proliferating MRC5 fibroblast, DDX58 CRISPR/Cas9; Replicate 2

description: Library name: SV08


title: Proliferating MRC5 fibroblast, IFIH1 CRISPR/Cas9; Replicate 2

description: Library name: SV05


title: Proliferating MRC5 fibroblast, IFIH1 CRISPR/Cas9; Replicate 3

description: Library name: SV06


title: Proliferating MRC5 fibroblast, IFIH1 CRISPR/Cas9; Replicate 1

description: Library name: SV04


title: Proliferating MRC5 fibroblast, pLenti CRISPR/Cas9; Replicate 3

description: Library name: SV03


title: Proliferating MRC5 fibroblast, pLenti CRISPR/Cas9; Replicate 2

description: Library name: SV02


title: Proliferating MRC5 fibroblast, DDX58 CRISPR/Cas9; Replicate 3

description: Library name: SV09


title: YFP-Parkin IMR90 human fibroblast; Senescent + CCCP (no mitochondria) + mtRNA 5

description: Library name: SV35

data_processing:
After investigating the quality of the raw data, sequence reads were trimmed to remove possible adapter sequences and nucleotides with poor quality using Trimmomatic v.0.36. The trimmed reads were mapped to the reference genome available on ENSEMBL using the STAR aligner v.2.5.2b. BAM files were generated as a result of this step. Unique gene hit counts were calculated by using feature Counts from the Subread package v.1.5.2. Only unique reads that fell within exon regions were counted. After extraction of gene hit counts, the gene hit counts table was used for downstream differential expression analysis. Using DESeq2, a comparison of gene expression between the groups of samples was performed. The Wald test was used to generate P values and Log2 fold changes. Genes with adjusted P values < 0.05 and absolute log2 fold changes >1 were called as differentially expressed genes for each comparison. Gene ontology analysis was performed on the statistically significant set of genes by implementing the software GeneSCF. The GO list was used to cluster the set of genes based on their biological process and determine their statistical significance.
Assembly: Data were aligned and quantified using the 10X Genomics Cell Ranger Software Suite (v6.1.1) against the human genome (hg38).
Supplementary files format and content: tab-delimited text file including raw counts for each sample

growth_protocol: For cell culture experiments, IMR90 fibroblasts were grown in DMEM supplemented with 10% FBS, 100 units/ml penicillin, 100 ug/ml streptomycin and 2mM glutamine. Cells were maintained at 37C with 5% CO2 and 3% O2.

treatment_protocol: For Parkin-mediated widespread mitochondrial clearance, irradiated Parkin-expressing IMR90 fibroblasts were treated with 12.5 uM CCCP (Sigma, C2759) (3 days after irradiation) for 48 hours (CCCP was refreshed every 24 hours). Mitochondria-depleted cells were then transfected with 2.5 ug of isolated mitochondrial RNA using Lipofectamine MessengerMax Reagent (Invitrogen, LMRNA003), according to the manufacturer’s instructions. Cells were incubated with mtRNA for 48 hours and then harvested for analysis.

value: YFP-Parkin expressing

value: 20Gy X-ray irradiation + CCCP + mtRNA


title: YFP-Parkin IMR90 human fibroblast; Senescent + CCCP (no mitochondria) + mtRNA 4

description: Library name: SV34


title: YFP-Parkin IMR90 human fibroblast; Senescent + CCCP (no mitochondria) + mtRNA 3

description: Library name: SV33


title: Proliferating MRC5 fibroblast, pLenti CRISPR/Cas9; Replicate 1

description: Library name: SV01


title: YFP-Parkin IMR90 human fibroblast; Senescent + CCCP (no mitochondria) + mtRNA 2

description: Library name: SV32


title: YFP-Parkin IMR90 human fibroblast; Senescent + CCCP (no mitochondria) + mtRNA 1

description: Library name: SV31


title: YFP-Parkin IMR90 human fibroblast; Senescent + CCCP (no mitochondria) 5

description: Library name: SV29

value: 20Gy X-ray irradiation + CCCP


title: YFP-Parkin IMR90 human fibroblast; Senescent + CCCP (no mitochondria) 3

description: Library name: SV27


title: YFP-Parkin IMR90 human fibroblast; Senescent + CCCP (no mitochondria) 2

description: Library name: SV26


title: YFP-Parkin IMR90 human fibroblast; Senescent + CCCP (no mitochondria) 4

description: Library name: SV28


title: YFP-Parkin IMR90 human fibroblast; Senescent + CCCP (no mitochondria) 6

description: Library name: SV30


title: YFP-Parkin IMR90 human fibroblast; Senescent + CCCP (no mitochondria) 1

description: Library name: SV25


title: YFP-Parkin IMR90 human fibroblast; Senescent control 3

description: Library name: SV24

value: 20Gy X-ray irradiation; control


title: YFP-Parkin IMR90 human fibroblast; Senescent control 2

description: Library name: SV23

value: Human lung fibroblast


title: YFP-Parkin IMR90 human fibroblast; Proliferating control 3

description: Library name: SV21

value: Proliferating control


title: YFP-Parkin IMR90 human fibroblast; Senescent control 1

description: Library name: SV22


title: YFP-Parkin IMR90 human fibroblast; Proliferating control 2

description: Library name: SV20


title: YFP-Parkin IMR90 human fibroblast; Proliferating control 1

description: Library name: SV19


title: YFP-Parkin IMR90 human fibroblast; Senescent + CCCP (no mitochondria) + mtRNA 6

description: Library name: SV36


title: RNaseP, 3 Nucleotide Deletion, unmodified

city: Chapel Hill

first: David

last: Qiu

state: NC

address: 104 Airport Dr

department: Chemistry

institute: UNC Chapel Hill

description: Library name: RNaseP_3NtDel_Control

library_source: other

data_processing:
ShapeMapper 2.1.5
DanceMapper
Supplementary files format and content: Files with format _profile.txt are from ShapeMapper v2.1.5. Each row in these tables describes per-nucleotide mutation rates and normalized reactivity profiles
Supplementary files format and content: Files with format_DanceMap.txt are reactivity files from DanceMapper. Each row in these tables describes the modelled per-nucleotide reactivity for each population in an ensemble.

molecule: other

organism: Geobacillus stearothermophilus

source_name: bacteria

extract_protocol:
RNA was generated by in vitro transcription with T7 polymerase
MaP-RT was performed using targeted primers. Two rounds of PCR were performed to add Illumina adapter sequences and barcodes.

value: bacteria

value: 3 Nucleotide Deletion

value: Control


title: RNaseP, 3 Nucleotide Deletion, TMO modified

description: Library name: RNaseP_3NtDel_TMO

value: TMO


title: RNaseP, Full Riboswitch, unmodified

description: Library name: RNaseP_LoopMutant_Control

value: Loop Mutant


title: RNaseP, 1 Nucleotide Deletion, TMO modified

description: Library name: RNaseP_1NtDel_TMO

value: 1 Nucleotide Deletion


title: RNaseP, 1 Nucleotide Deletion, unmodified

description: Library name: RNaseP_1NtDel_Control


title: RNaseP, Full Riboswitch, DMS modified

description: Library name: RNaseP_LoopMutant_DMS

value: DMS


title: RNaseP, U Substitution, unmodified

description: Library name: RNaseP_USub_Control

value: U Substitution


title: RNaseP, Full Riboswitch, TMO modified

description: Library name: RNaseP_LoopMutant_TMO


title: RNaseP, U Substitution, TMO modified

description: Library name: RNaseP_USub_TMO


title: RNaseP, native, unmodified

description: Library name: RNaseP_Native_Control

value: Native


title: BMDM, WT, PTX 4

city: Winston Salem

first: Ming

last: Leung

address: 1 Medical Center Blvd

department: Comprehensive Cancer Center

institute: Atrium Health Wake Forest

description: Library name: Sample 15

data_processing:
First, Trimmomatic 0.33 is used to trim and remove adapters. Second, STAR 2.5.2a is used to map the sequences. Third, picard 1.95 MarkDuplicates was used to remove duplicates. Fourth, featureCounts 1.4.5 is used to count the number of reads per gene.
Assembly: GRCm38
Supplementary files format and content: TSV (Tab separated value table) of the raw read counts per gene

growth_protocol: Mouse-derived bone marrow differentiated macrophages from Vav1creErn1f/f (conditional knockout - cKO) and  Ern1f/f (counterpart – WT) were generated by flushing the bone marrow and suspending the resulting cells in RPMI 1640. The cells were then incubated in DMEM/F12 1:1 media supplemented with 1% sodium pyruvate (Gibco), 10% FBS (Gibco), 1% penicillin-streptomycin (Gibco), 20 ng/ml murine recombinant M-CSF (R&D), and 1 ng/ml recombinant IL-3 (Peprotech) for 3 days in bacteriological dish plates. On day 4, approximately half of the media with non-adherent cells were removed and plated in a bacteriological dish plate in media containing 20 ng/ml recombinant M-CSF. Complete media (10 ml) with 20 ng/ml M-CSF was added to the original plates. On day 6, 5 ml of complete media containing 20 ng/ml M-CSF was added to all plates. On day 7, cells were harvested using Accutase (Invitrogen) and used immediately for subsequent in vitro assays. All treatments were performed in cells plated with a final concentration of 1x106 cell/ml in RPMI 1640 media with 10% FBS.

extract_protocol:
Total RNA was isolated using the PureLink RNA mini kit (Invitrogen). All samples achieved the quality controls conducted by the Agilent Bioanalyzer 2100.  1 ug of total RNA was used for the construction of sequencing libraries.
Illumina TruSeq Stranded Total RNA with Ribo-Zero Globin kit

treatment_protocol: Mouse-derived bone marrow differentiated macrophages from Vav1creErn1f/f (cKO) and  Ern1f/f (WT) were cultured at a density of 5×105 cells/ml and stimulated with paclitaxel (Sigma Aldrich; 10 µM) ordimethyl sulfoxide (DMSO; 0.5%) for 18 hours. After the treatments the supernatants were discarded, and the attached cells were washed with DPBS.

value: Bone marrow

value: Primary macrophage

value: Bone marrow differentiated macrophages

value: WT

value: Paclitaxel 10 µM


title: BMDM, WT, DMSO 4

description: Library name: Sample 16

value: DMSO


title: BMDM, WT, DMSO 3

description: Library name: Sample 14


title: RNaseP, native, TMO modified

description: Library name: RNaseP_Native_TMO


title: BMDM, WT, PTX 1

description: Library name: Sample 9


title: BMDM, WT, DMSO 1

description: Library name: Sample 10


title: BMDM, cKO, PTX 4

description: Library name: Sample 7

value: cKO


title: BMDM, cKO, DMSO 4

description: Library name: Sample 8


title: RNaseP, native, DMS modified

description: Library name: RNaseP_Native_DMS


title: BMDM, cKO, DMSO 3

description: Library name: Sample 6


title: BMDM, WT, DMSO 2

description: Library name: Sample 12


title: BMDM, cKO, PTX 3

description: Library name: Sample 5


title: BMDM, cKO, DMSO 2

description: Library name: Sample 4


title: BMDM, cKO, DMSO 1

description: Library name: Sample 2


title: BMDM, cKO, PTX 1

description: Library name: Sample 1


title: BMDM, WT, PTX 3

description: Library name: Sample 13


title: mESC-RBBP5FKBP, dTAG13 24hr, biological replicate 1, input, tech rep 1

city: La Jolla

first: Alon

last: Goren

state: CA

address: 9375 Gilman Dr

department: Department of Medicine

institute: University of California San Diego

description: Library name: LP101_RBBP5FKBP_dTAG13_24hr_1_input_1_S55

library_source: genomic

data_processing:
Samples were sequenced on Illumina NextSeq 550 or Nextseq 2000, samples were demultiplexed with Illumina bcl2fastq.
Adapters were removed with Trimmomatic, reads with Phred score <26 over a 4bp window were truncated.
Reads were aligned to a concatenated genome containing hg38 + sacCer3 + dm6, alignment files were separated by species. If UMIs are not present in adapter sequences, PCR duplicate reads were removed with samtools markdup. When present, UMIs were deduplicated with UMI-tools dedup, with method=unique. Alignments were additionally filtered to remove reads if paired-end reads aligned >1000bp apart, or on different chromosomes.
HOMER tag directories were made; by default read counts for each species were filtered for primary alignments with MAPQ > 10, reads were extended to model approximate ChIP-seq fragment size using the option -fragLength 150. Samples were converted to bed files for normalization. Details of spike-in normalization are described in Supplement, normalized data are in bedGraph format.
Read normalized bigwig files were created with HOMER makeBigWig.pl from tag directories.
Assembly: hg38, dm6, sacCer3
Supplementary files format and content: Normalized BigWig files for hg38 genome

molecule: genomic DNA

source_name: mESC

growth_protocol: HeLa-S3 cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1x penicillin-streptomycin, L-glutamine, and 110mg/L sodium pyruvate. Cells were incubated at 37°C and 5% CO2. HCT116-EF1a-OsTIR1-Rpb1 cells were cultured in McCoy’s 5A media (Thermo Scientific 16600082) with 10% FBS and 1x antibiotic-antimycotic. Cells were incubated at 37°C and 5% CO2. Cell lines mESc E14 Dpy30-miniAID-P2A-mTagBFP2 pB-hygro-OsTIR1-P2A-mCherry and mESc E14 Rbbp5-P2A-2xHA-P2A-NEO were provided from the Helin lab, and maintained in GMEM with 15% FBS, 10uM sodium pyruvate, 1x MEM-NEAA, 50uM B-mercaptoethanol, and antibiotic/antimycotic.

extract_protocol:
HCT116 or HeLa-S3 cells were double crosslinked on plates with 2 mM DSG for 30 minutes (Fisher Scientific NC1736108) and 1% formaldehyde (Pierce ThermoFisher PI28906) for 10 minutes. Crosslinking was quenched with 1/20th volume of 2.625 M glycine (EMD Millipore 357002) and 0.5% BSA (NEB B9000S). Cells were washed by resuspending 3 times with 0.5% BSA/PBS + Protease inhibitor (PIC, Roche 11697498001), spinning down at 1200g for 5 minutes at 4°C and removing the supernatant. Washed pellets were snap frozen in dry ice then stored in -80°C. Cells were lysed with LB3 lysis buffer + PIC, then chromatin was sheared on the PIXUL sonicator (Active Motif 53130) for 108 minutes. S2 cells were crosslinked in 1% formaldehyde for 10 minutes at room temperature, then quenched with glycine and 10% BSA to a final concentration of 0.5%, lysed in LB3 buffer, and sonicated on the PIXUL for 60 minutes. S. cerevisiae cultures in log phase were crosslinked with 0.86% formaldehyde for 30 minutes on a room temperature rotating shaker. The cell wall was digested with Zymolase at 37°C for 60 minutes, cells were washed by pelleting 3x at 5000g and washing in PBS + Protease inhibitor, then resuspended in NP-S buffer + PI. Sheared S. cerevisiae chromatin was sonicated on the PIXUL for 108 minutes. All PIXUL sonication was done with settings Pulse (N) = 50, PRF (kHz) = 1.00, Burst Rate (Hz) = 20, only Process Time was varied. Sheared chromatin from each species were clarified by centrifugation at 15000g, for 10 minutes at 4°C, then combined and inputs were taken before immunoprecipitation for each condition, to assess the relative ratios of chromatin. To HeLa-S3 cells, approximately 100k S2 and 500k S. cerevisiae cells were added per immunoprecipitation reaction. To HCT116 cells, approximately 500k S2 and 100k S. cerevisiae cells were added to each immunoprecipitation reaction. To each IP, 10uL of Protein A beads (Thermo Fisher Scientific 10002D), 1% Triton X-100 and 1x PIC were added. 1 million HCT116 cells or HeLa-S3 cells were used per immunoprecipitation reaction.
To each IP, 10uL of Protein A beads (Thermo Fisher Scientific 10002D), 1% Triton X-100 and 1x PIC were added. 1 million HCT116 cells or HeLa-S3 cells were used per immunoprecipitation reaction. Below are the volumes per IP of each antibody used: CST H3K27ac (D5E4): 1uL CST H3K4me3 (C42D8): 1uL CST H3K4me1 (D1A9): 1uL CST H3K36me3 (D5A7): 1uL CST Rpb1 NTD (D8L4Y): 4uL CST H3K9ac (C5B11): 4uL Samples were incubated overnight on a rotator at 4°C. IP Washes were performed: 3x with 180uL of WBI, 3x with 180uL of WBIII, and 2x with 185uL TET, then eluted in 25uL of cold TT Buffer. Input chromatin containing target and spike-in species’ chromatin from each biological condition were diluted 1:62.5x in TT buffer to a final volume of 25uL. Libraries were prepared with the NEB Ultra II Library Prep Kit (NEB E7645L). NEXTflex Dual-Barcode ChIP-seq Adapters (NOVA-514151) were added at 0.25uM or 0.5uM or 0.625uM. Libraries were then de-crosslinked at 55°C for 1hr, then 65°C overnight and cleaned with Speedbeads, 8.6% PEG and 0.8M NaCl and eluted in TT buffer. Next libraries were PCR amplified with 1uM Solexa 1GA and 1GB primers and NEB Ultra II Q5 Mastermix (PCR protocol: 98C for 30’’, then 12 or 13 cycles of 98C for 10’’, 60C for 15’’, 72C for 30’’, then hold at 72C for 2’). A final cleanup was done with Speedbeads, 8.5% PEG and 1M NaCl, washed twice with 80% EtOH, then the final libraries were eluted in 20uL TT. Library size was estimated by running 6uL of eluted library on a 1.8% agarose gel and concentration was quantified with Qubit HS dsDNA kit (Invitrogen Q32854). Libraries were pooled and sequenced on Illumina NextSeq 550 or NextSeq 2000.

treatment_protocol: HeLa-S3 cells were synchronized in G1/S by adding 2 mM thymidine for 16 hr, washed with PBS, released for 3 hr in fresh medium, and pro-metaphase arrested with 10uM S-trityl-L-cysteine (STC) for 12 hr then harvested. HeLa-S3 cells and HCT116 cells were treated with 500nM Triptolide for 1 or 4 hours. HCT116 cells were treated with 500uM auxin for 1 or 4 hours. mESCs were treated with either 500nM Auxin (3-indoleacetic acid, Sigma-Aldrich #I3750) for DPY30-mAID or 500nM dTAG13 (Tocris Biosciences #6605) for RBBP5-FKBP cells, for 0 and 24 hours.

tag: spike-in species

value: D. melanogaster, S. cerevisiae

value: mESC

value: mouse embryonic stem cell

value: dTAG13 24hr


title: BMDM, WT, PTX 2

description: Library name: Sample 11


title: mESC-DPY30mAID, auxin 24hr, biological replicate 1, input, tech rep 2

description: Library name: LP101_DPY30mAID_IAA_24hr_1_input_2_S52

value: auxin 24hr


title: mESC-RBBP5FKBP, dTAG13 24hr, biological replicate 1, H3K4me3, tech rep 1

description: Library name: LP101_RBBP5FKBP_dTAG13_24hr_1_H3K4me3_1_S47


title: mESC-RBBP5FKBP, dTAG13 24hr, biological replicate 1, H3K4me3, tech rep 2

description: Library name: LP101_RBBP5FKBP_dTAG13_24hr_1_H3K4me3_2_S48


title: mESC-RBBP5FKBP, dTAG13 0hr, biological replicate 1, H3K4me3, tech rep 1

description: Library name: LP101_RBBP5FKBP_dTAG13_0hr_1_H3K4me3_1_S45

value: dTAG13 0hr


title: mESC-DPY30mAID, auxin 24hr, biological replicate 1, H3K4me3, tech rep 1

description: Library name: LP101_DPY30mAID_IAA_24hr_1_H3K4me3_1_S43


title: mESC-RBBP5FKBP, dTAG13 0hr, biological replicate 1, input, tech rep 1

description: Library name: LP101_RBBP5FKBP_dTAG13_0hr_1_input_1_S53


title: mESC-DPY30mAID, auxin 24hr, biological replicate 1, input, tech rep 1

description: Library name: LP101_DPY30mAID_IAA_24hr_1_input_1_S51


title: BMDM, cKO, PTX 2

description: Library name: Sample 3


title: mESC-DPY30mAID, auxin 0hr, biological replicate 1, input, tech rep 2

description: Library name: LP101_DPY30mAID_IAA_0hr_1_input_2_S50

value: auxin 0hr


title: mESC-DPY30mAID, auxin 24hr, biological replicate 1, H3K4me3, tech rep 2

description: Library name: LP101_DPY30mAID_IAA_24hr_1_H3K4me3_2_S44


title: RescueRepl3 Rescue clon CD4/CD8 deplet rep3

city: Zurich

first: Francesc

last: Castro-Giner

address: Otto-Stern-Weg 7

country: Switzerland

institute: ETH Zurich

description:
Library name: RescueRepl3
dCas9 (i.e. no knockout)  - SgRNAs as barcodes for studying loss of clonality

data_processing:
Mapping using bowtie2 (v2.3.5.1; parameters: -L 11 -N 1 -I S,1,0.75 --norc), allowing for one mismatch.
Normalized abundance of individual sgRNAs were obtained using MAGeCK (v0.5.9.4).
Assembly: lentiviral libraries encompassing 2215 sgRNAs against mouse immune interactome genes  or 2180 sgRNAs against the homologue human genes
Supplementary files format and content: counts.txt.gz are tab-delimited files containing raw counts for each library
Library strategy: CRISPR Screen

extract_protocol:
All tissue samples were processed through a common DNA extraction protocol, as described (Scheidmann et al., 2022). Briefly, tumor and metastasis-carrying organs were lysed in a FastPrep tissue homogenizer (MP) using dedicated matrix beads-containing disposable tubes (MP) in presence of a DNA extraction buffer (50 mM Tris-Cl pH 8.0, 50 mM EDTA, 1% SDS). The lysate was then digested for at least 16 hours at 65 °C in presence of Proteinase K (Qiagen) and then for 30 minutes at 37 °C in presence of RNAase A (Qiagen). Separation of genomic DNA was carried out by first adding pre-chilled 7.5 M ammonium acetate, followed by isopropanol addition to the resulting supernatant, leading the DNA to precipitate. The pellet was then washed with 70% ethanol and the genomic DNA was resuspended in Tris-EDTA buffer (Ambion) and frozen at -20 °C. Finally, DNA extraction from cultivated cells was carried out using the Dneasy kit (Qiagen). For CRISPR genomics applications, DNA samples were quantified using NanoDrop ND-1000 UV-Vis Spectrophotometer (Thermo Fisher).
Samples were processed to reach a theoretical coverage of 500–1,000× per target. For mouse samples, 10-15 µg of genomic DNA were amplified in a one-step PCR with barcoded primers targeting the pLentiGuide vector backbone (P5 and P7). The primer sequences in the table below highlight an 8 bp barcode position in blue and vector binding site in bold, mapping between the sgRNA scaffold and the U6 promoter. All pPrimers were ordered as desalted Oligos oligos at IDT (Integrated DNA Technologies)(at Integrated DNA Technologies or at Microsynth) Microsynth and resuspended in nuclease-free water. For human samples, 10 µg of gDNA were pre-amplified in a nested PCR with the external primers targeting the huTHY1insert and the upstream portion of the U6 promoter (primer sequences in table below). In the second PCR step, barcoded primers targeting the pLentiguide vector backbone (P5 and P7) were used as internal primers (primer sequences in table above). For mouse samples, PCR conditions were as follows: 100 µl as reaction volume, KAPA HiFi Hot Start Ready Mix (2X; Roche)0.02 U/µl of Q5 polymerase (New England Biosciences), and 25-31 cycles of amplification. For human samples, only 23-27 cycles of amplification were applied in the first step using KAPA HiFi Hot Start Ready Mix (2X; Roche). Since some samples did not yield any clearly detectable product after the first PCR, all products were purified with AMPure XP Beads (Beckman Coulter) or Sera-Mag Select Beads (2X, Cytiva Europa GmbH) and used as input for a second PCR (23-27 cycles) with the same primers that were used for the first PCR. When loading a second PCR, pre-amplified fragments were re-quantified using NanoDrop. To reach the theoretical coverage after sequencing, 200 ng of pre-amplified product were re-amplified using nested barcoded primers (P5 and P7). PCR conditions for the second PCR were as follows: 200 ng of pre-amplified fragments per 25 µl reaction, 0.02 U/µl of Q5 polymerase (New England Biosciences), and 27 cycles of amplification. The amplified product containing the spacer sequence with a size of 202 bp was purified by SPRI bead cleanup and a right-sided size selection to remove the genomic DNA input. Using a dual barcoding system, the final NGS amplicons were multiplexed and sequenced on an Illumina platform with 21 dark cycles (DC) in Read 1 to skip the invariable region of the primer binding side (U6 promoter).

value: tumor

tag: origin

value: xenograft

tag: mouse model

value: BALBc

tag: crispr lib

value: Immuno Signalome (2215) sgRNA library

tag: sgrna library

tag: effect type

value: sgRNAs used as barcodes (no KO)

tag: vector

value: lentiGuide-Puro+ dCas9

tag: full library_complexity


title: RescueRepl5 Rescue clon CD4/CD8 deplet rep5

description:
Library name: RescueRepl5
dCas9 (i.e. no knockout)  - SgRNAs as barcodes for studying loss of clonality

tag: cell line


title: RescueRepl4 Rescue clon CD4/CD8 deplet rep4

description:
Library name: RescueRepl4
dCas9 (i.e. no knockout)  - SgRNAs as barcodes for studying loss of clonality

tag: cell type


title: mESC-DPY30mAID, auxin 0hr, biological replicate 1, H3K4me3, tech rep 1

description: Library name: LP101_DPY30mAID_IAA_0hr_1_H3K4me3_1_S41

tag: treatment


title: RescueRepl2 Rescue clon CD4/CD8 deplet rep2

description:
Library name: RescueRepl2
dCas9 (i.e. no knockout)  - SgRNAs as barcodes for studying loss of clonality


title: mESC-DPY30mAID, auxin 0hr, biological replicate 1, input, tech rep 1

description: Library name: LP101_DPY30mAID_IAA_0hr_1_input_1_S49


title: Replacement_8 Liver IV NSG Thy1 LLC1

description:
Library name: Replacement_8
dCas9 (i.e. no knockout)  - SgRNAs as barcodes for studying loss of clonality

value: NSG


title: Replacement_9bis Lungs NSG Figure 2D REPEAT rep9

description:
Library name: Replacement_9bis
dCas9 (i.e. no knockout)  - SgRNAs as barcodes for studying loss of clonality


title: Replacement_9 Lungs IV BL6 Thy1 CMT 167

description:
Library name: Replacement_9
dCas9 (i.e. no knockout)  - SgRNAs as barcodes for studying loss of clonality


title: Replacement_8bis Lungs NSG Figure 2D REPEAT rep8

description:
Library name: Replacement_8bis
dCas9 (i.e. no knockout)  - SgRNAs as barcodes for studying loss of clonality


title: Replacement_7bis Lungs NSG Figure 2D REPEAT rep7

description:
Library name: Replacement_7bis
dCas9 (i.e. no knockout)  - SgRNAs as barcodes for studying loss of clonality


title: Replacement_7 Lungs IV NSG Thy1 LLC1

description:
Library name: Replacement_7
dCas9 (i.e. no knockout)  - SgRNAs as barcodes for studying loss of clonality


title: Replacement_6bis Lungs NSG Figure 2D REPEAT rep6

description:
Library name: Replacement_6bis
dCas9 (i.e. no knockout)  - SgRNAs as barcodes for studying loss of clonality


title: RescueRepl1 Rescue clon CD4/CD8 deplet rep1

description:
Library name: RescueRepl1
dCas9 (i.e. no knockout)  - SgRNAs as barcodes for studying loss of clonality


title: Replacement_6 Liver IV BL6 Thy1 LLC1

description:
Library name: Replacement_6
dCas9 (i.e. no knockout)  - SgRNAs as barcodes for studying loss of clonality


title: Replacement_5bis Lungs NSG Figure 2D REPEAT rep5

description:
Library name: Replacement_5bis
dCas9 (i.e. no knockout)  - SgRNAs as barcodes for studying loss of clonality


title: Replacement_4bis CT26 StealTHY BALB Lungmets rep5

description:
Library name: Replacement_4bis
dCas9 (i.e. no knockout)  - SgRNAs as barcodes for studying loss of clonality

value: CRISPR KO


title: Replacement_4 CT26 StealTHY BALB TUMOR rep5

description:
Library name: Replacement_4
dCas9 (i.e. no knockout)  - SgRNAs as barcodes for studying loss of clonality


title: Replacement_3bis Liver IV NSG Thy1 CMT 167

description:
Library name: Replacement_3bis
dCas9 (i.e. no knockout)  - SgRNAs as barcodes for studying loss of clonality


title: Replacement_5 Lungs IV BL6 Thy1 LLC1

description:
Library name: Replacement_5
dCas9 (i.e. no knockout)  - SgRNAs as barcodes for studying loss of clonality


title: Replacement_3 CT26 StealTHY BALB Lung Mets rep4

description:
Library name: Replacement_3
dCas9 (i.e. no knockout)  - SgRNAs as barcodes for studying loss of clonality


title: Replacement_1 CT26 StealTHY BALB Lung Mets rep3

description:
Library name: Replacement_1
dCas9 (i.e. no knockout)  - SgRNAs as barcodes for studying loss of clonality


title: LLC1_StealTHY_Tum_2 LLC1 StealTHY IV BL6 TUMOR 2

description:
Library name: LLC1_StealTHY_Tum_2
dCas9 (i.e. no knockout)  - SgRNAs as barcodes for studying loss of clonality


title: Replacement_2 CT26 StealTHY BALB TUMOR rep4

description:
Library name: Replacement_2
dCas9 (i.e. no knockout)  - SgRNAs as barcodes for studying loss of clonality


title: LLC1_Thy1_baseline LLC1 Thy1 baseline

description:
Library name: LLC1_Thy1_baseline
Puro+dCas9 baseline


title: Replacement_2bis Lungs IV NSG Thy1 CMT 167

description:
Library name: Replacement_2bis
dCas9 (i.e. no knockout)  - SgRNAs as barcodes for studying loss of clonality


title: Replacement_1bis Liver IV BL6 Thy1 CMT 167

description:
Library name: Replacement_1bis
dCas9 (i.e. no knockout)  - SgRNAs as barcodes for studying loss of clonality


title: LLC1_StealTHY_Tum_5 LLC1 StealTHY IV BL6 TUMOR 5

description:
Library name: LLC1_StealTHY_Tum_5
dCas9 (i.e. no knockout)  - SgRNAs as barcodes for studying loss of clonality


title: LLC1_StealTHY_Tum_4 LLC1 StealTHY IV BL6 TUMOR 4

description:
Library name: LLC1_StealTHY_Tum_4
dCas9 (i.e. no knockout)  - SgRNAs as barcodes for studying loss of clonality


title: LLC1_StealTHY_Tum_1 LLC1 StealTHY IV BL6 TUMOR 1

description:
Library name: LLC1_StealTHY_Tum_1
dCas9 (i.e. no knockout)  - SgRNAs as barcodes for studying loss of clonality


title: LLC1_StealTHY_Tum_3 LLC1 StealTHY IV BL6 TUMOR 3

description:
Library name: LLC1_StealTHY_Tum_3
dCas9 (i.e. no knockout)  - SgRNAs as barcodes for studying loss of clonality


title: LLC1_StealTHY_Met_1 LLC1 StealTHY IV BL6 Liver Mets 1

description:
Library name: LLC1_StealTHY_Met_1
dCas9 (i.e. no knockout)  - SgRNAs as barcodes for studying loss of clonality


title: LLC1_StealTHY_Met_5 LLC1 StealTHY IV BL6 Liver Mets 5

description:
Library name: LLC1_StealTHY_Met_5
dCas9 (i.e. no knockout)  - SgRNAs as barcodes for studying loss of clonality


title: dC9BL6_LungIV_LLC1_4 Lungs IV BL6 dCas9 LLC1 rep4

description:
Library name: dC9BL6_LungIV_LLC1_4
dCas9 (i.e. no knockout)  - SgRNAs as barcodes for studying loss of clonality


title: dC9BL6_LungIV_LLC1_3 Lungs IV BL6 dCas9 LLC1 rep3

description:
Library name: dC9BL6_LungIV_LLC1_3
dCas9 (i.e. no knockout)  - SgRNAs as barcodes for studying loss of clonality


title: LLC1_StealTHY_Met_3 LLC1 StealTHY IV BL6 Liver Mets 3

description:
Library name: LLC1_StealTHY_Met_3
dCas9 (i.e. no knockout)  - SgRNAs as barcodes for studying loss of clonality


title: dC9BL6_LungIV_LLC1_2 Lungs IV BL6 dCas9 LLC1 rep2

description:
Library name: dC9BL6_LungIV_LLC1_2
dCas9 (i.e. no knockout)  - SgRNAs as barcodes for studying loss of clonality


title: dC9BL6_LungIV_C167_4 Lungs IV BL6 dCas9 CMT167 rep4

description:
Library name: dC9BL6_LungIV_C167_4
dCas9 (i.e. no knockout)  - SgRNAs as barcodes for studying loss of clonality


title: dC9BL6_LungIV_C167_3 Lungs IV BL6 dCas9 CMT167 rep3

description:
Library name: dC9BL6_LungIV_C167_3
dCas9 (i.e. no knockout)  - SgRNAs as barcodes for studying loss of clonality


title: dC9BL6_LungIV_LLC1_5 Lungs IV BL6 dCas9 LLC1 rep5

description:
Library name: dC9BL6_LungIV_LLC1_5
dCas9 (i.e. no knockout)  - SgRNAs as barcodes for studying loss of clonality


title: dC9BL6_LungIV_C167_1 Lungs IV BL6 dCas9 CMT167 rep1

description:
Library name: dC9BL6_LungIV_C167_1
dCas9 (i.e. no knockout)  - SgRNAs as barcodes for studying loss of clonality


title: dC9BL6_LiverIV_LLC1_5 Liver IV BL6 dCas9 LLC1 rep5

description:
Library name: dC9BL6_LiverIV_LLC1_5
dCas9 (i.e. no knockout)  - SgRNAs as barcodes for studying loss of clonality


title: dC9BL6_LiverIV_LLC1_4 Liver IV BL6 dCas9 LLC1 rep4

description:
Library name: dC9BL6_LiverIV_LLC1_4
dCas9 (i.e. no knockout)  - SgRNAs as barcodes for studying loss of clonality


title: dC9BL6_LiverIV_LLC1_3 Liver IV BL6 dCas9 LLC1 rep3

description:
Library name: dC9BL6_LiverIV_LLC1_3
dCas9 (i.e. no knockout)  - SgRNAs as barcodes for studying loss of clonality


title: dC9BL6_LiverIV_LLC1_1 Liver IV BL6 dCas9 LLC1 rep1

description:
Library name: dC9BL6_LiverIV_LLC1_1
dCas9 (i.e. no knockout)  - SgRNAs as barcodes for studying loss of clonality


title: dC9BL6_LiverIV_C167_5 Liver IV BL6 dCas9 CMT167 rep5

description:
Library name: dC9BL6_LiverIV_C167_5
dCas9 (i.e. no knockout)  - SgRNAs as barcodes for studying loss of clonality


title: ColoCT26_StealTHY_Tum_2 CT26 StealTHY BALB TUMOR rep2

description:
Library name: ColoCT26_StealTHY_Tum_2
dCas9 (i.e. no knockout)  - SgRNAs as barcodes for studying loss of clonality


title: ColoCT26_StealTHY_Tum_3 CT26 StealTHY BALB TUMOR rep3

description:
Library name: ColoCT26_StealTHY_Tum_3
dCas9 (i.e. no knockout)  - SgRNAs as barcodes for studying loss of clonality


title: LLC1_StealTHY_Met_4 LLC1 StealTHY IV BL6 Liver Mets 4

description:
Library name: LLC1_StealTHY_Met_4
dCas9 (i.e. no knockout)  - SgRNAs as barcodes for studying loss of clonality


title: ColoCT26_StealTHY_Tum_1 CT26 StealTHY BALB TUMOR rep1

description:
Library name: ColoCT26_StealTHY_Tum_1
dCas9 (i.e. no knockout)  - SgRNAs as barcodes for studying loss of clonality


title: C167_Thy1_baseline C167 Thy1 baseline

description:
Library name: C167_Thy1_baseline
Thy1 baseline


title: C167_StealTHY_Tum_5 C167 StealTHY IV BL6 TUMOR 5

description:
Library name: C167_StealTHY_Tum_5
dCas9 (i.e. no knockout)  - SgRNAs as barcodes for studying loss of clonality


title: dC9BL6_LiverIV_C167_3 Liver IV BL6 dCas9 CMT167 rep3

description:
Library name: dC9BL6_LiverIV_C167_3
dCas9 (i.e. no knockout)  - SgRNAs as barcodes for studying loss of clonality


title: ColoCT26_StealTHY_Met_2 CT26 StealTHY BALB Lung Mets rep2

description:
Library name: ColoCT26_StealTHY_Met_2
dCas9 (i.e. no knockout)  - SgRNAs as barcodes for studying loss of clonality


title: LLC1_StealTHY_Met_2 LLC1 StealTHY IV BL6 Liver Mets 2

description:
Library name: LLC1_StealTHY_Met_2
dCas9 (i.e. no knockout)  - SgRNAs as barcodes for studying loss of clonality


title: dC9BL6_LungIV_C167_5 Lungs IV BL6 dCas9 CMT167 rep5

description:
Library name: dC9BL6_LungIV_C167_5
dCas9 (i.e. no knockout)  - SgRNAs as barcodes for studying loss of clonality


title: IFNG_with_cellboundary

type: RNA

city: Munich

first: Ruoqing

last: Feng

address: Feodor-Lynen-Strasse 17

institute: German Center for Neurodegenerative Diseases (DZNE)

description: panel of 500 genes was measured by MERFISH

hyb_protocol: We stained our microglia with Vizgen’s MERSCOPE Cell Boundary Staining Kit to label cell membranes prior to MERFISH imaging. To perform MERFISH probe hybridization, first wash slides twice in Vizgen Sample Prep Wash Buffer (5 min each at room temperature), then pre‐hybridize in Formamide Hybridization Buffer (30% formamide, 2× SSC, 10% dextran sulfate) at 37 °C for 30 min. Apply 50 μL of the custom 500-gene MERFISH probe mix onto each slide (secure under a gasket or hybridization chamber) and incubate at 37 °C for 36–48 h. After hybridization, wash twice in Formamide Wash Buffer (30% formamide, 2× SSC) at 37 °C for 5 min each, rinse in 2× SSC, embed in polyacrylamide gel per the manufacturer’s instructions, clear with Proteinase K premix, then counterstain with DAPI and PolyT reagents for 15 min at room temperature prior to imaging on the MERSCOPE platform

scan_protocol: A low-resolution mosaic was acquired using a x10 objective and regions of interest were selected for high-resolution imaging with a x60 lens. For high-resolution imaging, the focus was locked to the fiducial fluorescent beads on the coverslip. Seven 1.5-μm-thick Z-planes were imaged for each field of view. Raw images were decoded to RNA spots with spatial coordinates and gene IDs using the Merlin software (Vizgen) on the MERSCOPE instrument.

data_processing: Cell segmentation was performed using the Cellpose algorithm based in the DAPI nuclear and polyT total RNA staining channels. for the experiment with cell boundary stain, we use cell boundary2 output to segment cells.

source_name: Primary mouse microglia

growth_protocol: Primary mouse microglia were isolated from mixed‐gender C57BL/6J pups (7–15 days old) by enzymatic digestion of whole brains using the Miltenyi Biotec Neuronal Tissue Dissociation Kit, followed by filtration through a 40 µm nylon strainer to generate a single‐cell suspension. Cells were then incubated with anti-CD11b magnetic microbeads and positively selected on LS columns to enrich for microglia. The purified cells were plated in DMEM supplemented with 10% fetal bovine serum, 1% penicillin–streptomycin, 1 mM sodium pyruvate, and 10 ng/mL CSF-1, and maintained at 37 °C in 5% CO₂ until confluent. One hour before any downstream assay, cultures were serum-depleted to synchronize cell state. For spatial transcriptomic profiling via MERFISH, microglia were seeded onto Vizgen glass slides pre-coated for 1 h with 0.1 mg/mL poly-L-lysine in PBS and cultured in serum-free DMEM (1% penicillin–streptomycin, 1 mM sodium pyruvate, 10 ng/mL CSF-1) .

extract_protocol: Microglia—after serum-depletion—were gently resuspended in serum-free DMEM (1% penicillin–streptomycin, 1 mM sodium pyruvate, 10 ng/ml CSF-1) and plated directly onto glass slides supplied by Vizgen that had been coated for 1 hour at room temperature with 0.1 mg/ml poly-L-lysine in PBS. Slides were then incubated at 37 °C/5% CO₂ for 1 hour to allow cells to adhere, followed by a gentle PBS wash to remove non-attached cells. Fresh serum-free medium was added prior to fixation (4% PFA) and permeabilization, after which the MERFISH workflow was carried out .

treatment_protocol: Recombinant murine IFN-γ was first reconstituted at 10 µg/mL in sterile PBS with 0.1% BSA and stored in aliquots at –80 °C. For MERFISH experiments, primary microglia—previously serum-depleted for 1 h—were maintained on poly-L-lysine–coated Vizgen slides in serum-free DMEM (1% penicillin–streptomycin, 1 mM sodium pyruvate, 10 ng/mL CSF-1) at 37 °C/5% CO₂. Cultures were exposed to 1 µg/mL purified myelin for 30 min, then supplemented with 1 nM IFN-γ and incubated for 8 h before washing, fixation and downstream processing

value: IFNG

value: C57BL/6J, wild type


title: C167_StealTHY_Tum_3 C167 StealTHY IV BL6 TUMOR 3

description:
Library name: C167_StealTHY_Tum_3
dCas9 (i.e. no knockout)  - SgRNAs as barcodes for studying loss of clonality


title: C167_StealTHY_Tum_1 C167 StealTHY IV BL6 TUMOR 1

description:
Library name: C167_StealTHY_Tum_1
dCas9 (i.e. no knockout)  - SgRNAs as barcodes for studying loss of clonality


title: dC9BL6_LungIV_C167_2 Lungs IV BL6 dCas9 CMT167 rep2

description:
Library name: dC9BL6_LungIV_C167_2
dCas9 (i.e. no knockout)  - SgRNAs as barcodes for studying loss of clonality


title: control_with_cellboundary

value: control


title: C167_StealTHY_Met_5 C167 StealTHY IV BL6 Liver Mets 5

description:
Library name: C167_StealTHY_Met_5
dCas9 (i.e. no knockout)  - SgRNAs as barcodes for studying loss of clonality


title: C167_StealTHY_Tum_2 C167 StealTHY IV BL6 TUMOR 2

description:
Library name: C167_StealTHY_Tum_2
dCas9 (i.e. no knockout)  - SgRNAs as barcodes for studying loss of clonality


title: dC9BL6_LungIV_LLC1_1 Lungs IV BL6 dCas9 LLC1 rep1

description:
Library name: dC9BL6_LungIV_LLC1_1
dCas9 (i.e. no knockout)  - SgRNAs as barcodes for studying loss of clonality


title: control_without_cellboundary


title: C167_StealTHY_Met_4 C167 StealTHY IV BL6 Liver Mets 4

description:
Library name: C167_StealTHY_Met_4
dCas9 (i.e. no knockout)  - SgRNAs as barcodes for studying loss of clonality


title: C167_StealTHY_Met_3 C167 StealTHY IV BL6 Liver Mets 3

description:
Library name: C167_StealTHY_Met_3
dCas9 (i.e. no knockout)  - SgRNAs as barcodes for studying loss of clonality


title: UntreatRepl5 Drop clon CD4/CD8 deplet replicate 5

description:
Library name: UntreatRepl5
dCas9 (i.e. no knockout)  - SgRNAs as barcodes for studying loss of clonality


title: C167_StealTHY_Met_1 C167 StealTHY IV BL6 Liver Mets 1

description:
Library name: C167_StealTHY_Met_1
dCas9 (i.e. no knockout)  - SgRNAs as barcodes for studying loss of clonality


title: ms16r0

hyb_protocol: The gene panel used in this study included 500 protein-coding genes, complemented by 50 blank probes. Within this gene panel, a diverse array of transcripts comprising recognized markers for glial and immune cell types was included based on previously published literature. Sections were post-fixed with 4%PFA in PBS for 15 min at room temperature, washed three times with PBS and then permeabilized in 70% ethanol at 4 °C overnight. For hybridization with the gene panel, samples were washed with Sample Prep Wash Buffer (Vizgen) and then incubated in Formamide Hybridization Buffer at 37 °C for 30 min before adding 50 μl of the gene panel mix on top of the tissue. Sections were hybridized at 37 °C for 36 to 48 h, washed, and embedded into a polyacrylamide gel according to the manufacturer’s instructions. Samples were cleared with Clearing Premix containing Proteinase K, washed and stained with DAPI and polyT reagent for 15 min at room temperature.

data_processing:
Cell segmentation was performed using the Cellpose algorithm based in the DAPI nuclear and polyT total RNA staining channels.
Encoded MERFISH images were processed according to manufacturer's pipeline (https://vizgen.com/technology/#merfish). The output of this pipeline are spatial positions of individual transcripts and segmented single cells based on DAPI and poly-A stainings. Single cell expression tables are produced by counting individual transcripts within segmented cell boundaries.

source_name: White Matter, fresh frozen

extract_protocol: The brain tissue were punched by a Brain Puncher, and were cut into 10-µm-thick sections then put onto glass slides supplied by Vizgen.

value: White Matter, fresh frozen

tag: gender

value: Female

tag: ms duration


title: ColoCT26_StealTHY_Met_1 CT26 StealTHY BALB Lung Mets rep1

description:
Library name: ColoCT26_StealTHY_Met_1
dCas9 (i.e. no knockout)  - SgRNAs as barcodes for studying loss of clonality


title: C167_StealTHY_Tum_4 C167 StealTHY IV BL6 TUMOR 4

description:
Library name: C167_StealTHY_Tum_4
dCas9 (i.e. no knockout)  - SgRNAs as barcodes for studying loss of clonality


title: UntreatRepl4 Drop clon CD4/CD8 control replicate 4

description:
Library name: UntreatRepl4
dCas9 (i.e. no knockout)  - SgRNAs as barcodes for studying loss of clonality


title: UntreatRepl3 Drop clon CD4/CD8 control replicate 3

description:
Library name: UntreatRepl3
dCas9 (i.e. no knockout)  - SgRNAs as barcodes for studying loss of clonality


title: ms15r1

value: Male

tag: age


title: ms12r1


title: ms15r0


title: UntreatRepl2 Drop clon CD4/CD8 control replicate 2

description:
Library name: UntreatRepl2
dCas9 (i.e. no knockout)  - SgRNAs as barcodes for studying loss of clonality


title: UntreatRepl1 Drop clon CD4/CD8 control replicate 1

description:
Library name: UntreatRepl1
dCas9 (i.e. no knockout)  - SgRNAs as barcodes for studying loss of clonality


title: ms11r1

description: panel of 500 genes was measured by MERFISH


title: ms12r0


title: MVT1_dCas9baseline MVT1 dCas9 baseline

description:
Library name: MVT1_dCas9baseline
Puro+dCas9 baseline


title: Py2T_dCas9baseline Py2T dCas9 baseline

description:
Library name: Py2T_dCas9baseline
Puro+dCas9 baseline


title: ms7r1


title: ms10r0


title: ms16r1


title: ms8r0


title: LLC1_StealTHY LLC1 StealTHY post-Cas9

description:
Library name: LLC1_StealTHY
Thy1 pre-Cas9 transfection


title: ms6r1


title: LLC1_dCas9baseline LLC1 dCas9 baseline

description:
Library name: LLC1_dCas9baseline
Puro+dCas9 baseline


title: dCas4T1Repl5_NSGTum Figure 2D, tumor NSG replicate 5

description:
Library name: dCas4T1Repl5_NSGTum
dCas9 (i.e. no knockout)  - SgRNAs as barcodes for studying loss of clonality


title: ms6r0


title: dCas4T1Repl3_NSGTum Figure 2D, tumor NSG replicate 3

description:
Library name: dCas4T1Repl3_NSGTum
dCas9 (i.e. no knockout)  - SgRNAs as barcodes for studying loss of clonality


title: dCas4T1Repl4_NSGTum Figure 2D, tumor NSG replicate 4

description:
Library name: dCas4T1Repl4_NSGTum
dCas9 (i.e. no knockout)  - SgRNAs as barcodes for studying loss of clonality


title: ms5r1


title: ms4r2


title: IFNG_without_cellboundary

tag: strain


title: dCas4T1Repl2_NSGTum Figure 2D, tumor NSG replicate 2

description:
Library name: dCas4T1Repl2_NSGTum
dCas9 (i.e. no knockout)  - SgRNAs as barcodes for studying loss of clonality


title: dCas4T1Repl1_NSGTum Figure 2D, tumor NSG replicate 1

description:
Library name: dCas4T1Repl1_NSGTum
dCas9 (i.e. no knockout)  - SgRNAs as barcodes for studying loss of clonality


title: 230712_Flex_HC_4

city: Ann Arbor

first: Poulami

last: Dey

address: 1301 Catherine St

institute: Michigan Medicine

description: Library name: 8911-JF-16

data_processing:
The CellRanger Pipeline (10X Genomics) was used to align reads and generate count matrices.”
Assembly: GRCm38
Supplementary files format and content: count matrix

source_name: skin

extract_protocol:
Protocol Overview: gentleMACS Octo Dissociator for Isolation of Cells from FFPE [https://cdn.10xgenomics.com/image/upload/v1721078231/support-documents/CG000632_DemonstratedProtocol_SamplePrep_from__FFPETissueSections_RevD.pdf]
Tissue Sections;  https://cdn.10xgenomics.com/image/upload/v1722287750/support-documents/CG000527_Chromium_FixedRNAProfiling_MultiplexedSamples_UserGuide_Rev_F.pdf

value: skin

tag: disease state

value: healthy control


title: ms4r1

tag: tissue


title: dC9NSG_LungIV_LLC1_4 Lungs IV NSG dCas9 LLC1 replicate 4

description:
Library name: dC9NSG_LungIV_LLC1_4
dCas9 (i.e. no knockout)  - SgRNAs as barcodes for studying loss of clonality


title: dC9NSG_LungIV_LLC1_5 Lungs IV NSG dCas9 LLC1 replicate 5

description:
Library name: dC9NSG_LungIV_LLC1_5
dCas9 (i.e. no knockout)  - SgRNAs as barcodes for studying loss of clonality


title: 230712_Flex_HC_3

description: Library name: 8911-JF-15


title: dC9NSG_LungIV_LLC1_2 Lungs IV NSG dCas9 LLC1 replicate 2

description:
Library name: dC9NSG_LungIV_LLC1_2
dCas9 (i.e. no knockout)  - SgRNAs as barcodes for studying loss of clonality


title: ms1r1


title: dC9NSG_LungIV_C167_5 Lungs IV NSG dCas9 CMT167 replicate 5

description:
Library name: dC9NSG_LungIV_C167_5
dCas9 (i.e. no knockout)  - SgRNAs as barcodes for studying loss of clonality


title: 230712_Flex_HC_1

description: Library name: 8911-JF-13


title: dC9NSG_LungIV_C167_4 Lungs IV NSG dCas9 CMT167 replicate 4

description:
Library name: dC9NSG_LungIV_C167_4
dCas9 (i.e. no knockout)  - SgRNAs as barcodes for studying loss of clonality


title: dC9NSG_LungIV_C167_3 Lungs IV NSG dCas9 CMT167 replicate 3

description:
Library name: dC9NSG_LungIV_C167_3
dCas9 (i.e. no knockout)  - SgRNAs as barcodes for studying loss of clonality


title: dC9NSG_LungIV_LLC1_1 Lungs IV NSG dCas9 LLC1 replicate 1

description:
Library name: dC9NSG_LungIV_LLC1_1
dCas9 (i.e. no knockout)  - SgRNAs as barcodes for studying loss of clonality


title: dC9NSG_LungIV_C167_2 Lungs IV NSG dCas9 CMT167 replicate 2

description:
Library name: dC9NSG_LungIV_C167_2
dCas9 (i.e. no knockout)  - SgRNAs as barcodes for studying loss of clonality


title: 230628_Flex_GPP_9

description: Library name: 8821-JF-9

value: GPP


title: Liver, FFC diet, AAV8-TBG-Cre (control) 4

description: Library name: SV36

source_name: Liver

value: Liver

tag: genotype

value: Bak -/- Bax -/-

value: FFC diet


title: 230712_Flex_HC_2

description: Library name: 8911-JF-14


title: Liver, FFC diet, AAV8-TBG-Cre (control) 3

description: Library name: SV35


title: Liver, FFC diet, AAV8-TBG-Cre (control) 2

description: Library name: SV34


title: 230628_Flex_GPP_7

description: Library name: 8821-JF-7


title: dC9NSG_LiverIV_LLC1_2 Liver IV NSG dCas9 LLC1 replicate 2

description:
Library name: dC9NSG_LiverIV_LLC1_2
dCas9 (i.e. no knockout)  - SgRNAs as barcodes for studying loss of clonality

value: tumor

tag: origin

value: xenograft

value: Immuno Signalome (2215) sgRNA library


title: dC9NSG_LiverIV_LLC1_3 Liver IV NSG dCas9 LLC1 replicate 3

description:
Library name: dC9NSG_LiverIV_LLC1_3
dCas9 (i.e. no knockout)  - SgRNAs as barcodes for studying loss of clonality

tag: mouse model

value: NSG

value: sgRNAs used as barcodes (no KO)

value: lentiGuide-Puro+ dCas9


title: dC9NSG_LiverIV_C167_5 Liver IV NSG dCas9 CMT167 replicate 5

description:
Library name: dC9NSG_LiverIV_C167_5
dCas9 (i.e. no knockout)  - SgRNAs as barcodes for studying loss of clonality

tag: crispr lib


title: 230628_Flex_GPP_6

description: Library name: 8821-JF-6


title: Liver, FFC diet, AAV8-GFP (control) 3

description: Library name: SV32

value: Bak -/- Bax fl/fl


title: dC9NSG_LungIV_LLC1_3 Lungs IV NSG dCas9 LLC1 replicate 3

description:
Library name: dC9NSG_LungIV_LLC1_3
dCas9 (i.e. no knockout)  - SgRNAs as barcodes for studying loss of clonality

tag: sgrna library


title: ms1r2

description: panel of 500 genes was measured by MERFISH


title: dC9NSG_LiverIV_C167_4 Liver IV NSG dCas9 CMT167 replicate 4

description:
Library name: dC9NSG_LiverIV_C167_4
dCas9 (i.e. no knockout)  - SgRNAs as barcodes for studying loss of clonality

tag: effect type


title: Liver, FFC diet, AAV8-GFP (control) 2

description: Library name: SV31


title: dC9NSG_LiverIV_C167_3 Liver IV NSG dCas9 CMT167 replicate 3

description:
Library name: dC9NSG_LiverIV_C167_3
dCas9 (i.e. no knockout)  - SgRNAs as barcodes for studying loss of clonality

tag: vector


title: 230628_Flex_GPP_5

description: Library name: 8821-JF-5


title: dC9NSG_LiverIV_C167_2 Liver IV NSG dCas9 CMT167 replicate 2

description:
Library name: dC9NSG_LiverIV_C167_2
dCas9 (i.e. no knockout)  - SgRNAs as barcodes for studying loss of clonality

tag: full library_complexity


title: dC9NSG_LiverIV_LLC1_4 Liver IV NSG dCas9 LLC1 replicate 4

description:
Library name: dC9NSG_LiverIV_LLC1_4
dCas9 (i.e. no knockout)  - SgRNAs as barcodes for studying loss of clonality

tag: cell line


title: 230628_Flex_GPP_8

description: Library name: 8821-JF-8


title: dC9NSG_LiverIV_LLC1_5 Liver IV NSG dCas9 LLC1 replicate 5

description:
Library name: dC9NSG_LiverIV_LLC1_5
dCas9 (i.e. no knockout)  - SgRNAs as barcodes for studying loss of clonality

tag: cell type


title: Liver, FFC diet, AAV8-GFP (control) 1

description: Library name: SV30

tag: treatment


title: 230628_Flex_GPP_3

description: Library name: 8821-JF-3


title: C167_dCas9baseline C167 dCas9 baseline

description:
Library name: C167_dCas9baseline
Puro+dCas9 baseline


title: DENV2, Native, Unmodified Rep 2

description: Library name: DENV2_Control_R2

organism: Orthoflavivirus denguei

extract_protocol:
Dengue virions were collected from infected cells and then sujected to a sucrose gradient followed by ultracentrifugation. The resultant fraction was collected and RNA extraction was performed using Trizol.
MaP-RT was performed using random priming. Two rounds of PCR were performed to add Illumina adapter sequences and barcodes.

value: Native

value: Control


title: C167_StealTHY C167 StealTHY post-Cas9

description:
Library name: C167_StealTHY
Thy1 pre-Cas9 transfection

value: CRISPR KO


title: 230628_Flex_GPP_2

description: Library name: 8821-JF-2


title: 4T1_dCas9baseline 4T1 dCas9 baseline

description:
Library name: 4T1_dCas9baseline
Puro+dCas9 baseline


title: dC9NSG_LiverIV_LLC1_1 Liver IV NSG dCas9 LLC1 replicate 1

description:
Library name: dC9NSG_LiverIV_LLC1_1
dCas9 (i.e. no knockout)  - SgRNAs as barcodes for studying loss of clonality


title: Liver, FFC diet, AAV8-TBG-Cre (control) 1

description: Library name: SV33


title: PlatePositionH7

description: PlatePositionH7 StealTHY novel CRISPR KO - whole lungs using 4T1 into BALB (Imm.competent) mouse, with vector lentiGuide-Puro plus transient SpCas9 transduction Replicate 4

data_processing:
Mapping using bowtie2 (v2.3.5.1; parameters: -L 11 -N 1 -I S,1,0.75 --norc), allowing for one mismatch.
Normalized abundance of individual sgRNAs were obtained using MAGeCK (v0.5.9.4).
Assembly: lentiviral libraries encompassing 2215 sgRNAs against mouse immune interactome genes  or 2180 sgRNAs against the homologue human genes
Supplementary files format and content: counts.txt.gz are tab-delimited files containing raw counts for each library
Library strategy: CRISPR-seq

source_name: CRISPR Knockout (by Cas9 transfection) through the StealTHY system

value: BALBc

value: lentiGuide-Puro


title: PlatePositionH9

description: PlatePositionH9 StealTHY novel CRISPR KO - whole lungs using Py2T into FVB (Imm.competent) mouse, with vector lentiGuide-Puro plus transient SpCas9 transduction Replicate 4

value: FVB


title: PlatePositionH5

description: PlatePositionH5 GoldStandard CRISPR KO - whole lungs using 4T1 into BALB (Imm.competent) mouse, with vector lentiGuide-Puro plus stable SpCas9 transduction, Replicate 5

source_name: CRISPR Knockout GoldStandard

value: lentiGuide-Puro + EF1alpha_SpCas9


title: 230628_Flex_GPP_13

description: Library name: 8821-JF-13

value: skin

value: GPP


title: DENV2, Native, Unmodified Rep 1

description: Library name: DENV2_Control_R1


title: 230628_Flex_GPP_12

description: Library name: 8821-JF-12


title: PlatePositionH4

description: PlatePositionH4 GoldStandard CRISPR KO - whole lungs using 4T1 into BALB (Imm.competent) mouse, with vector lentiGuide-Puro plus stable SpCas9 transduction, Replicate 1


title: PlatePositionH3

description: PlatePositionH3 GoldStandard CRISPR KO - whole lungs using 4T1 into NSG (Imm.deficient) mouse, with vector lentiGuide-Puro plus stable SpCas9 transduction, Replicate 2


title: DENV2, Native, DMS modified Rep 1

description: Library name: DENV2_DMS_R1

value: DMS


title: 230628_Flex_GPP_4

description: Library name: 8821-JF-4


title: PlatePositionH2

description: PlatePositionH2 dCas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality - whole lungs using 4T1 into BALB (Imm.competent) mouse, with vector lentiGuide-Puro plus stable dCas9 transduction, Replicate 3

value: tumor

value: lentiGuide-Puro + EF1alpha_dCas9


title: dC9NSG_LiverIV_C167_1 Liver IV NSG dCas9 CMT167 replicate 1

description:
Library name: dC9NSG_LiverIV_C167_1
dCas9 (i.e. no knockout)  - SgRNAs as barcodes for studying loss of clonality

value: xenograft

value: Immuno Signalome (2215) sgRNA library


title: CT26_StealTHY CT26 StealTHY post-Cas9

description:
Library name: CT26_StealTHY
Thy1 pre-Cas9 transfection


title: DENV2, Native, TMO modified Rep 1

description: Library name: DENV2_TMO_R1

value: TMO


title: PlatePositionH10

description: PlatePositionH10 4T1 Thy1 post-Cas9 transfection, Replicate 3

source_name: Thy1 post-Cas9 transfection

value: In vitro


title: DENV2, Native, DMS modified Rep 2

description: Library name: DENV2_DMS_R2


title: PlatePositionH1

description: PlatePositionH1 dCas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality - whole lungs using 4T1 into NSG (Imm.deficient) mouse, with vector lentiGuide-Puro plus stable dCas9 transduction, Replicate 4

value: NSG

value: sgRNAs used as barcodes (no KO)


title: 230628_Flex_GPP_10

description: Library name: 8821-JF-10


title: PlatePositionG8

description: PlatePositionG8 StealTHY novel CRISPR KO - whole lungs using MVT1 into FVB (Imm.competent) mouse, with vector lentiGuide-Puro plus transient SpCas9 transduction Replicate 3


title: PlatePositionG7

description: PlatePositionG7 StealTHY novel CRISPR KO - whole lungs using 4T1 into BALB (Imm.competent) mouse, with vector lentiGuide-Puro plus transient SpCas9 transduction Replicate 3


title: PlatePositionG6

description: PlatePositionG6 GoldStandard CRISPR KO - whole tumor using 4T1 into BALB (Imm.competent) mouse, with vector lentiGuide-Puro plus stable SpCas9 transduction, Replicate 9


title: 230628_Flex_GPP_1

description: Library name: 8821-JF-1


title: untreated motor neurons, treated, GFP negative, bio rep 3

city: Middletown

first: Alison

middle: Linsley

last: ONeil

state: CT

address: 52 Lawn Ave

institute: Wesleyan University

description:
Library name: T3N
Column name in CCMN_Featurecounts_FINAL.csv: Treated_GFP_neg_3

data_processing:
Galaxy 2018 Update
The sample libraries for replicates run in separate lanes were concatenated into individual technical replicates using Concatenate datasets
Reads were mapped to the built-in hg38 human genome on Galaxy using HISAT2
Assembly: Homo_sapiens.GRCh38.103.gtf.gz
Supplementary files format and content: Featurecounts

source_name: embryonic stem cell

growth_protocol: human embryonic stem cells were differentiated to motor neurons following a 16 day protocol (Rigamonti, 2016), dissociated to single cells, and plated on matrigel coated dishes in complete medium with Rock inhibitor. After 24 hours, Rock was removed and the cells were incubated for an additional 48 hours. Media was then changed to contain 10uM cis-chlordane or a matching volume of DMSO (solvent) and incubated for 16 hours. After treatment, cells were lifted and dissociated from their plates using trypsin and then FACS sorted based on GFP signal.

extract_protocol:
Trizol extraction
Illumina Stranded mRNA library Prep workflow

treatment_protocol: 16 hours, at 37C, 5% Co2

value: embryonic stem cell

value: motor neuron

value: wild-type

value: 10 uM cis-chlordane 16 hours


title: PlatePositionG4

description: PlatePositionG4 GoldStandard CRISPR KO - whole tumor using 4T1 into BALB (Imm.competent) mouse, with vector lentiGuide-Puro plus stable SpCas9 transduction, Replicate 1

value: BALBc

value: CRISPR KO


title: untreated motor neurons, treated, GFP positive, bio rep 3

description:
Library name: T3P
Column name in CCMN_Featurecounts_FINAL.csv: Treated_GFP_pos_3


title: PlatePositionG9

description: PlatePositionG9 StealTHY novel CRISPR KO - whole lungs using Py2T into FVB (Imm.competent) mouse, with vector lentiGuide-Puro plus transient SpCas9 transduction Replicate 3

value: FVB

value: lentiGuide-Puro


title: DENV2, Native, TMO modified Rep 2

description: Library name: DENV2_TMO_R2

value: Native


title: PlatePositionG5

description: PlatePositionG5 GoldStandard CRISPR KO - whole tumor using 4T1 into BALB (Imm.competent) mouse, with vector lentiGuide-Puro plus stable SpCas9 transduction, Replicate 5

value: tumor

value: lentiGuide-Puro + EF1alpha_SpCas9


title: PlatePositionH6

description: PlatePositionH6 GoldStandard CRISPR KO - whole lungs using 4T1 into BALB (Imm.competent) mouse, with vector lentiGuide-Puro plus stable SpCas9 transduction, Replicate 9

value: xenograft


title: PlatePositionG3

description: PlatePositionG3 GoldStandard CRISPR KO - whole tumor using 4T1 into NSG (Imm.deficient) mouse, with vector lentiGuide-Puro plus stable SpCas9 transduction, Replicate 2

value: Immuno Signalome (2215) sgRNA library


title: untreated motor neurons, treated, GFP negative, bio rep 2

description:
Library name: T2N
Column name in CCMN_Featurecounts_FINAL.csv: Treated_GFP_neg_2


title: PlatePositionG1

description: PlatePositionG1 dCas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality - whole tumor using 4T1 into NSG (Imm.deficient) mouse, with vector lentiGuide-Puro plus stable dCas9 transduction, Replicate 4

value: lentiGuide-Puro + EF1alpha_dCas9


title: untreated motor neurons, treated, GFP negative, bio rep 1

description:
Library name: T1N
Column name in CCMN_Featurecounts_FINAL.csv: Treated_GFP_neg_1


title: untreated motor neurons, treated, GFP positive, bio rep 1

description:
Library name: T1P
Column name in CCMN_Featurecounts_FINAL.csv: Treated_GFP_pos_1


title: PlatePositionF8

description: PlatePositionF8 StealTHY novel CRISPR KO - whole lungs using MVT1 into FVB (Imm.competent) mouse, with vector lentiGuide-Puro plus transient SpCas9 transduction Replicate 2


title: 230628_Flex_GPP_11

description: Library name: 8821-JF-11

value: skin

value: GPP


title: PlatePositionG10

description: PlatePositionG10 4T1 Thy1 post-Cas9 transfection, Replicate 2

value: In vitro


title: PlatePositionF4

description: PlatePositionF4 GoldStandard CRISPR KO - whole lungs using 4T1 into NSG (Imm.deficient) mouse, with vector lentiGuide-Puro plus stable SpCas9 transduction, Replicate 5

value: NSG


title: PlatePositionF9

description: PlatePositionF9 StealTHY novel CRISPR KO - whole lungs using Py2T into FVB (Imm.competent) mouse, with vector lentiGuide-Puro plus transient SpCas9 transduction Replicate 2


title: untreated motor neurons, control, GFP negative, bio rep 3

description:
Library name: C3N
Column name in CCMN_Featurecounts_FINAL.csv: Control_GFP_neg_3

value: embryonic stem cell

value: motor neuron

value: control


title: untreated motor neurons, control, GFP positive, bio rep 3

description:
Library name: C3P
Column name in CCMN_Featurecounts_FINAL.csv: Control_GFP_pos_3

value: wild-type


title: PlatePositionF2

description: PlatePositionF2 dCas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality - whole lungs using 4T1 into BALB (Imm.competent) mouse, with vector lentiGuide-Puro plus stable dCas9 transduction, Replicate 2

value: BALBc

value: sgRNAs used as barcodes (no KO)


title: PlatePositionF1

description: PlatePositionF1 dCas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality - whole lungs using 4T1 into NSG (Imm.deficient) mouse, with vector lentiGuide-Puro plus stable dCas9 transduction, Replicate 3


title: PlatePositionF10

description: PlatePositionF10 dCas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality - whole lungs using 4T1 into BALB (Imm.competent) mouse, with vector lentiGuide-Puro plus stable dCas9 transduction, IgG treatment, Replicate 3

source_name: No Cas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality

value: lentiGuide-Thy1 + EF1alpha_dCas9


title: PlatePositionF5

description: PlatePositionF5 GoldStandard CRISPR KO - whole lungs using 4T1 into BALB (Imm.competent) mouse, with vector lentiGuide-Puro plus stable SpCas9 transduction, Replicate 4

value: CRISPR KO


title: PlatePositionF6

description: PlatePositionF6 GoldStandard CRISPR KO - whole lungs using 4T1 into BALB (Imm.competent) mouse, with vector lentiGuide-Puro plus stable SpCas9 transduction, Replicate 8

value: tumor


title: untreated motor neurons, control, GFP positive, bio rep 2

description:
Library name: C2P
Column name in CCMN_Featurecounts_FINAL.csv: Control_GFP_pos_2


title: untreated motor neurons, control, GFP negative, bio rep 2

description:
Library name: C2N
Column name in CCMN_Featurecounts_FINAL.csv: Control_GFP_neg_2


title: PlatePositionE9

description: PlatePositionE9 StealTHY novel CRISPR KO - whole lungs using Py2T into FVB (Imm.competent) mouse, with vector lentiGuide-Puro plus transient SpCas9 transduction Replicate 1

value: FVB

value: lentiGuide-Puro


title: untreated motor neurons, treated, GFP positive, bio rep 2

description:
Library name: T2P
Column name in CCMN_Featurecounts_FINAL.csv: Treated_GFP_pos_2

value: 10 uM cis-chlordane 16 hours


title: untreated motor neurons, control, GFP positive, bio rep 1

description:
Library name: C1P
Column name in CCMN_Featurecounts_FINAL.csv: Control_GFP_pos_1


title: PlatePositionE8

description: PlatePositionE8 StealTHY novel CRISPR KO - whole lungs using MVT1 into FVB (Imm.competent) mouse, with vector lentiGuide-Puro plus transient SpCas9 transduction Replicate 1

value: xenograft

value: Immuno Signalome (2215) sgRNA library


title: PlatePositionG2

description: PlatePositionG2 dCas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality - whole tumor using 4T1 into BALB (Imm.competent) mouse, with vector lentiGuide-Puro plus stable dCas9 transduction, Replicate 3

value: lentiGuide-Puro + EF1alpha_dCas9


title: PlatePositionE6

description: PlatePositionE6 GoldStandard CRISPR KO - whole tumor using 4T1 into BALB (Imm.competent) mouse, with vector lentiGuide-Puro plus stable SpCas9 transduction, Replicate 8

value: lentiGuide-Puro + EF1alpha_SpCas9


title: PlatePositionF3

description: PlatePositionF3 GoldStandard CRISPR KO - whole lungs using 4T1 into BALBc (Imm.competent) mouse, with vector lentiGuide-Puro plus stable SpCas9 transduction, Replicate 1


title: untreated motor neurons, control, GFP negative, bio rep 1

description:
Library name: C1N
Column name in CCMN_Featurecounts_FINAL.csv: Control_GFP_neg_1


title: PlatePositionE7

description: PlatePositionE7 StealTHY novel CRISPR KO - whole lungs using 4T1 into BALB (Imm.competent) mouse, with vector lentiGuide-Puro plus transient SpCas9 transduction Replicate 1


title: PlatePositionE5

description: PlatePositionE5 GoldStandard CRISPR KO - whole tumor using 4T1 into BALB (Imm.competent) mouse, with vector lentiGuide-Puro plus stable SpCas9 transduction, Replicate 4


title: Ctrl3_snRNA-seq

description: Library name: Ctrl3

data_processing:
Sequencing was carried out on an Illumina NovaSeq 6000, aiming for a depth of 30,000 reads per nucleus. Sequencing reads were aligned to the human reference genome (GRCh38) using the CellRanger pipeline (v7.1.0) with the --include-introns option to account for intronic reads. This process yielded a snRNA-seq dataset comprising around 20,000 nuclei per sample.
Assembly: GRCh38
Supplementary files format and content: sparse matrix h5

source_name: Brain

extract_protocol:
Fresh frozen human tissue samples were sectioned into 80- to 100-µm-thick slices using a cryostat at -20 °C to prepare them for single-nucleus isolation. For each sample, tissue slices were weighed until the total weight was approximately 25 mg, ensuring consistent processing across samples. White matter generates significantly more debris than other tissue types, necessitating additional processing steps for efficient nuclei isolation. Tissue dissociation and nuclei isolation were conducted using the 10x Genomics Chromium Nuclei Isolation Kit with RNase Inhibitor (PN-1000494) following the manufacturer’s protocol, with modifications tailored to optimize yield and purity. The frozen 1.5 ml tube containing the tissue was placed directly on ice. 200 µl of lysis buffer were immediately added to the tube to prevent tissue degradation. Tissue was dissociated using a plastic pestle until the mixture was homogeneous. For samples weighing around 25 mg, ~20 up-and-down strokes with the pestle were sufficient. 300 µl of additional lysis buffer were added and the solution was pipette-mixed 10 times. The dissociated tissue was incubated on ice for 10 minutes and transferred to a pre-chilled nuclei isolation column assembled with a collection tube. The column was centrifuged at 16,000 × g for 20 seconds at 4 °C. The flowthrough was vortexed at maximum speed for 10 seconds to resuspend nuclei. The collection tube was centrifuged at 500 × g for 3 minutes at 4 °C. The supernatant was carefully discarded, leaving ~200 µl behind. Two-step debris removal was performed by resuspending the nuclei pellet in 310 µl of washing buffer and 90 µl of debris removal solution (Miltenyi Biotech), overlaying with 400 µl of cold washing buffer, and centrifugation at 3,000 × g for 10 minutes. The debris layer and the liquid above were discarded (the pellet may not be visible for white matter tissues). Nuclei pellets were resuspended in 500 µl of debris removal buffer, gently pipette-mixed at least 15 times until no pellet was visible and centrifuged at 700 × g for 10 minutes at 4 °C. The supernatant was discarded, leaving ~200 µl behind, and resuspended in 1 ml of wash and resuspension buffer. After centrifugation at 500 × g for 5 minutes at 4°C, the supernatant was discarded, and the pellet resuspended in a low volume (50 µl). Propidium iodide (PI) staining was used for nuclei counting before proceeding with further steps.
Nucleis were further processed using the Chromium Single Cell 3’Reagent Kit v3.1 (10x Genomics). Following the manufacturer's protocol, we performed cell capture, barcoding, reverse transcription, cDNA amplification, and library preparation.

value: Brain


title: PlatePositionE4

description: PlatePositionE4 GoldStandard CRISPR KO - whole tumor using 4T1 into NSG (Imm.deficient) mouse, with vector lentiGuide-Puro plus stable SpCas9 transduction, Replicate 5

value: NSG


title: PlatePositionE3

description: PlatePositionE3 GoldStandard CRISPR KO - whole tumor using 4T1 into NSG (Imm.deficient) mouse, with vector lentiGuide-Puro plus stable SpCas9 transduction, Replicate 1


title: PlatePositionF7

description: PlatePositionF7 StealTHY novel CRISPR KO - whole lungs using 4T1 into BALB (Imm.competent) mouse, with vector lentiGuide-Puro plus transient SpCas9 transduction Replicate 2


title: PlatePositionE2

description: PlatePositionE2 dCas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality - whole tumor using 4T1 into BALB (Imm.competent) mouse, with vector lentiGuide-Puro plus stable dCas9 transduction, Replicate 2


title: PlatePositionE10

description: PlatePositionE10 dCas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality - whole lungs using 4T1 into BALB (Imm.competent) mouse, with vector lentiGuide-Puro plus stable dCas9 transduction, CD4/CD8 treatment, Replicate 2


title: Ctrl2_snRNA-seq

description: Library name: Ctrl2


title: PlatePositionE1

description: PlatePositionE1 dCas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality - whole tumor using 4T1 into NSG (Imm.deficient) mouse, with vector lentiGuide-Puro plus stable dCas9 transduction, Replicate 3


title: Ctrl1_snRNA-seq

description: Library name: Ctrl1


title: PlatePositionD9

description: PlatePositionD9 StealTHY novel CRISPR KO - whole tumor using Py2T into FVB (Imm.competent) mouse, with vector lentiGuide-Puro plus transient SpCas9 transduction Replicate 4


title: PlatePositionD7

description: PlatePositionD7 StealTHY novel CRISPR KO - whole tumor using 4T1 into BALB (Imm.competent) mouse, with vector lentiGuide-Puro plus transient SpCas9 transduction Replicate 4


title: MS14_snRNA-seq

description: Library name: MS14


title: PlatePositionD5

description: PlatePositionD5 GoldStandard CRISPR KO - whole lungs using 4T1 into BALB (Imm.competent) mouse, with vector lentiGuide-Puro plus stable SpCas9 transduction, Replicate 3


title: PlatePositionD6

description: PlatePositionD6 GoldStandard CRISPR KO - whole lungs using 4T1 into BALB (Imm.competent) mouse, with vector lentiGuide-Puro plus stable SpCas9 transduction, Replicate 7


title: MS12_snRNA-seq

description: Library name: MS12


title: MS13_snRNA-seq

description: Library name: MS13


title: PlatePositionD2

description: PlatePositionD2 dCas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality - whole lungs using 4T1 into BALB (Imm.competent) mouse, with vector lentiGuide-Puro plus stable dCas9 transduction, Replicate 1


title: PlatePositionD11

description: PlatePositionD11 Py2T Thy1 post-Cas9 transfection, Replicate 3

value: In vitro


title: PlatePositionD3

description: PlatePositionD3 dCas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality - whole lungs using 4T1 into BALB (Imm.competent) mouse, with vector lentiGuide-Puro plus stable dCas9 transduction, Replicate 5


title: MS11_snRNA-seq

description: Library name: MS11


title: MS10_snRNA-seq

description: Library name: MS10


title: PlatePositionC8

description: PlatePositionC8 StealTHY novel CRISPR KO - whole tumor using MVT1 into FVB (Imm.competent) mouse, with vector lentiGuide-Puro plus transient SpCas9 transduction Replicate 3


title: PlatePositionC9

description: PlatePositionC9 StealTHY novel CRISPR KO - whole tumor using Py2T into FVB (Imm.competent) mouse, with vector lentiGuide-Puro plus transient SpCas9 transduction Replicate 3


title: MS9_snRNA-seq

description: Library name: MS9


title: PlatePositionC6

description: PlatePositionC6 GoldStandard CRISPR KO - whole tumor using 4T1 into BALB (Imm.competent) mouse, with vector lentiGuide-Puro plus stable SpCas9 transduction, Replicate 7


title: PlatePositionC7

description: PlatePositionC7 StealTHY novel CRISPR KO - whole tumor using 4T1 into BALB (Imm.competent) mouse, with vector lentiGuide-Puro plus transient SpCas9 transduction Replicate 3


title: PlatePositionD10

description: PlatePositionD10 dCas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality - whole lungs using 4T1 into BALB (Imm.competent) mouse, with vector lentiGuide-Puro plus stable dCas9 transduction, IgG treatment, Replicate 3


title: MS8_snRNA-seq

description: Library name: MS8


title: PlatePositionC3

description: PlatePositionC3 dCas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality - whole tumor using 4T1 into BALB (Imm.competent) mouse, with vector lentiGuide-Puro plus stable dCas9 transduction, Replicate 5


title: PlatePositionC10

description: PlatePositionC10 dCas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality - whole lungs using 4T1 into BALB (Imm.competent) mouse, with vector lentiGuide-Puro plus stable dCas9 transduction, CD4/CD8 treatment, Replicate 2


title: PlatePositionC4

description: PlatePositionC4 GoldStandard CRISPR KO - whole tumor using 4T1 into NSG (Imm.deficient) mouse, with vector lentiGuide-Puro plus stable SpCas9 transduction, Replicate 4


title: PlatePositionC11

description: PlatePositionC11 Py2T Thy1 post-Cas9 transfection, Replicate 2


title: PlatePositionC2

description: PlatePositionC2 dCas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality - whole tumor using 4T1 into BALB (Imm.competent) mouse, with vector lentiGuide-Puro plus stable dCas9 transduction, Replicate 1


title: MS7_snRNA-seq

description: Library name: MS7


title: MS5_snRNA-seq

description: Library name: MS5


title: MS6_snRNA-seq

description: Library name: MS6


title: PlatePositionB8

description: PlatePositionB8 StealTHY novel CRISPR KO - whole tumor using MVT1 into FVB (Imm.competent) mouse, with vector lentiGuide-Puro plus transient SpCas9 transduction Replicate 2


title: PlatePositionD4

description: PlatePositionD4 GoldStandard CRISPR KO - whole lungs using 4T1 into NSG (Imm.deficient) mouse, with vector lentiGuide-Puro plus stable SpCas9 transduction, Replicate 4


title: PlatePositionB9

description: PlatePositionB9 StealTHY novel CRISPR KO - whole tumor using Py2T into FVB (Imm.competent) mouse, with vector lentiGuide-Puro plus transient SpCas9 transduction Replicate 2


title: PlatePositionC1

description: PlatePositionC1 dCas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality - whole tumor using 4T1 into NSG (Imm.deficient) mouse, with vector lentiGuide-Puro plus stable dCas9 transduction, Replicate 2


title: PlatePositionD1

description: PlatePositionD1 dCas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality - whole lungs using 4T1 into NSG (Imm.deficient) mouse, with vector lentiGuide-Puro plus stable dCas9 transduction, Replicate 2


title: MS3_snRNA-seq

description: Library name: MS3


title: PlatePositionB6

description: PlatePositionB6 GoldStandard CRISPR KO - whole lungs using 4T1 into NSG (Imm.deficient) mouse, with vector lentiGuide-Puro plus stable SpCas9 transduction, Replicate 6


title: PlatePositionB4

description: PlatePositionB4 GoldStandard CRISPR KO - whole lungs using 4T1 into NSG (Imm.deficient) mouse, with vector lentiGuide-Puro plus stable SpCas9 transduction, Replicate 3


title: MS1_snRNA-seq

description: Library name: MS1


title: PlatePositionB5

description: PlatePositionB5 GoldStandard CRISPR KO - whole lungs using 4T1 into BALB (Imm.competent) mouse, with vector lentiGuide-Puro plus stable SpCas9 transduction, Replicate 2


title: PlatePositionB2

description: PlatePositionB2 dCas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality - whole lungs using 4T1 into NSG (Imm.deficient) mouse, with vector lentiGuide-Puro plus stable dCas9 transduction, Replicate 5


title: MS4_snRNA-seq

description: Library name: MS4


title: MS2_snRNA-seq

description: Library name: MS2


title: PlatePositionB7

description: PlatePositionB7 StealTHY novel CRISPR KO - whole tumor using 4T1 into BALB (Imm.competent) mouse, with vector lentiGuide-Puro plus transient SpCas9 transduction Replicate 2


title: PlatePositionC5

description: PlatePositionC5 GoldStandard CRISPR KO - whole tumor using 4T1 into BALB (Imm.competent) mouse, with vector lentiGuide-Puro plus stable SpCas9 transduction, Replicate 3


title: PlatePositionB10

description: PlatePositionB10 dCas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality - whole lungs using 4T1 into BALB (Imm.competent) mouse, with vector lentiGuide-Puro plus stable dCas9 transduction, IgG treatment, Replicate 1


title: PlatePositionB11

description: PlatePositionB11 MVT1 Thy1 post-Cas9 transfection, Replicate 3


title: PlatePositionB1

description: PlatePositionB1 dCas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality - whole lungs using 4T1 into NSG (Imm.deficient) mouse, with vector lentiGuide-Puro plus stable dCas9 transduction, Replicate 1


title: H3K27me3_PKO_gOE

city: Montréal

first: Vincent

last: Giguère

state: QC

address: 1160 Pine Ave West, McIntyre Bldg Room 710

department: Goodman Cancer Centre

country: Canada

institute: McGill University

description: Library name: Sample 8

data_processing:
ChIP-seq reads were first trimmed for adapter sequences and low-quality score bases using Trimmomatic v0.36. The resulting reads were mapped to the human reference genome (hg38) using BWA-MEM v0.7.12 in paired-end mode at default parameters. Only reads that had a unique alignment (mapping quality > 20) were retained and PCR duplicates were removed using Picard tools v2.0.1.
ERRγ peaks in PTEN-ERRγ+ (sgPTEN+ERRg) DU145 cells were called using MACS2 software suite v2.1.1.20160309 at an FDR < 0.05 using sequenced libraries of ERRγ ChIP'd DNA from PTEN-ERRγlow (sgPTEN) DU145 cells as control. H3K27ac and H3K27me3 peaks in PTEN-ERRγ+ (sgPTEN+ERRg) and PTEN-ERRγlow (sgPTEN) DU145 cells were called using MACS2 software suite v2.1.1.20160309 at an FDR < 0.05 using sequenced libraries of input DNA from the respective DU145 cell lines. Peaks in mitochondrial chromosome and scaffold regions were removed.
Peak annotations, intersections, bed files and de novo motif enrichment analyses were performed using HOMER v4.11.1 and binding intensity heatmaps were generated using deepTools v3.5.0.
Assembly: hg38
Supplementary files format and content: Bedgraph and bigwig for ChIP-seq samples as well as ERRγ annotated ChIP-seq peak text file for PTEN-ERRγ+ (sgPTEN+ERRγ) DU145 cells and H3K27ac and H3K27me3 annotated ChIP-seq test files for both PTEN-ERRγ+ (sgPTEN+ERRγ) and PTEN-ERRγlow (sgPTEN) DU145 cells

growth_protocol: The engineerred DU145 cell lines were maintained in Phenol Red-free RPMI-1640 medium (Wisent, Cat#350-046-CL) supplemented with 10% FBS (Thermo Fisher Scientific, Cat#12483020), 2 mM sodium pyruvate, 100 U/mL penicillin, and 100 μg/mL streptomycin.

extract_protocol:
ChIP samples for ChIP-seq were prepared using the ChIP-IT High Sensitivity kit (Active Motif, Cat#53040).
Libraries were prepared at Novogene Bioinformatics Technology Co., Ltd using the NEBNext Ultra II DNA Library Prep kit according to manufacturer's recommendations and sequenced as paired-end 150bp reads on an Illumina NovaSeq 6000 sequencer.

treatment_protocol: For PTEN knockout using the CRISPR/Cas9 system, sgRNA plasmids targeting human control CRISPR KO plasmid and human PTEN were purchased from Santa Cruz Biotechnology (Santa Cruz, Cat#sc-418922 and Cat#sc-400103, respectively). PTEN knockout was achieved to establish a PTEN-ERRγlow (sgPTEN) cell line from the parental DU145 cell line (PTEN+ERRγlow, sgC). ERRγ was overexpressed in the PTEN-ERRγlow DU145 cell line using lentivirus to establish a PTEN-ERRγ+ (sgPTEN+ERRg) cell line.

value: Human prostate cancer

value: sgPTEN+ERRg

value: CRISPR/Cas9-mediated PTEN knockout + lentivirus-mediated overexpression of ERRγ

tag: chip antibody

value: H3K27me3 antibody:Abcam, Cat# ab6002


title: H3K27ac_PKO_gOE

description: Library name: Sample 7

value: H3K27ac antibody:Abcam, Cat# ab4729


title: PlatePositionA9

description: PlatePositionA9 StealTHY novel CRISPR KO - whole tumor using Py2T into FVB (Imm.competent) mouse, with vector lentiGuide-Puro plus transient SpCas9 transduction Replicate 1

value: CRISPR KO


title: input_PKO_gOE

description: Library name: Sample 6