title: PlatePositionA6

description: PlatePositionA6 GoldStandard CRISPR KO - whole tumor using 4T1 into BALB (Imm.competent) mouse, with vector lentiGuide-Puro plus stable SpCas9 transduction, Replicate 6

value: tumor

value: BALBc


title: PlatePositionA7

status: Public on Oct 29 2025

type: SRA

city: Zurich

first: Francesc

last: Castro-Giner

address: Otto-Stern-Weg 7

country: Switzerland

institute: ETH Zurich

description: PlatePositionA7 StealTHY novel CRISPR KO - whole tumor using 4T1 into BALB (Imm.competent) mouse, with vector lentiGuide-Puro plus transient SpCas9 transduction Replicate 1

library_source: genomic

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

molecule: genomic DNA

organism: Mus musculus

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

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).

tag: tissue

tag: cell line

tag: cell type

tag: genotype

value: xenograft

tag: treatment

tag: crispr lib

value: Immuno Signalome (2215) sgRNA library

tag: sgrna library

tag: effect type

value: CRISPR KO

tag: vector

value: lentiGuide-Puro

tag: full library_complexity


title: PlatePositionA4

description: PlatePositionA4 GoldStandard CRISPR KO - whole tumor using 4T1 into NSG (Imm.deficient) mouse, with vector lentiGuide-Puro plus stable SpCas9 transduction, Replicate 3

source_name: CRISPR Knockout GoldStandard

value: NSG

value: lentiGuide-Puro + EF1alpha_SpCas9


title: PlatePositionA5

description: PlatePositionA5 GoldStandard CRISPR KO - whole tumor using 4T1 into BALB (Imm.competent) mouse, with vector lentiGuide-Puro plus stable SpCas9 transduction, Replicate 2


title: PlatePositionA8

description: PlatePositionA8 StealTHY novel CRISPR KO - whole tumor using MVT1 into FVB (Imm.competent) mouse, with vector lentiGuide-Puro plus transient SpCas9 transduction Replicate 1

value: FVB


title: H3K27ac_PTEN_KO

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 4

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

organism: Homo sapiens

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

value: CRISPR/Cas9-mediated PTEN knockout

tag: chip antibody

value: H3K27ac antibody:Abcam, Cat# ab4729


title: PlatePositionA2

description: PlatePositionA2 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 5

value: sgRNAs used as barcodes (no KO)

value: lentiGuide-Puro + EF1alpha_dCas9


title: PlatePositionA1

description: PlatePositionA1 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 1


title: ERRg_PKO_gOE

description: Library name: Sample 2

value: sgPTEN+ERRg

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

value: Custom ERRγ antibody (gift from Dr. Anastasia Kralli, Johns Hopkins University, USA)


title: PlatePositionA3

description: PlatePositionA3 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 4

value: tumor

value: BALBc


title: PlatePositionA11

description: PlatePositionA11 MVT1 Thy1 post-Cas9 transfection, Replicate 2

source_name: Thy1 post-Cas9 transfection

value: In vitro

value: Immuno Signalome (2215) sgRNA library


title: H3K27me3_PTEN_KO

description: Library name: Sample 5

value: H3K27me3 antibody:Abcam, Cat# ab6002


title: SPC KP tumors

city: Rochester

first: Cheng

last: Zhang

state: MN

address: 200 1st street SW

country: USA

institute: Mayo Clinic

description:
Library name: SPC_KP
10X Genomics

library_source: transcriptomic single cell

data_processing:
Sequencing output was demultiplexed and aligned to the mouse reference genome GRCm38 using Cell Ranger.
Assembly: GRCm38
Supplementary files format and content: filtered feature-barcode matrix in hdf5 format

molecule: total RNA

source_name: LUAD tumor

extract_protocol:
Individual tumors were microdissected from ~30-week KP and ~40-week KPI animals (n = 5 tumors/animal; 2 animals/genotype). Microdissected tumors were dissociated using the Lung Dissociation Kit (Miltenyi Biotech, 130-095-927) according to the manufacturer’s instructions. Dissociated cells were filtered using 70-μm and 30-μm strainers. CD31+ and TER-119+ cells were further removed using the EasySep Mouse Biotin Positive Selection Kit II (StemCell Technologies, 17665), followed by removal of dead cells using the EasySep Dead Cell Removal (Annexin V) Kit (StemCell Technologies, 17899).
scRNA-seq was performed on purified suspensions of epithelial and immune cells using the Chromium Next GEM Automated Single Cell 3' Library & Gel Bead Kit v3.1 (10X Genomics, 1000147) according to the manufacturer’s instructions. Briefly, cell number and viability were determined using 0.2% (w/v) Trypan Blue staining. 10,000 cells were then loaded onto the Chromium Connect for an automated process of GEM generation, post GEM-RT cleanup, cDNA amplification, and 3' gene expression library construction. Single-cell libraries were then sequenced on the Illumina NovaSeq S4 by the Mayo Clinic Genome Analysis Core.

treatment_protocol: Intratracheal administration of Ad-SPC-Cre in KP and KPI animals

value: LUAD tumor

value: KP


title: SPC KPI tumors

description:
Library name: SPC_KPI
10X Genomics

value: KPI


title: PY_TumoP_F1

description: PY_TumoP_F1 No Cas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality -whole tumor using Py2T into FVB (Imm.competent) mouse, with vector lentiGuide-Puro

value: xenograft

value: FVB

value: lentiGuide-Puro


title: Replicate 2, Low JAM-A expression

city: Solna

first: Bernhard

last: Schmierer

state: (optional)

address: Biomedicum 9B, Solnavagen 9

department: Medical Biochemistry and Biophysics

country: Sweden

institute: Karolinska Institutet

description:
Library name: R2_Low
20nt(Read1)-10nt(Index1)-6nt(Index2) setup using 'SP' flowcell

library_source: other

data_processing:
Readcounts were mapped to the library sequences using MAGeCK/0.5.9.4
Assembly: GRCh38
Supplementary files format and content: tab-delimited .txt file with mapped readcounts
Library strategy: CRISPR

molecule: other

source_name: Pancreas

growth_protocol: DMEM/F12; 8%FCS/100U/ml Pen/Strep

extract_protocol:
QIAamp DNA Blood Mini (Qiagen) according to manufacturer's instructions
PCR one amplicon with adaptor-containing primers

value: Pancreas

value: RLT-PSC

value: Pancreatic stellate cell

value: Untreated


title: ERRg_PTEN_KO

description: Library name: Sample 1

value: Human prostate cancer

value: sgPTEN

value: CRISPR/Cas9-mediated PTEN knockout


title: sample_5

description: sample_5 No Cas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality -tbd using 4T1 into BALB (Imm.competent) mouse, with vector lentiGuide-Puro

value: tumor

value: BALBc

value: sgRNAs used as barcodes (no KO)


title: PY_LungsP_F2

description: PY_LungsP_F2 No Cas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality -whole lungs using Py2T into FVB (Imm.competent) mouse, with vector lentiGuide-Puro

value: Immuno Signalome (2215) sgRNA library


title: PY_LungsP_F5

description: PY_LungsP_F5 No Cas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality -whole lungs using Py2T into FVB (Imm.competent) mouse, with vector lentiGuide-Puro


title: Replicate 2_Unsorted

description:
Library name: R2_Unsorted
20nt(Read1)-10nt(Index1)-6nt(Index2) setup using 'SP' flowcell


title: Lung, IL-1β KO, VEH

city: La Jolla,

first: Samuel

last: Bertin

state: CA

address: 9500 Gilman Drive

department: Division of Rheumatology, Autoimmunity, and Inflammation

institute: UC San Diego Department of Medicine

description:
Library name: KO_VEH
10x genomics 3' v3.1

data_processing:
Raw base calls from the NovaSeq 6000 sequencer were converted from BCL to FASTQ and demultiplexed with unique I5 and I7 barcodes using an in-house pipeline. Data were processed by CellRanger 7.1.0 (10X Genomics) with `cellranger count` pipeline with default parameters for murine reference genome alignment and transcriptome annotation (mm10-2020-A), barcoded processing with UMI deduplication and cell barcodes, and gene count as explained in manufacter's website (https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/3.0/algorithms/overview)
Assembly: mm10-2020-A
Supplementary files format and content: Tab-separated values files and matrix files

molecule: polyA RNA

source_name: Lung

extract_protocol:
Mice lungs were perfused by cardiac perfusion with 5 mL HBSS/1mM EDTA. Five lung lobes/mouse were harvested, manually minced with scissors, and then enzymatically digested in 2 mL of digestion buffer containing 500 μL Collagenase IA (Sigma, Cat# C9891), 100 μL DNAse I Type IV (Sigma, Cat# D5025), 250 uL Penicillin-Streptomycin (Sigma, Cat# P4333), and 5% FCS. RBCs were lysed using ACK buffer (0.5 mL, 1 minute on ice) and the single-cell suspensions from n = 3-4 mice/group were pooled together. The cells were resuspended in PBS/2% FCS, stained with an Annexin V-PE antibody (BioLegend, Cat# 640908) and DAPI (Thermo Fisher Scientific, Cat# 62248) according to the manufacturer’s instructions, and sorted on a BD FACSAria cell sorter (BD Biosciences) to remove transcriptional noise from dead/dying cells. Live single cells (Annexin V and DAPI double negative cells, purity >98%) were counted, resuspended in PBS/0.04% BSA (Thermo Fisher Scientific, Cat# AM2616) in 1.5 mL RNA/DNA LoBind Eppendorf tubes (Thermo Fisher Scientific, Cat# 13-698-791)
Library was constructed using the manufacter's instruction (Single Cell 3' kit v3.1 Dual Index; 10X Genomics). Briefly, total 16,500 cells per sample were loaded in duplicate on 10X Chrominium platform for Gel beads-in-Emulsion eneration (GEM). The poly-A RNA encapsulated in GEM were barcoded with unique molecular index, 10x barcode, and Illumina read 1 during reverse transcription. After post GEM-RT cleanup, cDNA amplification, enzymatic fragmentation, end repair, A-tail, ligation for Illumina Read 2 sequence, sample index PCR was conducted to add Illumina I5, P5, I7, and P7 for pair-end library construction.

value: Lung

tag: age

value: 3-5 months

tag: Sex

value: Male

tag: strain

value: VEH

tag: batch


title: MVT_TumoP_F3

description: MVT_TumoP_F3 No Cas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality -whole tumor using MVT1 into FVB (Imm.competent) mouse, with vector lentiGuide-Puro

value: xenograft

value: FVB


title: PY_TumoP_F2

description: PY_TumoP_F2 No Cas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality -whole tumor using Py2T into FVB (Imm.competent) mouse, with vector lentiGuide-Puro

value: lentiGuide-Puro


title: PY_TumoP_F5

description: PY_TumoP_F5 No Cas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality -whole tumor using Py2T into FVB (Imm.competent) mouse, with vector lentiGuide-Puro


title: Lung, WT, HDM

description:
Library name: WT_HDM
10x genomics 3' v3.1

value: WT

value: HDM


title: Lung, IL-1β KO, HDM

description:
Library name: KO_HDM
10x genomics 3' v3.1


title: PY_LungsP_F1

description: PY_LungsP_F1 No Cas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality -whole lungs using Py2T into FVB (Imm.competent) mouse, with vector lentiGuide-Puro


title: Replicate 2, High JAM-A expression

description:
Library name: R2_High
20nt(Read1)-10nt(Index1)-6nt(Index2) setup using 'SP' flowcell

value: Pancreas

value: RLT-PSC

value: Pancreatic stellate cell

value: Untreated


title: Lung, WT, VEH

description:
Library name: WT_VEH
10x genomics 3' v3.1


title: MVT_TumoP_F4

description: MVT_TumoP_F4 No Cas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality -whole tumor using MVT1 into FVB (Imm.competent) mouse, with vector lentiGuide-Puro

value: tumor


title: MVT_LungsP_F4

description: MVT_LungsP_F4 No Cas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality -whole lungs using MVT1 into FVB (Imm.competent) mouse, with vector lentiGuide-Puro


title: CRISPRKO_Clone_8

description: CRISPRKO_Clone_8 CRISPR Knockout (by Cas9 transfection) through the StealTHY system -whole lungs using Py2T into FVB (Imm.competent) mouse, with vector lentiGuide-Thy1

value: NSG

value: CRISPR KO


title: InVivo_Control_sgRNA_rep2 Bulk fat pad tumor, Py2T tumor cells carrying control sgRNA

description: Library name: InVivo_Control_sgRNA_rep2

library_source: transcriptomic

data_processing:
Trim Galore! (v0.6.6; parameters: –q 20 –length 20)
Mapping using STAR (v 2.5.9a) to mouse (GRCm39)  with splice junctions from the human GENCODE annotation (release M29)
The gene-level expression counts were computed with featureCounts from the subread package (v.2.0.3; parameters: -t exon -g gene_id --minOverlap 10 -Q 10) using the human gene annotations from GENCODE (release 40)
Samples were retained for further analyses if they had at least 500’000 reads, at least 5’000 genes with non-zero expression and less than 50% of reads mapping to mitochondrial genes
Assembly: GRCm39 and GENCODE release M29
Supplementary files format and content: counts.txt.gz are tab-delimited files containing raw counts for each library

extract_protocol:
FACS-purified cells were lysed immediately upon direct sorting in 150 µL of extraction buffer from the Arcturus PicoPure RNA isolation kit (Thermo Scientific). The quality of the RNA isolated through the PicoPure kit was determined with a TapeStation RNA High sensitivity Screen Tape (Agilent).
Due to the low concentration of total RNA the SMARTer® Stranded Total RNA-Seq Kit v3 -Pico Input Mammalian (Takara Bio) was used to prepare sequencing libraries. Briefly, total RNA samples (0.27–8 ng) were mechanically fragmented (4 minutes) and reverse-transcribed using random priming into double-stranded cDNA in the presence of a template switch oligo (TSO). When the reverse transcriptase reaches the 5’ end of the RNA fragment, the enzyme’s terminal transferase activity adds non-templated nucleotides to the 3’ end of the cDNA, then the TSO pairs with the added non-templated nucleotide, enabling the reverse transcriptase to continue replicating to the end of the oligonucleotide. This procedure results in a cDNA fragment that contains sequences derived from the random priming oligo and the TSO. So, PCR amplification using primers binding to the latter sequences was performed so to add full-length Illumina adapters, including dual barcodes for multiplexing. Ribosomal cDNA was cleaved by ZapR in the presence of the mammalian-specific R-Probes. Remaining fragments were enriched with a second round of PCR amplification using primers designed to match Illumina adapters. Since RNA input concentrations was highly variable the samples were grouped into two sets of samples and PCR cycle numbers were adjusted (Group 1 = 6.5-8ng input, 12 cycles; Group 2 = 0.27-4 ng, 16 cycles). The quality and quantity of the enriched libraries were validated using a TapeStation analyzer (Agilent). The product presented as a smear with an average fragment size of approximately 300 - 360 bp. The libraries were normalized to 5nM in Tris-Cl 10 mM, pH8.5 with 0.1% Tween 20.

tag: number of_cells

tag: origin

value: allograft

tag: mouse model

value: no


title: CRISPRKO_Clone_71

description: CRISPRKO_Clone_71 CRISPR Knockout (by Cas9 transfection) through the Lentiguide-Puro standard system -Bulk in vitro mouse cells, antibiotic-selected as Puro-resistant

tag: tissue

value: bulk

tag: cell line

value: In vitro

value: Immuno Signalome (2215) sgRNA library

value: lentiguide-Puro


title: GG3 cells_biol rep3

city: Genova

first: Kiril

last: Tuntevski

address: Via Morego 30

country: Italy

institute: Istituto Italiano di Tecnologia

description: Library name: IIT_PIR_3_GBM4_GG3_CTL_3_S1_R1_001.fastq.gz

data_processing:
Matlab
Sequence reads were trimmed for adaptor sequence/low-quality sequence, using Cutadapt (v4.9)
The composite files were processed for tetramers, as per library manufacturer instructions, and quantified with miRge3.0
Outputs were normalized to obtain TPM expression relative to each sample, for each detected miRNA species.
Assembly: miRge3.0 (human)
Supplementary files format and content: miRNA_expression_TPM.txt: tab delimited file that includes TPM for all genes in all samples

source_name: patient-derived glioma cells

extract_protocol:
RNA was harvested using Trizol according to the manufacturer’s protocol. 1 ug of total RNA was used for the construction of sequencing libraries.
RNA libraries for RNA-seq were prepared using NEXTFLEX Small RNA-Seq Kit v3 following manufacturer's protocols.

value: patient-derived glioma cells

value: male

tag: time

value: untreated


title: CRISPRKO_Clone_70

description: CRISPRKO_Clone_70 CRISPR Knockout (by Cas9 transfection) through the Lentiguide-Puro standard system -Bulk in vitro mouse cells, antibiotic-selected as Puro-resistant

tag: cell type

tag: genotype

value: xenograft

tag: treatment


title: Replicate 1, Low JAM-A expression

description:
Library name: R1_Low
20nt(Read1)-10nt(Index1)-6nt(Index2) setup using 'SP' flowcell

value: Pancreas


title: InVivo_Control_sgRNA_rep1 Bulk fat pad tumor, Py2T tumor cells carrying control sgRNA

description: Library name: InVivo_Control_sgRNA_rep1

value: FVB


title: GG3 cells_biol rep2

description: Library name: IIT_PIR_2_GBM4_GG3_CTL_2_S26_R1_001.fastq.gz


title: MVT_LungsP_F3

description: MVT_LungsP_F3 No Cas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality -whole lungs using MVT1 into FVB (Imm.competent) mouse, with vector lentiGuide-Puro

tag: crispr lib

tag: sgrna library

tag: effect type

value: sgRNAs used as barcodes (no KO)

value: lentiGuide-Puro


title: input_PTEN_KO

description: Library name: Sample 3

value: Human prostate cancer

value: sgPTEN

value: CRISPR/Cas9-mediated PTEN knockout

tag: chip antibody


title: CRISPRKO

description: CRISPRKO CRISPR Knockout (by Cas9 transfection) through the StealTHY system -whole tumor using Py2T into FVB (Imm.competent) mouse, with vector lentiGuide-Thy1

value: tumor

value: CRISPR KO

tag: vector

tag: full library_complexity


title: Replicate 1, High JAM-A expression

description:
Library name: R1_High
20nt(Read1)-10nt(Index1)-6nt(Index2) setup using 'SP' flowcell

value: RLT-PSC

value: Pancreatic stellate cell

value: Untreated


title: PlatePositionB3

description: PlatePositionB3 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 4

value: BALBc

value: Immuno Signalome (2215) sgRNA library

value: lentiGuide-Puro + EF1alpha_dCas9


title: CRISPRKO_Clone_9

description: CRISPRKO_Clone_9 CRISPR Knockout (by Cas9 transfection) through the StealTHY system -whole lungs using Py2T into FVB (Imm.competent) mouse, with vector lentiGuide-Thy1

value: NSG


title: Replicate 1_Unsorted

description:
Library name: R1_Unsorted
20nt(Read1)-10nt(Index1)-6nt(Index2) setup using 'SP' flowcell


title: InVivo_Control_sgRNA_rep3 Bulk fat pad tumor, Py2T tumor cells carrying control sgRNA

description: Library name: InVivo_Control_sgRNA_rep3

value: allograft

value: no


title: CRISPRKO_Clone_68

description: CRISPRKO_Clone_68 CRISPR Knockout (by Cas9 transfection) through the StealTHY system -Bulk In vitro mouse cells MACS-selected for Thy1

value: bulk

value: xenograft

value: In vitro


title: CRISPRKO_Clone_65

description: CRISPRKO_Clone_65 CRISPR Knockout (by Cas9 transfection) through the StealTHY system -Bulk In vitro mouse cells MACS-selected for Thy1


title: CRISPRKO_Clone_66

description: CRISPRKO_Clone_66 CRISPR Knockout (by LV Cas9 transduction) through the Lentiguide-Puro standard system -Bulk in vitro mouse cells, antibiotic-selected as Puro-resistant

source_name: CRISPR Knockout (by Cas9 transfection) through the Lentiguide-Puro standard system


title: InVivo_AMHR2_KO_rep1 Bulk fat pad tumor, Py2T tumor cells carrying Amhr2 KO

description: Library name: InVivo_AMHR2_KO_rep1

value: FVB


title: CRISPRKO_Clone_7

description: CRISPRKO_Clone_7 CRISPR Knockout (by Cas9 transfection) through the StealTHY system -whole lungs using Py2T into FVB (Imm.competent) mouse, with vector lentiGuide-Thy1


title: CRISPRKO_Clone_69

description: CRISPRKO_Clone_69 CRISPR Knockout (by LV Cas9 transduction) through the Lentiguide-Puro standard system -Bulk in vitro mouse cells, antibiotic-selected as Puro-resistant


title: CRISPRKO_Clone_62

description: CRISPRKO_Clone_62 CRISPR Knockout (by LV Cas9 transduction) through the Lentiguide-Puro standard system -Bulk in vitro mouse cells, antibiotic-selected as Puro-resistant


title: CRISPRKO_Clone_67

description: CRISPRKO_Clone_67 CRISPR Knockout (by Cas9 transfection) through the StealTHY system -Bulk In vitro mouse cells MACS-selected for Thy1


title: InVivo_AMHR2_KO_rep3 Bulk fat pad tumor, Py2T tumor cells carrying Amhr2 KO

description: Library name: InVivo_AMHR2_KO_rep3


title: GG3 cells_biol rep1

description: Library name: IIT_PIR_1_GBM4_GG3_CTL_1_S41_R1_001.fastq.gz

value: patient-derived glioma cells

tag: Sex

value: male

value: untreated


title: InVivo_AMHR2_KO_rep2 Bulk fat pad tumor, Py2T tumor cells carrying Amhr2 KO

description: Library name: InVivo_AMHR2_KO_rep2

value: tumor


title: GBM2 cells_ biol rep3

description: Library name: IIT_PIR_48_GBM2_1740R_48_17_G23_CTL_3_S3_R1_001.fastq.gz

value: female


title: CRISPRKO_Clone_64

description: CRISPRKO_Clone_64 CRISPR Knockout (by Cas9 transfection) through the StealTHY system -Bulk In vitro mouse cells MACS-selected for Thy1

value: CRISPR KO


title: AMHR2_Overexpression_rep2 Cultivated Py2T cells carrying Amhr2 overexpression

description: Library name: AMHR2_Overexpression_rep2

value: 1 MiO


title: CRISPRKO_Clone_63

description: CRISPRKO_Clone_63 CRISPR Knockout (by Cas9 transfection) through the Lentiguide-Puro standard system -Bulk in vitro mouse cells, antibiotic-selected as Puro-resistant

value: lentiguide-Puro


title: AMHR2_Overexpression_rep1 Cultivated Py2T cells carrying Amhr2 overexpression

description: Library name: AMHR2_Overexpression_rep1


title: GBM1 cells_biol rep1

description: Library name: IIT_PIR_43_GBM1_1740R_48_11_G3_CTL_1_S8_R1_001.fastq.gz


title: CRISPRKO_Clone_56

description: CRISPRKO_Clone_56 CRISPR Knockout (by Cas9 transfection) through the StealTHY system -whole lungs using MVT1 into NSG (Imm.deficient) mouse, with vector lentiGuide-Thy1

value: Immuno Signalome (2215) sgRNA library


title: AMHR2_KO_noTGFbeta_rep3 Cultivated Py2T cells carrying Amhr2 KO and blocked TGF-beta pathway

description: Library name: AMHR2_KO_noTGFbeta_rep3


title: AMHR2_Overexpression_rep3 Cultivated Py2T cells carrying Amhr2 overexpression

description: Library name: AMHR2_Overexpression_rep3


title: GBM2 cells_biol rep2

description: Library name: IIT_PIR_47_GBM2_1740R_48_17_G23_CTL_2_S4_R1_001.fastq.gz


title: CRISPRKO_Clone_54

description: CRISPRKO_Clone_54 CRISPR Knockout (by Cas9 transfection) through the StealTHY system -tbd using Py2T into NSG (Imm.deficient) mouse, with vector lentiGuide-Thy1


title: CRISPRKO_Clone_60

description: CRISPRKO_Clone_60 CRISPR Knockout (by Cas9 transfection) through the StealTHY system -Bulk In vitro mouse cells MACS-selected for Thy1


title: CRISPRKO_Clone_61

description: CRISPRKO_Clone_61 CRISPR Knockout (by Cas9 transfection) through the StealTHY system -Bulk In vitro mouse cells MACS-selected for Thy1


title: GBM1 cells_biol rep2

description: Library name: IIT_PIR_45_GBM1_1740R_48_11_G3_CTL_3_S6_R1_001.fastq.gz


title: CRISPRKO_Clone_6

description: CRISPRKO_Clone_6 CRISPR Knockout (by Cas9 transfection) through the StealTHY system -whole lungs using Py2T into FVB (Imm.competent) mouse, with vector lentiGuide-Thy1


title: CRISPRKO_Clone_59

description: CRISPRKO_Clone_59 CRISPR Knockout (by Cas9 transfection) through the StealTHY system -whole lungs using MVT1 into NSG (Imm.deficient) mouse, with vector lentiGuide-Thy1


title: GBM2 cells_biol rep1

description: Library name: IIT_PIR_46_GBM2_1740R_48_17_G23_CTL_1_S5_R1_001.fastq.gz


title: CRISPRKO_Clone_53

description: CRISPRKO_Clone_53 CRISPR Knockout (by Cas9 transfection) through the StealTHY system -tbd using Py2T into NSG (Imm.deficient) mouse, with vector lentiGuide-Thy1


title: CRISPRKO_Clone_58

description: CRISPRKO_Clone_58 CRISPR Knockout (by Cas9 transfection) through the StealTHY system -whole lungs using MVT1 into NSG (Imm.deficient) mouse, with vector lentiGuide-Thy1


title: GBM2 POOL3

description: Library name: IIT_P2W_GBM2_POOL3_S23

data_processing:
CLC Genomic Workbench 24.0.1 - "RNA-seq Analysis" tool
Assembly: For tumor xenograft samples, a mouse (mm10) and human (hg19) hybrid reference genome was created with CLC Workbench. Max n° of hits for a read was set 1 and mouse genes were filtered out.
Supplementary files format and content: CSV files. Contain tabulated output from the CLC analysis software with gene information, associated ENSEMBL gene codes, number of reads, and normalised quantification.

growth_protocol: GBM cells (tumor ID, GBM1: GSC#Ge003 1740R-48-11; GBM2: GSC#Ge023 1740R-48-17) were established and cultured as previously described (Griffero et al., 2009). 5E5 GBM2 Luc+ cells were injected in the striatum of NOD/SCID female mice and tumor growth monitored for 42-49 days.

extract_protocol:
RNA was exctracted using Rneasy kit, according to the manufacturer’s protocol. 1 ug of total RNA was used for the construction of sequencing libraries.
Libraries were prepared using Illumina Stranded mRNA Prep kit following manufacturer's protocols.

treatment_protocol: GBM1 and GBM2 cells were transfected (Lipofectamine) with the 11 miRNA pool (25 nM each) or scrambled control (250 nM tot), detached and kept in spheroid-permissive medium for 3 days. Orthotopic GBM xenograft (NOD/SCID mice) were intratumorally injected with ApoE-coated LNP loaded with either the 11 miRNA pool or scrambled control and sacrificed after 14 days. Brains were dissected and tumor tissue withdrawn and fresh-frozen.

value: 3 days post treatment

value: pool


title: CRISPRKO_Clone_5

description: CRISPRKO_Clone_5 CRISPR Knockout (by Cas9 transfection) through the StealTHY system -whole lungs using Py2T into FVB (Imm.competent) mouse, with vector lentiGuide-Thy1

value: NSG


title: CRISPRKO_Clone_57

description: CRISPRKO_Clone_57 CRISPR Knockout (by Cas9 transfection) through the StealTHY system -whole lungs using MVT1 into NSG (Imm.deficient) mouse, with vector lentiGuide-Thy1


title: GBM2 SCRAMBLE 3

description: Library name: IIT_P2W_GBM2_SCRAMBLE_3_S9

value: scrambled


title: AMHR2_KO_noTGFbeta_rep2 Cultivated Py2T cells carrying Amhr2 KO and blocked TGF-beta pathway

description: Library name: AMHR2_KO_noTGFbeta_rep2

value: no


title: CRISPRKO_Clone_51

description: CRISPRKO_Clone_51 CRISPR Knockout (by Cas9 transfection) through the StealTHY system -tbd using Py2T into NSG (Imm.deficient) mouse, with vector lentiGuide-Thy1

value: xenograft


title: AMHR2_KO_noTGFbeta_rep1 Cultivated Py2T cells carrying Amhr2 KO and blocked TGF-beta pathway

description: Library name: AMHR2_KO_noTGFbeta_rep1


title: GBM2 SCRAMBLE 2

description: Library name: IIT_P2W_GBM2_SCRAMBLE_2_S27


title: AMHR2_KO_culture_rep1 Cultivated Py2T cells carrying Amhr2 KO

description: Library name: AMHR2_KO_culture_rep1


title: CRISPRKO_Clone_55

description: CRISPRKO_Clone_55 CRISPR Knockout (by Cas9 transfection) through the StealTHY system -whole lungs using MVT1 into NSG (Imm.deficient) mouse, with vector lentiGuide-Thy1


title: CRISPRKO_Clone_52

description: CRISPRKO_Clone_52 CRISPR Knockout (by Cas9 transfection) through the StealTHY system -tbd using Py2T into NSG (Imm.deficient) mouse, with vector lentiGuide-Thy1


title: GBM2 SCRAMBLE 1

description: Library name: IIT_P2W_GBM2_SCRAMBLE_1_S13


title: AMHR2_KO_culture_rep3 Cultivated Py2T cells carrying Amhr2 KO

description: Library name: AMHR2_KO_culture_rep3


title: GBM2 POOL1

description: Library name: IIT_P2W_GBM2_POOL1_S10


title: AMHR2_Control_culture_rep3 Cultivated Py2T cells carrying control sgRNA

description: Library name: AMHR2_Control_culture_rep3


title: CRISPRKO_Clone_50

description: CRISPRKO_Clone_50 CRISPR Knockout (by Cas9 transfection) through the StealTHY system -tbd using Py2T into NSG (Imm.deficient) mouse, with vector lentiGuide-Thy1


title: GBM1 SCRAMBLE3

description: Library name: IIT_P2W_GBM1_SCRAMBLE3_S20


title: CRISPRKO_Clone_44

description: CRISPRKO_Clone_44 CRISPR Knockout (by Cas9 transfection) through the StealTHY system -whole lungs using 4T1 into NSG (Imm.deficient) mouse, with vector lentiGuide-Thy1


title: GBM1 SCRAMBLE2

description: Library name: IIT_P2W_GBM1_SCRAMBLE2_S8


title: CRISPRKO_Clone_43

description: CRISPRKO_Clone_43 CRISPR Knockout (by Cas9 transfection) through the StealTHY system -whole lungs using 4T1 into NSG (Imm.deficient) mouse, with vector lentiGuide-Thy1


title: CRISPRKO_Clone_42

description: CRISPRKO_Clone_42 CRISPR Knockout (by Cas9 transfection) through the StealTHY system -whole lungs using 4T1 into NSG (Imm.deficient) mouse, with vector lentiGuide-Thy1


title: AMHR2_KO_culture_rep2 Cultivated Py2T cells carrying Amhr2 KO

description: Library name: AMHR2_KO_culture_rep2


title: CRISPRKO_Clone_49

description: CRISPRKO_Clone_49 CRISPR Knockout (by Cas9 transfection) through the StealTHY system -whole tumor using 4T1 into NSG (Imm.deficient) mouse, with vector lentiGuide-Thy1


title: GBM2 POOL2

description: Library name: IIT_P2W_GBM2_POOL2_S16


title: CRISPRKO_Clone_48

description: CRISPRKO_Clone_48 CRISPR Knockout (by Cas9 transfection) through the StealTHY system -whole tumor using 4T1 into NSG (Imm.deficient) mouse, with vector lentiGuide-Thy1


title: CRISPRKO_Clone_41

description: CRISPRKO_Clone_41 CRISPR Knockout (by Cas9 transfection) through the StealTHY system -whole lungs using 4T1 into NSG (Imm.deficient) mouse, with vector lentiGuide-Thy1


title: GBM1 SCRAMBLE1

description: Library name: IIT_P2W_GBM1_SCRAMBLE1_S17


title: CRISPRKO_Clone_40

description: CRISPRKO_Clone_40 CRISPR Knockout (by Cas9 transfection) through the StealTHY system -whole lungs using 4T1 into NSG (Imm.deficient) mouse, with vector lentiGuide-Thy1


title: CRISPRKO_Clone_47

description: CRISPRKO_Clone_47 CRISPR Knockout (by Cas9 transfection) through the StealTHY system -whole tumor using 4T1 into NSG (Imm.deficient) mouse, with vector lentiGuide-Thy1


title: CRISPRKO_Clone_39

description: CRISPRKO_Clone_39 CRISPR Knockout (by Cas9 transfection) through the StealTHY system -whole tumor using MVT1 into NSG (Imm.deficient) mouse, with vector lentiGuide-Thy1


title: GBM1 POOL2

description: Library name: IIT_P2W_GBM1POOL2_S26


title: CRISPRKO_Clone_37

description: CRISPRKO_Clone_37 CRISPR Knockout (by Cas9 transfection) through the StealTHY system -whole tumor using MVT1 into NSG (Imm.deficient) mouse, with vector lentiGuide-Thy1


title: AMHR2_Control_culture_rep2 Cultivated Py2T cells carrying control sgRNA

description: Library name: AMHR2_Control_culture_rep2


title: GBM1 POOL1

description: Library name: IIT_P2W_GBM1_POOL1_S22


title: CRISPRKO_Clone_46

description: CRISPRKO_Clone_46 CRISPR Knockout (by Cas9 transfection) through the StealTHY system -whole tumor using 4T1 into NSG (Imm.deficient) mouse, with vector lentiGuide-Thy1


title: CRISPRKO_Clone_45

description: CRISPRKO_Clone_45 CRISPR Knockout (by Cas9 transfection) through the StealTHY system -whole tumor using 4T1 into NSG (Imm.deficient) mouse, with vector lentiGuide-Thy1


title: CRISPRKO_Clone_33

description: CRISPRKO_Clone_33 CRISPR Knockout (by Cas9 transfection) through the StealTHY system -whole tumor using Py2T into NSG (Imm.deficient) mouse, with vector lentiGuide-Thy1


title: AMHR2_Control_culture_rep1 Cultivated Py2T cells carrying control sgRNA

description: Library name: AMHR2_Control_culture_rep1


title: CRISPRKO_Clone_38

description: CRISPRKO_Clone_38 CRISPR Knockout (by Cas9 transfection) through the StealTHY system -whole tumor using MVT1 into NSG (Imm.deficient) mouse, with vector lentiGuide-Thy1


title: GBM1 POOL3

description: Library name: IIT_P2W_GBM1_POOL3_S11


title: scRNA_WT_Control3

city: New York

first: Prokopios Alexandros

last: Polyzos

address: 413 E 69th St

institute: Weill Cornell Medicine

description:
Library name: CTRL_3-GEX_S9
10x Single Cell 5' gene expression

data_processing:
Cellranger 3.0.0
Seurat 4.0
Assembly: mm10
Supplementary files format and content: Integrated Seurat Object containing all samples and additional published samples

source_name: spleen

extract_protocol:
Single-cell RNA-seq libraries were prepared using the Chromium Single Cell according to 10x Genomics specifications.
10x Single-cell RNA-seq

treatment_protocol: WT mice were immunized with SRBC for 9 days before sorting cells by FACS.

value: spleen

value: GCB and GCB-derived populations cells (YFP+ IgD-)

value: WT (Cg1Cre +/-, Rosa26 YFP)

value: SRBC 9 days

tag: batch


title: scRNA_N_d0

description:
Library name: N_D0_S1
10x Single Cell 5' gene expression

value: Naive B cells (B220+ Fas- Gl7- IgD+)

value: WT


title: scRNA_GC_d0

description:
Library name: GC_D0_S2
10x Single Cell 5' gene expression

value: GCB cells (B220+ Fas+ Gl7+)


title: 80 SCRAMBLE

description: Library name: IIT_P2W_80_SCRAMBLE_S5

source_name: human glioblastoma xenograft from mouse striatum

value: GBM2 Luc+

value: female

value: 14 days post treatment

value: ApoE-LNP scrambled


title: CRISPRKO_Clone_36

description: CRISPRKO_Clone_36 CRISPR Knockout (by Cas9 transfection) through the StealTHY system -whole tumor using MVT1 into NSG (Imm.deficient) mouse, with vector lentiGuide-Thy1

value: tumor

value: FVB

value: Immuno Signalome (2215) sgRNA library

value: CRISPR KO


title: N_A

description: Library name: LS_R21

data_processing:
Alignment with STAR aligner (version 2.7.10a)
fliter and assign reads to genes mm10 – (GRCm38.95) with featureCounts (v2.0.6)
Differential analysis was performed with DESeq2 in R4.2.2
Assembly: mm10 and mm10.GRCm38.95.gtf
Supplementary files format and content: tab-delimited text file with raw counts for all genes

growth_protocol: primary naive B cells were maintained in Embryonic Stem cell media (KO-DMEM) in humidified atmosphere with 5% CO2 at 37C and activated for 5 days with 80ng/ml of mIL4 and 5 μg/ml of anti-CD40, of 2μM of CpG or 25μg/ml of LPS

extract_protocol:
RNA was harvested using Rneasy mini plus kit (Qiagen). 1ug of total RNA was used for the construction of sequencing libraries.
RNA-seq libraries were prepared and sequenced by Novogene Bioinformatics Technology Co. Ltd.

treatment_protocol: Cytokines and fresh media were provided at Day 0 and 3

value: naive B cells


title: CRISPRKO_Clone_35

description: CRISPRKO_Clone_35 CRISPR Knockout (by Cas9 transfection) through the StealTHY system -whole tumor using MVT1 into NSG (Imm.deficient) mouse, with vector lentiGuide-Thy1

value: xenograft


title: scRNA_WT_Control2

description:
Library name: CTRL_2-GEX_S8
10x Single Cell 5' gene expression

value: spleen


title: CRISPRKO_Clone_4

description: CRISPRKO_Clone_4 CRISPR Knockout (by Cas9 transfection) through the StealTHY system -whole tumor using Py2T into FVB (Imm.competent) mouse, with vector lentiGuide-Thy1


title: CRISPRKO_Clone_34

description: CRISPRKO_Clone_34 CRISPR Knockout (by Cas9 transfection) through the StealTHY system -whole tumor using Py2T into NSG (Imm.deficient) mouse, with vector lentiGuide-Thy1


title: GC_C

description: Library name: LS_R20

value: GC B cells


title: scRNA_WT_Control1

description:
Library name: CTRL_1-GEX_S7
10x Single Cell 5' gene expression

value: GCB and GCB-derived populations cells (YFP+ IgD-)

value: WT (Cg1Cre +/-, Rosa26 YFP)


title: 86 POOL

description: Library name: IIT_P2W_86_POOL_S6

value: human glioblastoma xenograft from mouse striatum

value: ApoE-LNP pool


title: CRISPRKO_Clone_32

description: CRISPRKO_Clone_32 CRISPR Knockout (by Cas9 transfection) through the StealTHY system -whole tumor using Py2T into NSG (Imm.deficient) mouse, with vector lentiGuide-Thy1


title: 76 SCRAMBLE

description: Library name: IIT_P2W_76_SCRAMBLE_S24


title: GC_B

description: Library name: LS_R19

value: WT


title: CRISPRKO_Clone_24

description: CRISPRKO_Clone_24 CRISPR Knockout (by Cas9 transfection) through the StealTHY system -tbd using MVT1 into FVB (Imm.competent) mouse, with vector lentiGuide-Thy1


title: CRISPRKO_Clone_23

description: CRISPRKO_Clone_23 CRISPR Knockout (by Cas9 transfection) through the StealTHY system -tbd using MVT1 into FVB (Imm.competent) mouse, with vector lentiGuide-Thy1


title: CRISPRKO_Clone_31

description: CRISPRKO_Clone_31 CRISPR Knockout (by Cas9 transfection) through the StealTHY system -whole tumor using Py2T into NSG (Imm.deficient) mouse, with vector lentiGuide-Thy1


title: N_C

description: Library name: LS_R23


title: CRISPRKO_Clone_30

description: CRISPRKO_Clone_30 CRISPR Knockout (by Cas9 transfection) through the StealTHY system -whole tumor using Py2T into NSG (Imm.deficient) mouse, with vector lentiGuide-Thy1


title: CRISPRKO_Clone_22

description: CRISPRKO_Clone_22 CRISPR Knockout (by Cas9 transfection) through the StealTHY system -tbd using MVT1 into FVB (Imm.competent) mouse, with vector lentiGuide-Thy1


title: 81 SCRAMBLE

description: Library name: IIT_P2W_81_SCRAMBLE_S3

value: GBM2 Luc+


title: CRISPRKO_Clone_3

description: CRISPRKO_Clone_3 CRISPR Knockout (by Cas9 transfection) through the StealTHY system -whole tumor using Py2T into FVB (Imm.competent) mouse, with vector lentiGuide-Thy1


title: N_B

description: Library name: LS_R22


title: CRISPRKO_Clone_28

description: CRISPRKO_Clone_28 CRISPR Knockout (by Cas9 transfection) through the StealTHY system -whole lungs using tbd into FVB (Imm.competent) mouse, with vector lentiGuide-Thy1

value: NSG


title: CRISPRKO_Clone_29

description: CRISPRKO_Clone_29 CRISPR Knockout (by Cas9 transfection) through the StealTHY system -whole lungs using tbd into FVB (Imm.competent) mouse, with vector lentiGuide-Thy1


title: CRISPRKO_Clone_2

description: CRISPRKO_Clone_2 CRISPR Knockout (by Cas9 transfection) through the StealTHY system -whole tumor using Py2T into FVB (Imm.competent) mouse, with vector lentiGuide-Thy1


title: Nact D5 CpG_C

description: Library name: LS_R16

value: Activated naive B cells


title: CRISPRKO_Clone_19

description: CRISPRKO_Clone_19 CRISPR Knockout (by Cas9 transfection) through the StealTHY system -whole lungs using 4T1 into BALB (Imm.competent) mouse, with vector lentiGuide-Thy1

value: BALBc


title: CRISPRKO_Clone_18

description: CRISPRKO_Clone_18 CRISPR Knockout (by Cas9 transfection) through the StealTHY system -whole lungs using 4T1 into BALB (Imm.competent) mouse, with vector lentiGuide-Thy1

value: tumor


title: 69 POOL

description: Library name: IIT_P2W_69_POOL_S18

value: female

value: 14 days post treatment


title: CRISPRKO_Clone_27

description: CRISPRKO_Clone_27 CRISPR Knockout (by Cas9 transfection) through the StealTHY system -whole lungs using tbd into FVB (Imm.competent) mouse, with vector lentiGuide-Thy1

value: Immuno Signalome (2215) sgRNA library

value: CRISPR KO


title: 78 SCRAMBLE

description: Library name: IIT_P2W_78_SCRAMBLE_S12

value: ApoE-LNP scrambled


title: CRISPRKO_Clone_17

description: CRISPRKO_Clone_17 CRISPR Knockout (by Cas9 transfection) through the StealTHY system -whole lungs using 4T1 into BALB (Imm.competent) mouse, with vector lentiGuide-Thy1

value: xenograft


title: CRISPRKO_Clone_26

description: CRISPRKO_Clone_26 CRISPR Knockout (by Cas9 transfection) through the StealTHY system -whole lungs using tbd into FVB (Imm.competent) mouse, with vector lentiGuide-Thy1


title: CRISPRKO_Clone_25

description: CRISPRKO_Clone_25 CRISPR Knockout (by Cas9 transfection) through the StealTHY system -whole lungs using tbd into FVB (Imm.competent) mouse, with vector lentiGuide-Thy1


title: 84 POOL

description: Library name: IIT_P2W_84_POOL_S14


title: GC_A

description: Library name: LS_R18

value: spleen

value: GC B cells


title: 73 POOL

description: Library name: IIT_P2W_73_POOL_S2


title: Nact D5 LPS_B

description: Library name: LS_R14


title: 71 SCRAMBLE

description: Library name: IIT_P2W_71_SCRAMBLE_S25


title: Nact D5 CpG_B

description: Library name: LS_R13


title: R882C_2_EPO

city: London

first: Hector

last: Huerga Encabo

address: The Francis Crick Institute, 1 Midland Rd

country: United Kingdom

institute: The Francis Crick Institute

description: Library name: HUE6313A30

data_processing:
Obtained FASTQ files were aligned and QC using the nf-core/rnaseq pipeline version 3.7,using '-profile crick --aligner star_rsem" as extra parameters.
Obtained counts were then uploaded and analyzed using Deseq2 on R.
Assembly: Ensembl GRCh38 release 95
Supplementary files format and content: tab separated gene counts per gene per sample

extract_protocol:
RNA was isolated using RNAeasy microkit (Qiagen)
Libraries were prepared using KAPA Stranded with RiboErase RNA-seq kit (according to the manufacturer’s instructions). Briefly, 17–25 ng of starting RNA were subjected first to cytoplasmic and mitochondrial ribosomal RNA (rRNA) depletion by hybridization of complementary DNA oligonucleotides, followed by treatment with RNase H and DNase to remove rRNA duplexed to DNA and original DNA oligonucleotides. Samples depleted of rRNA were then subjected to 94 °C for 6 min in the 2×Fragment, Prime, and Elute Buffer in order to obtain 200–300 bp fragments. cDNA synthesis was run in two steps following the manufacturer’s instructions. The ligation step consisted of a final volume of 110 μl of the adaptor ligation reaction mixture with 60 μl of input cDNA, 5μl of diluted adaptor, and 45 μl of ligation mix (50 μl of ligation buffer + 10 μl of DNA ligase). The Kapa Dual- Indexed Adaptors stock concentration was diluted to 1.5mM to get the best adaptor concentration for library construction. The ligation cycle was run according to the manufacturer’s instructions. To remove short fragments such as adaptor dimers, 2X AMPure XP bead clean-ups were done (0.63 SPRI and 0.7 SPRI).

value: EPO


title: CRISPRKO_Clone_21

description: CRISPRKO_Clone_21 CRISPR Knockout (by Cas9 transfection) through the StealTHY system -tbd using MVT1 into FVB (Imm.competent) mouse, with vector lentiGuide-Thy1

value: FVB


title: Nact D5 CD40_C

description: Library name: LS_R15


title: CRISPRKO_Clone_20

description: CRISPRKO_Clone_20 CRISPR Knockout (by Cas9 transfection) through the StealTHY system -tbd using MVT1 into FVB (Imm.competent) mouse, with vector lentiGuide-Thy1


title: CRISPRKO_Clone_15

description: CRISPRKO_Clone_15 CRISPR Knockout (by Cas9 transfection) through the StealTHY system -whole lungs using 4T1 into BALB (Imm.competent) mouse, with vector lentiGuide-Thy1


title: Nact D5 LPS_C

description: Library name: LS_R17


title: CRISPRKO_Clone_10

description: CRISPRKO_Clone_10 CRISPR Knockout (by Cas9 transfection) through the StealTHY system -whole tumor using 4T1 into BALB (Imm.competent) mouse, with vector lentiGuide-Thy1


title: 70POOL

description: Library name: IIT_P2W_70_POOL_S19

value: human glioblastoma xenograft from mouse striatum

value: ApoE-LNP pool


title: CRISPRKO_Clone_1

description: CRISPRKO_Clone_1 CRISPR Knockout (by Cas9 transfection) through the StealTHY system -whole tumor using Py2T into FVB (Imm.competent) mouse, with vector lentiGuide-Thy1


title: Nact D5 LPS_A

description: Library name: LS_R11

value: WT


title: R882C_1_EPO

description: Library name: HUE6313A29


title: PY_TumoT_N5

description: PY_TumoT_N5 No Cas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality -whole tumor using Py2T into NSG (Imm.deficient) mouse, with vector lentiGuide-Thy1

value: NSG (Imm.deficient)

value: sgRNAs used as barcodes (no KO)


title: CRISPRKO_Clone_16

description: CRISPRKO_Clone_16 CRISPR Knockout (by Cas9 transfection) through the StealTHY system -whole lungs using 4T1 into BALB (Imm.competent) mouse, with vector lentiGuide-Thy1

value: BALBc


title: R882C_4_EPO

description: Library name: HUE6313A32


title: R882C_3_CTRL

description: Library name: HUE6313A27

value: UNTREATED


title: CRISPRKO_Clone_14

description: CRISPRKO_Clone_14 CRISPR Knockout (by Cas9 transfection) through the StealTHY system -whole tumor using 4T1 into BALB (Imm.competent) mouse, with vector lentiGuide-Thy1

value: tumor


title: R882C_3_EPO

description: Library name: HUE6313A31


title: PY_TumoT_N2

description: PY_TumoT_N2 No Cas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality -whole tumor using Py2T into NSG (Imm.deficient) mouse, with vector lentiGuide-Thy1


title: CRISPRKO_Clone_13

description: CRISPRKO_Clone_13 CRISPR Knockout (by Cas9 transfection) through the StealTHY system -whole tumor using 4T1 into BALB (Imm.competent) mouse, with vector lentiGuide-Thy1


title: R882C_2_CTRL

description: Library name: HUE6313A26


title: PY_TumoT_F5

description: PY_TumoT_F5 No Cas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality -whole tumor using Py2T into FVB (Imm.competent) mouse, with vector lentiGuide-Thy1

value: FVB (Imm.competent)


title: R882C_1_CTRL

description: Library name: HUE6313A25


title: PY_TumoT_F2

description: PY_TumoT_F2 No Cas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality -whole tumor using Py2T into FVB (Imm.competent) mouse, with vector lentiGuide-Thy1

value: Immuno Signalome (2215) sgRNA library


title: Nact D5 CD40_B

description: Library name: LS_R12

value: Activated naive B cells


title: CRISPRKO_Clone_11

description: CRISPRKO_Clone_11 CRISPR Knockout (by Cas9 transfection) through the StealTHY system -whole tumor using 4T1 into BALB (Imm.competent) mouse, with vector lentiGuide-Thy1

value: CRISPR KO


title: PY_TumoP_N3

description: PY_TumoP_N3 No Cas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality -whole tumor using Py2T into NSG (Imm.deficient) mouse, with vector lentiGuide-Puro

value: xenograft

value: lentiGuide-Puro


title: H3K27me3_D18-2

city: Wenzhou

first: zhengbiao

last: zhu

state: Zhejiang

address: Ouhai District

department: The Affiliated Eye Hospital of Wenzhou Medical University

country: China

institute: Wenzhou Medical University

description:
Library name: H3K27me3_D18-2
H3K27me3

data_processing:
For data analysis, we performed quality control to raw data using FastQC (v0.12.1). Then the reads were trimmed using TrimGalore (v0.6.10) to remove detected adapters automatically. The clean reads were mapped to mouse reference genome mm10 using Bowtie2 (v2.3.4.2). After converting and sorting the output SAM files to BAM files using Samtools (v1.9), we removed the duplicated reads and reads with low mapping quality using Picard and Samtools. Next, we downsample the deduplicated BAM files to the same number of bases and perform quantile normalization using DANPOS.
Assembly: GRCm38.102
Supplementary files format and content: bw file (BigWig file)
Library strategy: CUT&Tag

source_name: E13.5 mouse embryos

extract_protocol:
Approximately 1×105 cells from each sample were processed using the Hyperactive Universal CUT&Tag Assay Kit for Illumina (Vazyme) following the manufacturer's protocol. Briefly, cells were harvested, and 1×105 cells from each sample were counted. The cells were then incubated with Concanavalin A beads at room temperature for 10 minutes. After removing the liquid, primary antibody was added in ice-cold Antibody Buffer, and the mixture was incubated overnight at 4°C. The following day, the liquid was discarded, and a secondary antibody (Guinea Pig Anti-Rabbit Antibody) in Dig-Wash Buffer was added, followed by a 1-hour incubation at room temperature.
After sufficient washing, CUT&Tag pA/G-Tn5 Transposomes were added and incubated at room temperature for 1 hour. Following a final wash, tagmentation was carried out at 37°C for 1 hour. DNA was then extracted using DNA extraction beads. PCR amplification was performed using i7 and i5 Indexed Primers, followed by library cleanup, quantification, and Illumina sequencing. All components except for primary antibody (summarized in key resources table) are included in CUT&Tag Kit.

treatment_protocol: Generation of ciRPE cells from MEFs via a two-stage chemically defined reprogramming protocol M7 reprogramming medium (RM) preparation Equal volumes of Neurobasal (Gibco) and DMEM/F12/GlutaMAX supplemented with 1% N2 (Gibco), 1% B27 supplement without vitamin A (Gibco), 7.5% BSA (Solarbio), 1% NEAA, 1% P/S, bFGF (PeproTech, 10 ng/ml) and the small molecules CHIR-99021 (3 μM, MCE), LDN193189 (0.1 mM, Selleckchem), A 83-01 (0.5 mM, Selleckchem), Hh-Ag1.5 (0.5 mM, Xcess Biosciences), RG108 (10 μM, Selleckchem), BMS-345541 (0.2 mM, MCE) and CKI-7 (5 μM, Sigma-Aldrich). M3 differentiation and maturation medium (DM) preparation DMEM/F12/GlutaMAX supplemented with 10% KSR (Gibco), 1% NEAA, 1% P/S, 0.055 mM 2-mercaptoethanol, Y27632 (10 μM, Selleckchem) and Nicotinamide (NIC, 10 mM, Sigma-Aldrich), Retinoic acid (RA, 1 μM, Sigma-Aldrich), Activin A (0.2 mM, MCE). ciRPE maintenance medium preparation ciRPE maintenance medium includes proliferation medium and function-maintaining medium. The proliferation medium consisted of DMEM/F12/GlutaMAX supplemented with 1% N2, 2% B27 without vitamin A, 1% NEAA, 1% P/S, 0.1 mM 2-mercaptoethanol, 10 ng/mL bFGF, 20 ng/mL EGF (Gibco), 10 μM Y27632, and 0.5 μM A-83-01. The function-maintaining medium consisted of DMEM/F12/GlutaMAX supplemented with 1% N2, 2% B27 with vitamin A, 1% NEAA, 1% P/S, 0.1 mM 2-mercaptoethanol, 0.2 μM Activin A, 0.5 μM RA, 1 μM BMP4, and 10 mM NIC. All chemical components are listed in the key resources table. Small molecules were dissolved and diluted in DMSO (Sigma-Aldrich) following the manufacturer’s instructions and are detailed in Table S13. Stage I: Chemical conversion of eye-field cells from MEFs (1) Four days before the chemical conversion, Matrigel was thawed overnight at 4°C. The following day, 6-well plates were pre-cooled at 4°C for a minimum of 1 hour. Matrigel was diluted 1:40 in DMEM/F12/GlutaMAX and 1 mL of this diluted Matrigel solution was added to each pre-cooled well of the 6-well plate, which was then incubated at 4°C overnight. Note: To ensure optimal performance, Matrigel must be thawed at 4°C and should not be subjected to repeated freeze-thaw cycles. (2) Two days before chemical induction, thaw the cryopreserved primary MEFs in a 37°C water bath and centrifuge to remove the cryoprotectant. Resuspend the cells in MEF medium and seed them into 6-well plates at a density of 20×104 cells per well. Prior to seeding the MEFs, pre-warm the Matrigel-coated 6-well plates at 37°C for at least 30 minutes. Note: The density of MEFs is crucial for the success of chemical induction. Ensure that cells are evenly distributed; too few cells can lead to poor proliferation and cell health, while overly dense cultures may affect the success rate of induction. Regularly monitor the health and growth of MEFs after seeding to ensure they adhere well and proliferate as expected. Proper pre-warming of Matrigel-coated plates is essential for optimal cell attachment and performance. (3) One day before chemical induction, replace the culture medium of MEFs with fresh medium and allow the cells to proliferate for one day. Regularly check the growth and condition of the MEFs to ensure that the cell health and density are optimal. Note: The health of MEFs is crucial for successful conversion. Long-term storage of MEFs in liquid nitrogen may also reduce conversion efficiency. This study used freshly prepared MEFs or those stored in liquid nitrogen for no more than 6 months. (4) On the day of chemical induction, prepare the fresh RM of Stage I, working in the dark to ensure complete dissolution of all components, particularly the chemical agents. After incubating MEFs overnight in MEF medium, wash the cells twice with 1× PBS and replace the medium with the freshly prepared RM. Refresh the RM the next day. (5) During Stage I induction, cells will rapidly proliferate within the first 1-4 days, with their morphology transitioning from elongated spindle-shaped to small oval-shaped. After one week, proliferation of the clones will slow. At this point, mechanically remove surrounding non-clonal cells using a yellow pipette tip to facilitate the continued expansion of the clones. About one week later, cells will begin to adopt a cobblestone-like morphology. Thereafter, refresh the medium daily until EF-like cells are formed. Stage II: Generation of ciRPE cells from eye-field cells For Stage II induction, the cells exhibit initial RPE cell morphology but still lack maturity and pigmentation. At this stage, replace the medium with DM of Stage II to induce pigmentation and promote further maturation of ciRPE cells. The medium is changed daily. After two weeks of treatment, the cells reach near-full maturity and show significant pigmentation. Subsequently, ciRPE cells are passaged and expanded in maintenance medium.

value: E13.5 mouse embryos

value: Reprogramming intermediate

value: M7 reprogramming medium


title: PY_TumoP_N2

description: PY_TumoP_N2 No Cas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality -whole tumor using Py2T into NSG (Imm.deficient) mouse, with vector lentiGuide-Puro


title: PY_TumoP_N1

description: PY_TumoP_N1 No Cas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality -whole tumor using Py2T into NSG (Imm.deficient) mouse, with vector lentiGuide-Puro


title: PY_TumoT_F4

description: PY_TumoT_F4 No Cas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality -whole tumor using Py2T into FVB (Imm.competent) mouse, with vector lentiGuide-Thy1


title: 4T_LungsP_B5

description: 4T_LungsP_B5 No Cas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality -tbd using 4T1 into BALB (Imm.competent) mouse, with vector lentiGuide-Puro


title: R882H_3_EPO

description: Library name: HUE6313A23

value: EPO


title: Nact D5 CpG_A

description: Library name: LS_R10

value: spleen


title: PY_TumoT_N4

description: PY_TumoT_N4 No Cas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality -whole tumor using Py2T into NSG (Imm.deficient) mouse, with vector lentiGuide-Thy1


title: MVT_TumoT_N4

description: MVT_TumoT_N4 No Cas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality -whole tumor using MVT1 into NSG (Imm.deficient) mouse, with vector lentiGuide-Thy1


title: H3K27me3_D7-2

description:
Library name: H3K27me3_D7-2
H3K27me3


title: R882H_1_EPO

description: Library name: HUE6313A21


title: H3K27me3_D7-1

description:
Library name: H3K27me3_D7-1
H3K27me3


title: MVT_TumoT_N1

description: MVT_TumoT_N1 No Cas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality -whole tumor using MVT1 into NSG (Imm.deficient) mouse, with vector lentiGuide-Thy1


title: CRISPRKO_Clone_12

description: CRISPRKO_Clone_12 CRISPR Knockout (by Cas9 transfection) through the StealTHY system -whole tumor using 4T1 into BALB (Imm.competent) mouse, with vector lentiGuide-Thy1


title: R882H_2_CTRL

description: Library name: HUE6313A18


title: MVT_TumoT_F2

description: MVT_TumoT_F2 No Cas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality -whole tumor using MVT1 into FVB (Imm.competent) mouse, with vector lentiGuide-Thy1


title: W305_4_EPO

description: Library name: HUE6313A16


title: H3K27me3_MEF-2

description:
Library name: H3K27me3_MEF-2
H3K27me3

value: MEF


title: MVT_TumoT_F4

description: MVT_TumoT_F4 No Cas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality -whole tumor using MVT1 into FVB (Imm.competent) mouse, with vector lentiGuide-Thy1


title: PY_TumoT_N3

description: PY_TumoT_N3 No Cas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality -whole tumor using Py2T into NSG (Imm.deficient) mouse, with vector lentiGuide-Thy1

value: NSG (Imm.deficient)


title: Nact D5 CD40_A

description: Library name: LS_R9

value: WT


title: H3K4me3_P_PRE-2

description:
Library name: H3K4me3_P_PRE-2
H3K4me3

value: RPE


title: MVT_TumoP_N4

description: MVT_TumoP_N4 No Cas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality -whole tumor using MVT1 into NSG (Imm.deficient) mouse, with vector lentiGuide-Puro

value: sgRNAs used as barcodes (no KO)


title: H3K27me3_P_PRE-2

description:
Library name: H3K27me3_P_PRE-2
H3K27me3


title: H3K27me3_P_PRE-1

description:
Library name: H3K27me3_P_PRE-1
H3K27me3


title: H3K4me3_P_PRE-1

description:
Library name: H3K4me3_P_PRE-1
H3K4me3


title: PY_TumoT_F3

description: PY_TumoT_F3 No Cas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality -whole tumor using Py2T into FVB (Imm.competent) mouse, with vector lentiGuide-Thy1

value: tumor


title: R882C_4_CTRL

description: Library name: HUE6313A28

value: UNTREATED


title: H3K27me3_D32-2

description:
Library name: H3K27me3_D32-2
H3K27me3

value: M3 differentiation and maturation medium


title: R882H_4_EPO

description: Library name: HUE6313A24


title: W305_1_EPO

description: Library name: HUE6313A13


title: PY_TumoT_F1

description: PY_TumoT_F1 No Cas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality -whole tumor using Py2T into FVB (Imm.competent) mouse, with vector lentiGuide-Thy1

value: FVB (Imm.competent)

value: Immuno Signalome (2215) sgRNA library


title: MVT_TumoP_F5

description: MVT_TumoP_F5 No Cas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality -whole tumor using MVT1 into FVB (Imm.competent) mouse, with vector lentiGuide-Puro

value: xenograft


title: PY_TumoP_N5

description: PY_TumoP_N5 No Cas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality -whole tumor using Py2T into NSG (Imm.deficient) mouse, with vector lentiGuide-Puro

value: lentiGuide-Puro


title: PY_TumoP_N4

description: PY_TumoP_N4 No Cas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality -whole tumor using Py2T into NSG (Imm.deficient) mouse, with vector lentiGuide-Puro


title: R882H_2_EPO

description: Library name: HUE6313A22


title: PY_TumoT_N1

description: PY_TumoT_N1 No Cas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality -whole tumor using Py2T into NSG (Imm.deficient) mouse, with vector lentiGuide-Thy1


title: H3K27me3_D18-1

description:
Library name: H3K27me3_D18-1
H3K27me3

value: E13.5 mouse embryos


title: H3K27me3_D32-1

description:
Library name: H3K27me3_D32-1
H3K27me3

value: Reprogramming intermediate


title: MVT_TumoP_F2

description: MVT_TumoP_F2 No Cas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality -whole tumor using MVT1 into FVB (Imm.competent) mouse, with vector lentiGuide-Puro


title: W305_4_CTRL

description: Library name: HUE6313A12


title: MVT_TumoT_N3

description: MVT_TumoT_N3 No Cas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality -whole tumor using MVT1 into NSG (Imm.deficient) mouse, with vector lentiGuide-Thy1


title: R882H_3_CTRL

description: Library name: HUE6313A19


title: MVT_TumoP_F1

description: MVT_TumoP_F1 No Cas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality -whole tumor using MVT1 into FVB (Imm.competent) mouse, with vector lentiGuide-Puro


title: MVT_TumoT_N2

description: MVT_TumoT_N2 No Cas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality -whole tumor using MVT1 into NSG (Imm.deficient) mouse, with vector lentiGuide-Thy1


title: 4T_TumoT_N5

description: 4T_TumoT_N5 No Cas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality -whole tumor using 4T1 into NSG (Imm.deficient) mouse, with vector lentiGuide-Thy1


title: R882H_4_CTRL

description: Library name: HUE6313A20


title: W305_3_CTRL

description: Library name: HUE6313A11


title: 4T_TumoT_N4

description: 4T_TumoT_N4 No Cas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality -whole tumor using 4T1 into NSG (Imm.deficient) mouse, with vector lentiGuide-Thy1


title: MVT_TumoT_F5

description: MVT_TumoT_F5 No Cas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality -whole tumor using MVT1 into FVB (Imm.competent) mouse, with vector lentiGuide-Thy1


title: R882H_1_CTRL

description: Library name: HUE6313A17


title: W305_2_CTRL

description: Library name: HUE6313A10


title: H3K4me3_D32-2

description:
Library name: H3K4me3_D32-2
H3K4me3


title: H3K27me3_MEF-1

description:
Library name: H3K27me3_MEF-1
H3K27me3

value: M7 reprogramming medium


title: MVT_TumoT_F1

description: MVT_TumoT_F1 No Cas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality -whole tumor using MVT1 into FVB (Imm.competent) mouse, with vector lentiGuide-Thy1


title: W305_1_CTRL

description: Library name: HUE6313A9


title: 4T_TumoT_N1

description: 4T_TumoT_N1 No Cas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality -whole tumor using 4T1 into NSG (Imm.deficient) mouse, with vector lentiGuide-Thy1


title: 4T_TumoT_B5

description: 4T_TumoT_B5 No Cas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality -whole tumor using 4T1 into BALB (Imm.competent) mouse, with vector lentiGuide-Thy1

value: BALB (Imm.competent)


title: H3K4me3_D18-2

description:
Library name: H3K4me3_D18-2
H3K4me3


title: MVT_TumoT_N5

description: MVT_TumoT_N5 No Cas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality -whole tumor using MVT1 into NSG (Imm.deficient) mouse, with vector lentiGuide-Thy1


title: MVT_TumoP_N5

description: MVT_TumoP_N5 No Cas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality -whole tumor using MVT1 into NSG (Imm.deficient) mouse, with vector lentiGuide-Puro


title: MVT_TumoT_F3

description: MVT_TumoT_F3 No Cas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality -whole tumor using MVT1 into FVB (Imm.competent) mouse, with vector lentiGuide-Thy1


title: H3K4me3_P_PRE-3

description:
Library name: H3K4me3_P_PRE-3
H3K4me3


title: MVT_TumoP_N3

description: MVT_TumoP_N3 No Cas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality -whole tumor using MVT1 into NSG (Imm.deficient) mouse, with vector lentiGuide-Puro


title: W305_3_EPO

description: Library name: HUE6313A15

value: EPO


title: WT2_EPO

description: Library name: HUE6313A6


title: 4T_TumoP_N4

description: 4T_TumoP_N4 No Cas9 (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


title: W305_2_EPO

description: Library name: HUE6313A14


title: WT1_EPO

description: Library name: HUE6313A5


title: H3K4me3_D7-2

description:
Library name: H3K4me3_D7-2
H3K4me3


title: 4T_TumoP_N2

description: 4T_TumoP_N2 No Cas9 (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


title: H3K4me3_D7-1

description:
Library name: H3K4me3_D7-1
H3K4me3


title: MVT_TumoP_N2

description: MVT_TumoP_N2 No Cas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality -whole tumor using MVT1 into NSG (Imm.deficient) mouse, with vector lentiGuide-Puro


title: MVT_TumoP_N1

description: MVT_TumoP_N1 No Cas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality -whole tumor using MVT1 into NSG (Imm.deficient) mouse, with vector lentiGuide-Puro


title: H3K4me3_D32-3

description:
Library name: H3K4me3_D32-3
H3K4me3


title: 4T_TumoP_B4

description: 4T_TumoP_B4 No Cas9 (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


title: H3K4me3_MEF-3

description:
Library name: H3K4me3_MEF-3
H3K4me3

value: MEF


title: H3K4me3_MEF-2

description:
Library name: H3K4me3_MEF-2
H3K4me3


title: 4T_TumoP_B2

description: 4T_TumoP_B2 No Cas9 (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


title: WT2_CTRL

description: Library name: HUE6313A2


title: WT1_CTRL

description: Library name: HUE6313A1


title: H3K4me3_D32-1

description:
Library name: H3K4me3_D32-1
H3K4me3


title: 4T_TumoT_N3

description: 4T_TumoT_N3 No Cas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality -whole tumor using 4T1 into NSG (Imm.deficient) mouse, with vector lentiGuide-Thy1

value: NSG (Imm.deficient)


title: H3K4me3_D18-3

description:
Library name: H3K4me3_D18-3
H3K4me3


title: WT4_EPO

description: Library name: HUE6313A8

value: WT


title: 4T_TumoT_B3

description: 4T_TumoT_B3 No Cas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality -whole tumor using 4T1 into BALB (Imm.competent) mouse, with vector lentiGuide-Thy1


title: H3K4me3_D18-1

description:
Library name: H3K4me3_D18-1
H3K4me3


title: 4T_TumoT_B2

description: 4T_TumoT_B2 No Cas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality -whole tumor using 4T1 into BALB (Imm.competent) mouse, with vector lentiGuide-Thy1


title: WT3_EPO

description: Library name: HUE6313A7


title: 4T_TumoP_B3

description: 4T_TumoP_B3 No Cas9 (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


title: 4T_TumoT_N2

description: 4T_TumoT_N2 No Cas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality -whole tumor using 4T1 into NSG (Imm.deficient) mouse, with vector lentiGuide-Thy1


title: H3K4me3_D7-3

description:
Library name: H3K4me3_D7-3
H3K4me3


title: H3K27ac_P_PRE-2

description:
Library name: H3K27ac_P_PRE-2
H3K27ac

value: RPE


title: 4T_TumoT_B1

description: 4T_TumoT_B1 No Cas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality -whole tumor using 4T1 into BALB (Imm.competent) mouse, with vector lentiGuide-Thy1

value: sgRNAs used as barcodes (no KO)


title: PY_LungsT_N1

description: PY_LungsT_N1 No Cas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality -whole lungs using Py2T into NSG (Imm.deficient) mouse, with vector lentiGuide-Thy1

value: tumor


title: 4T_TumoP_N5

description: 4T_TumoP_N5 No Cas9 (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


title: PY_LungsT_F5

description: PY_LungsT_F5 No Cas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality -whole lungs using Py2T into FVB (Imm.competent) mouse, with vector lentiGuide-Thy1


title: 4T_TumoT_B4

description: 4T_TumoT_B4 No Cas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality -whole tumor using 4T1 into BALB (Imm.competent) mouse, with vector lentiGuide-Thy1


title: PY_LungsT_N3

description: PY_LungsT_N3 No Cas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality -whole lungs using Py2T into NSG (Imm.deficient) mouse, with vector lentiGuide-Thy1


title: Immune_progeny_WT_LPS

city: LONDON

first: Giuseppe

middle: Alessandro

last: D'Agostino

state: ENGLAND

address: 1 Midland Road

description:
Library name: Progeny_WT_LPS
10X Multiome GEX

data_processing:
For the HSPC single cell RNA-seq data, FASTQ files from different lanes were merged and STAR v. 2.7.10b was used to align each of them to the Ensembl h38 genome, release 108, using the following parameters: --runMode alignReads, --soloType CB_UMI_Simple, --soloUMIlen 12, --runRNGseed 1. For the myeloid progeny single cell RNA-seq data, cellranger v. 7.1.0 was used using pre-computed references (“refdata-cellranger-GRCh38-3.0.0”) and chemistry=ARC-v1.
Assembly: Ensembl h38 genome, release 108
Supplementary files format and content: tar.gz archives of the filtered gene counts from STARSolo and HDF5 files from the CellRanger v. 7.0 output (multiome gene expression)

molecule: cytoplasmic RNA

source_name: Bone Marrow

extract_protocol:
HSPCs samples were prepared using 10X 3’ V3.1 and sequenced using the HiSeq4000, for immune progeny samples we used the 10X multiome kit and were sequenced in NovaSeq6000.
The concentration and viability of the single cell suspension was measured using trypan blue using Eve Automatic Cell Counter. Approximately 3,000-10,000 cells were loaded on Chromium Chip and partitioned in nanolitre scale droplets using the Chromium Controller and Chromium Next GEM Single Cell Reagents (Chromium Single Cell 3' Reagent Kits User Guide (v3.1)). Within each droplet the cells were lysed, and the RNA was reverse transcribed. All the resulting cDNA within a droplet shared the same cell barcode. Illumina compatible libraries were generated from the cDNA using Chromium Next GEM Single Cell library reagents in accordance with the manufacturer’s instructions (10x Genomics, CG000315 Chromium Single Cell 3' Reagent Kits User Guide (v3.1 - Dual Index)). For QC of libraries, we used the Agilent TapeStation and sequenced using the Illumina HiSeq4000. Sequencing read configuration: 28-10-10-90. For the immune progeny, we sorted human hematopoietic cell subsets from humanized mice reconstituted with either TET2WT or TET2Mut HSCs as described in Figure S5A. The concentration and viability of the single nuclei suspension was measured using acridine orange (AO) and propidium iodide (PI) and the Luna-FX7 Automatic Cell Counter. Approximately 15,000-20,000 nuclei were transposed, then loaded on Chromium Chip and partitioned in nanolitre scale droplets using the Chromium Controller and Chromium Next GEM Single Cell Reagents (Chromium Next GEM Single Cell Multiome ATAC + Gene Expression Reagent Kits User Guide, User Guide, CG000338). Sequencing read configuration: 28-10-10-90 (GEX). The 10x Barcodes in each library type are used to associate individual reads back to the individual partitions, and thereby, to each individual nucleus.
scRNA-Seq 10X  3' Gene Expression and 10X Multiome Gene Expression

value: Bone Marrow

value: Human Immune cells

value: LPS

tag: library type

value: mRNA


title: PY_LungsT_F4

description: PY_LungsT_F4 No Cas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality -whole lungs using Py2T into FVB (Imm.competent) mouse, with vector lentiGuide-Thy1

value: xenograft

value: FVB (Imm.competent)

value: Immuno Signalome (2215) sgRNA library


title: 4T_TumoP_N1

description: 4T_TumoP_N1 No Cas9 (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

value: lentiGuide-Puro


title: WT4_CTRL

description: Library name: HUE6313A4

value: UNTREATED


title: 4T_TumoP_N3

description: 4T_TumoP_N3 No Cas9 (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


title: WT3_CTRL

description: Library name: HUE6313A3


title: 4T_TumoP_B1

description: 4T_TumoP_B1 No Cas9 (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

value: BALB (Imm.competent)


title: PY_LungsT_N5

description: PY_LungsT_N5 No Cas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality -whole lungs using Py2T into NSG (Imm.deficient) mouse, with vector lentiGuide-Thy1


title: PY_LungsT_N4

description: PY_LungsT_N4 No Cas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality -whole lungs using Py2T into NSG (Imm.deficient) mouse, with vector lentiGuide-Thy1


title: H3K27ac_P_PRE-3

description:
Library name: H3K27ac_P_PRE-3
H3K27ac

value: E13.5 mouse embryos


title: H3K27ac_D32-3

description:
Library name: H3K27ac_D32-3
H3K27ac

value: Reprogramming intermediate

value: M3 differentiation and maturation medium


title: PY_LungsT_N2

description: PY_LungsT_N2 No Cas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality -whole lungs using Py2T into NSG (Imm.deficient) mouse, with vector lentiGuide-Thy1

value: NSG (Imm.deficient)


title: PY_LungsT_F1

description: PY_LungsT_F1 No Cas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality -whole lungs using Py2T into FVB (Imm.competent) mouse, with vector lentiGuide-Thy1


title: PY_LungsP_N5

description: PY_LungsP_N5 No Cas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality -whole lungs using Py2T into NSG (Imm.deficient) mouse, with vector lentiGuide-Puro


title: Immune_progeny_TET2_LPS

description:
Library name: Progeny_KO_LPS
10X Multiome GEX


title: PY_LungsP_N4

description: PY_LungsP_N4 No Cas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality -whole lungs using Py2T into NSG (Imm.deficient) mouse, with vector lentiGuide-Puro


title: PY_LungsP_N3

description: PY_LungsP_N3 No Cas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality -whole lungs using Py2T into NSG (Imm.deficient) mouse, with vector lentiGuide-Puro


title: P2- Vehicle

first: Trinidad

last: Montero

address: Charterhouse Square

department: Biochemical Pharmacology

institute: Whilliam Harvey Research Institute, Bart's and The London School of Medicine; Queen Mary University of London

data_processing:
Data were analysed using Partek Flow Genomic Analysis Software (release no. 10.0.23.0720), inlcuding reads trimming, alignment, quentification to transcriptome and normalization.
Base trimming was based on quality score (end min quality level (Phred) 20) from both ends
Reads were aligned to the human genome (hg38) using STAR 2.7.8a
Quanfitication to transcriptome was conducted using assemby Homo sapines (human) hg38, annotation model RefSeq Transcripts 93 - 2020 - 02 - 03
Transcript quantification was conducted with TPM normalisation method
Assembly: Human Genome G38
Supplementary files format and content: RPKM values are provided for each sample in a txt file

source_name: Skin

growth_protocol: Fibroblasts were maintained in Dulbecco’s modified eagle medium (DMEM) supplemented with 10% heat inactivated foetal bovine serum (FBS), 2 mM glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin (‘complete medium’) until ~80% confluence

extract_protocol:
RNA was extracted using Direct-zol RNA MiniPrep  (Zymo Research)
RNAseq library was prepared using the KAPA mRNA HyperPrep Kit (Roche) and xGen UDI+UMI adapter set (Integrated DNA Technologies). Sequencing was performed on a NovaSeq 6000 Sequencing System (Illumina), using an S1 flow-cell and 100 cycle SBS sequencing kit.

treatment_protocol: Cells were plated on day 0 and drug treatments (BMS-470539 -10uM-, alphaMSH -10uM-, itraconazole -1uM- or vehicle -PBS-) applied on days 1, 3 and 5. Cells were collected on day 6 and RNA extracted.

value: Skin

value: Primary

value: Fibroblast

tag: individual

value: Patient 2

value: Vehicle


title: H3K27ac_D32-1

description:
Library name: H3K27ac_D32-1
H3K27ac


title: P1- Vehicle

value: Patient 1


title: PY_LungsP_N2

description: PY_LungsP_N2 No Cas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality -whole lungs using Py2T into NSG (Imm.deficient) mouse, with vector lentiGuide-Puro

value: tumor

value: sgRNAs used as barcodes (no KO)


title: H3K4me3_MEF-1

description:
Library name: H3K4me3_MEF-1
H3K4me3

value: MEF

value: M7 reprogramming medium


title: H3K27ac_D18-3

description:
Library name: H3K27ac_D18-3
H3K27ac


title: H3K27ac_P_PRE-1

description:
Library name: H3K27ac_P_PRE-1
H3K27ac

value: RPE


title: P4- Itraconazole

value: Patient 4

value: Itraconazole


title: P4- Vehicle


title: PY_LungsP_N1

description: PY_LungsP_N1 No Cas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality -whole lungs using Py2T into NSG (Imm.deficient) mouse, with vector lentiGuide-Puro

value: xenograft

value: Immuno Signalome (2215) sgRNA library

value: lentiGuide-Puro


title: H3K27ac_D18-2

description:
Library name: H3K27ac_D18-2
H3K27ac

value: E13.5 mouse embryos

value: Reprogramming intermediate


title: H3K27ac_D32-2

description:
Library name: H3K27ac_D32-2
H3K27ac

value: M3 differentiation and maturation medium


title: PY_LungsT_F3

description: PY_LungsT_F3 No Cas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality -whole lungs using Py2T into FVB (Imm.competent) mouse, with vector lentiGuide-Thy1

value: FVB (Imm.competent)


title: MVT_LungsT_N5

description: MVT_LungsT_N5 No Cas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality -whole lungs using MVT1 into NSG (Imm.deficient) mouse, with vector lentiGuide-Thy1

value: NSG (Imm.deficient)


title: PY_LungsT_F2

description: PY_LungsT_F2 No Cas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality -whole lungs using Py2T into FVB (Imm.competent) mouse, with vector lentiGuide-Thy1


title: PY_LungsP_F4

description: PY_LungsP_F4 No Cas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality -whole lungs using Py2T into FVB (Imm.competent) mouse, with vector lentiGuide-Puro


title: P3- Vehicle

value: Skin

value: Primary

value: Fibroblast

value: Patient 3

value: Vehicle


title: PY_LungsP_F3

description: PY_LungsP_F3 No Cas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality -whole lungs using Py2T into FVB (Imm.competent) mouse, with vector lentiGuide-Puro

value: tumor

value: sgRNAs used as barcodes (no KO)


title: H3K27ac_D7-3

description:
Library name: H3K27ac_D7-3
H3K27ac

value: M7 reprogramming medium


title: MVT_LungsT_N1

description: MVT_LungsT_N1 No Cas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality -whole lungs using MVT1 into NSG (Imm.deficient) mouse, with vector lentiGuide-Thy1


title: P2- Itraconazole

value: Patient 2

value: Itraconazole


title: P1- Itraconazole

value: Patient 1


title: H3K27ac_D7-2

description:
Library name: H3K27ac_D7-2
H3K27ac


title: P3- Itraconazole


title: MVT_LungsT_N3

description: MVT_LungsT_N3 No Cas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality -whole lungs using MVT1 into NSG (Imm.deficient) mouse, with vector lentiGuide-Thy1

value: xenograft

value: Immuno Signalome (2215) sgRNA library


title: MVT_LungsT_N2

description: MVT_LungsT_N2 No Cas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality -whole lungs using MVT1 into NSG (Imm.deficient) mouse, with vector lentiGuide-Thy1


title: MVT_LungsT_F1

description: MVT_LungsT_F1 No Cas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality -whole lungs using MVT1 into FVB (Imm.competent) mouse, with vector lentiGuide-Thy1


title: H3K27ac_D18-1

description:
Library name: H3K27ac_D18-1
H3K27ac

value: E13.5 mouse embryos

value: Reprogramming intermediate


title: P2- BMS-470539


title: MVT_LungsP_N5

description: MVT_LungsP_N5 No Cas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality -whole lungs using MVT1 into NSG (Imm.deficient) mouse, with vector lentiGuide-Puro

value: lentiGuide-Puro


title: MVT_LungsT_N4

description: MVT_LungsT_N4 No Cas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality -whole lungs using MVT1 into NSG (Imm.deficient) mouse, with vector lentiGuide-Thy1

value: NSG (Imm.deficient)


title: MVT_LungsP_N4

description: MVT_LungsP_N4 No Cas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality -whole lungs using MVT1 into FVB (Imm.competent) mouse, with vector lentiGuide-Puro


title: H3K27ac_MEF-2

description:
Library name: H3K27ac_MEF-2
H3K27ac

value: MEF


title: H3K27ac_MEF-1

description:
Library name: H3K27ac_MEF-1
H3K27ac


title: MVT_LungsP_F5

description: MVT_LungsP_F5 No Cas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality -whole lungs using MVT1 into FVB (Imm.competent) mouse, with vector lentiGuide-Puro

value: FVB (Imm.competent)


title: MVT_LungsT_F5

description: MVT_LungsT_F5 No Cas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality -whole lungs using MVT1 into FVB (Imm.competent) mouse, with vector lentiGuide-Thy1


title: P4- Alpha-MSH

value: Skin

value: Primary

value: Fibroblast

value: Patient 4

value: alpha-MSH


title: MVT_LungsT_F4

description: MVT_LungsT_F4 No Cas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality -whole lungs using MVT1 into FVB (Imm.competent) mouse, with vector lentiGuide-Thy1

value: tumor

value: sgRNAs used as barcodes (no KO)


title: MVT_LungsP_F1

description: MVT_LungsP_F1 No Cas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality -whole lungs using MVT1 into FVB (Imm.competent) mouse, with vector lentiGuide-Puro


title: MVT_LungsP_F2

description: MVT_LungsP_F2 No Cas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality -whole lungs using MVT1 into FVB (Imm.competent) mouse, with vector lentiGuide-Puro


title: P3- Alpha-MSH

value: Patient 3


title: P4- BMS-470539


title: MVT_LungsT_F3

description: MVT_LungsT_F3 No Cas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality -whole lungs using MVT1 into FVB (Imm.competent) mouse, with vector lentiGuide-Thy1


title: H3K27ac_D7-1

description:
Library name: H3K27ac_D7-1
H3K27ac

value: M7 reprogramming medium


title: 4T_LungsT_N5

description: 4T_LungsT_N5 No Cas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality -whole lungs using 4T1 into NSG (Imm.deficient) mouse, with vector lentiGuide-Thy1

value: xenograft


title: P2- Alpha-MSH

value: Patient 2


title: 4T_LungsT_N4

description: 4T_LungsT_N4 No Cas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality -whole lungs using 4T1 into FVB (Imm.competent) mouse, with vector lentiGuide-Thy1

value: Immuno Signalome (2215) sgRNA library


title: P3- BMS-470539


title: MVT_LungsT_F2

description: MVT_LungsT_F2 No Cas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality -whole lungs using MVT1 into FVB (Imm.competent) mouse, with vector lentiGuide-Thy1


title: H3K27ac_MEF-3

description:
Library name: H3K27ac_MEF-3
H3K27ac

value: E13.5 mouse embryos


title: 4T_LungsT_N3

description: 4T_LungsT_N3 No Cas9 (i.e. no knockout) - SgRNAs as barcodes for studying loss of clonality -whole lungs using 4T1 into FVB (Imm.competent) mouse, with vector lentiGuide-Thy1


title: D7_scRNA

description:
Library name: D7_scRNA
SeekOne® Digital Droplet

data_processing:
For scRNA-seq data analysis, the demultiplexing, barcoded processing, gene counting and aggregation were performed using the SeekSoulTools (v1.2.2) software with the default parameters. All scRNA-seq reads were aligned to the mouse reference genome (mm10). The scRNA-seq expression profiles were analyzed using R package Seurat (v4.1.0). The cells within a minimum of 200 genes expressed and the percentage of mitochondrial gene counts < 10% were kept for subsequent analysis. We followed the standard Seurat pipeline by running function FindVariableFeatures, ScaleData, RunPCA, FindNeighbors, and FindClusters in order. We performed single-cell regulatory network inference and clustering (SCENIC) analysis using python package pySCENIC (0.12.1) to identify activated TFs and subnetwork of each TF (regulons) for each cell type. To calculate RNA velocity, both velocyto (v0.17) and scVelo (0.2.5)  were used to analyze reads that passed the quality control after clustering as instructed. First, the standard velocyto pipeline was run to count spliced and unspliced reads for each sample based on the filtered SeekSoulTools-generated bam files. Then the output loom file was used as input for scVelo based on the dynamic model to estimate velocity embedding.
Assembly: GRCm38.102
Supplementary files format and content: RDS data for saving single-cell data

extract_protocol:
For scRNA-seq sample collection, we scraped all cells with different induction phenotypes at each time point, aiming to analyze all cell types involved in the induction process. Cells were washed in ice-cold PBS and dissociated using SeekMate Tissue Dissociation Kits (SeekGene). DNase I treatment was applied if needed. Cell viability was assessed using a Fluorescence Cell Analyzer (Countstar® Rigel, S2), after removing erythrocytes and filtering out debris and dead cells. Fresh cells were resuspended in RPMI1640 with 2% FBS at 1×106 cells/mL.
Single-cell RNA-Seq libraries were prepared using the SeekOne® Digital Droplet Single Cell 3' Kit (SeekGene). Cells were mixed with reverse transcription reagents and loaded into SeekOne® chip S3 for droplet generation. Reverse transcription was performed at 42°C for 90 minutes, followed by inactivation at 85°C for 5 minutes. cDNA was purified, amplified, fragmented, end-repaired, and ligated to sequencing adaptors. Indexed PCR was performed to amplify the 3' polyA regions, including Cell Barcodes and Unique Molecular Indices (UMIs). Libraries were cleaned, quantified, and sequenced on an Illumina NovaSeq 6000 or DNBSEQ-T7 platform.

value: Reprogramming intermediate


title: P1- BMS-470539

value: Patient 1