title: mCherry_positive_tumor_WTA

description: DMBA/TPA-induced skin tumor mCherry-positive cells scRNA-seq WTA

data_processing:
The raw sequencing data comprising the BCL files were demultiplexed using the Illumina bcl2fastq program v2.20 with default options allowing one mismatch of the sample barcode sequences. The resulting FASTQ files were processed using the BD Rhapsody™ Whole Transcriptome Analysis (WTA) Pipeline (v1.11.1) hosted at the Seven Bridge’s cloud platform (https://www.sevenbridges.com/). We utilized a custom mouse reference genome based on the Gencode version 25 (mm10) combined with our 500 sgRNA sequences. Since the BD pipeline uses STAR aligner in the backend, the custom STAR reference genome was generated by the genomeGenerate command in STAR (version 2.5.2b). Unique Molecular Identifier (UMI) count errors that are single-base substitution errors were identified and adjusted to the parent UMI barcode in the pipeline using RSEC (Recursive Substitution Error Correction) method, developed by BD Biosciences. The output consisted of thus corrected UMI counts, and was used for further downstream analysis.
Assembly: mm10
Supplementary files format and content: *_MolsPerCell.csv.gz compressed files contain a comma-separated (csv) expression matrix with molecule (UMI) counts per cells. Cells are in rows, gene names are in columns. Cells are identified with a unique cell index number. *.h5 files are HDF5 files containing RSEC molecule counts with only the final subset of cells used in our publication.

source_name: skin

extract_protocol:
Tumors and 1-2 mm of surrounding tissue from DMBA/TPA treated mice were excised and finely minced with a scalpel. Tumor pieces were submerged in 5 mL prewarmed DMEM with 0.25% Trypsin/EDTA and 3 U/mL DNAse I at 37°C on an orbital shaker with rigorous resuspension every 5 minutes for a total of 30 minutes. Reaction was quenched with 1 ml FBS and strained through a 70 µM mesh, pelleted at 1400rpm for 10 minutes with excess PBS and resuspended in 0.5 mL FACS buffer before mCherry-positive sorting. Single-Cell capture was performed with BD Rhapsody using Rhapsody Enhanced Cartridge Kit (BD Biosciences 664887).
Reverse transcription from beads and sequencing library production was carried out according to manufacturer's instructions (BD Biosciences, Doc ID: 210967 Rev. 1.0). Whole Transcriptome Amplification Analysis (WTAs) from BD Rhapsody Cartridges were sequenced in full SP flowcells at 1.8nM concentration with 10% PhiX, dual indexing i5 - 8bp, i7 - 8bp and Read1 64 cycles.

value: mouse epidermis

value: skin cells

value: JAX #026179, CD1 wildtype x Tg(B6J.129(Cg)-Gt(ROSA)26Sortm1.1(CAG-cas9*,-EGFP)Fezh/J

value: 144 days post natal

value: DMBA/TPA carcinogenesis


title: primary cortical neurons, KO+I39V biol rep 3

status: Public on Jul 17 2024

type: SRA

city: Philadelphia

first: Emily

last: Feierman

address: 3400 Civic Center Blvd

country: USA

institute: University of Pennsylvania

library_source: genomic

data_processing:
Fastq files were aligned by Bowtie2
bam files from the three sequencing runs were merged for each biological replicate, all biological replicates from the same group were merged, and 80 million reads were subset for each group SAMTools for downstream analysis
NGSplot was used to generate metaplots
deepTools was used to generate bigwig files
Assembly: mm10
Supplementary files format and content: bigwig files generated by deepTools from bam files aligned to bowtie2

molecule: genomic DNA

organism: Mus musculus

source_name: primary cortical neurons

extract_protocol:
Cells were collected in cell lysis buffer (10mM TrisHCl ph7.5, 10mM NaCl, 3mM MgCl2, 0.1% NP-40, 0.1% Tween20, 0.1% Digitonin)
Transposition reaction mix from Nextera Library Prep kit was used (Tagment DNA Enzyme 1 and Tagment DNA Buffer)
Nextera primer sets with NEBNext Master Mix was used to generate libraries with AMPure beads used for cleanup

tag: age

tag: cell type

value: primary cortical neurons

tag: genotype

tag: infection


title: iPSC-NSCs cells, Rep2

city: nanjing

first: Jie

last: Liu

state: Jiang Su

address: 101 Longmian Avenue, Jiangning District, Nanjing

country: China

institute: Nanjing Medical University

library_source: transcriptomic

data_processing:
first of all, ligated the 3′ SR Adaptor. Mixed 3′ SR Adaptor for Illumina, RNA and Nuclease-Free Water, mixture system after incubation for 2 minutes at 70 degrees in a preheated thermal cycler. Tube was transfer to ice.
Then, add 3′ Ligation Reaction Buffer (2X) and 3′ Ligation Enzyme Mix ligate the 3′ SR Adaptor. Incubated for 1 hour at 25°C in a thermal cycler. To prevent adaptor-dimer formation, the SR RT Primer hybridizes to the excess of 3′ SR Adaptor (that remains free after the 3′ ligation reaction) and transforms the single stranded DNA adaptor into a double-stranded DNA molecule. dsDNAs are not substrates for ligation mediated.
The second, ligated the 5′ SR Adaptor. Then, reverse transcription synthetic first chain. Last, PCR amplification and Size Selection. PAGE gel was used to electrophoresis fragment screening purposes, rubber cutting recycling as the pieces get small RNA libraries.
At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system.
Assembly: hg38
Supplementary files format and content: tab-delimited text file includes RPM amd Count values  for each sample

molecule: total RNA

organism: Homo sapiens

source_name: Induced pluripotent stem cells-derived neural stem cells

growth_protocol: iPSC-NSCs were cultured in NeuroEasy recovery and maintaining medium (Cellapy Biotechnology Co. Beijing, China) under 5% CO2 at 37°C

extract_protocol:
A total amount of 2.5 ng RNA per sample was used as input material for the RNA sample preparations.
Sequencing libraries were generated using NEBNextR UltraTM small RNA Sample Library Prep Kit for Illumina R(NEB, USA) following manufacturer’s recommendations and index codes were added to attribute sequences to each sample.
first of all, ligated the 3′ SR Adaptor. Mixed 3′ SR Adaptor for Illumina, RNA and Nuclease-Free Water, mixture system after incubation for 2 minutes at 70 degrees in a preheated thermal cycler. Tube was transfer to ice.
Then, add 3′ Ligation Reaction Buffer (2X) and 3′ Ligation Enzyme Mix ligate the 3′ SR Adaptor. Incubated for 1 hour at 25°C in a thermal cycler. To prevent adaptor-dimer formation, the SR RT Primer hybridizes to the excess of 3′ SR Adaptor (that remains free after the 3′ ligation reaction) and transforms the single stranded DNA adaptor into a double-stranded DNA molecule. dsDNAs are not substrates for ligation mediated.
The second, ligated the 5′ SR Adaptor. Then, reverse transcription synthetic first chain. Last, PCR amplification and Size Selection. PAGE gel was used to electrophoresis fragment screening purposes, rubber cutting recycling as the pieces get small RNA libraries.
At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system.

value: WT

tag: treatment

value: iPSC-NSCs cells


title: DMBA/TPA-induced skin tumor Visium Spatial Transcriptomics, replicate 1 (A1_230331)

city: Basel

first: Simona

last: Baghai Sain

address: Schanzenstrasse 44

department: Department of Biosystems Science and Engineering

country: Switzerland

institute: ETH Zurich

data_processing:
The raw sequencing data comprising the BCL files were demultiplexed into FASTQ files using the Illumina bcl2fastq program v2.20 with default options allowing one mismatch of the sample barcode sequences. FASTQ files were processed using Space Ranger software (version 2.0.1, 10x Genomics). Reads were aligned with the ‘spaceranger count’ pipeline to the pre-built mouse reference genome provided by 10x Genomics version 2020-A (comprising the STAR-indexed mm10 mouse genome and the GTF gene annotation from GENCODE version M23 (Ensembl 98)), and the probe set reference CSV file (Visium Mouse Transcriptome Probe Set v1.0) was provided for filtering of valid genes. The resulting filtered count matrix of features (i.e. genes) per spot barcodes were employed for the downstream analysis.
Assembly: mm10
Supplementary files format and content: *_barcodes.tsv.gz:  list of spatial barcodes; *_features.tsv.gz:  list of gene IDs; *_matrix.mtx.gz:  gene-barcode expression count data in Matrix Market Exchange Format; *_aligned_fiducials.jpg:  aligned fiducials of the tissue image; *_detected_tissue_image.jpg:  image of tissue and spots; *_scalefactors_json.json:  scalefactors in JSON format; *_tissue_hires_image.png:  hi-res image of tissue; *_tissue_lowres_image.png:  low-res image of tissue; *_tissue_positions_list.csv.gz:  list of spatial barcodes and the coordinates specifying spots
Library strategy: Visium Spatial Transcriptomics

extract_protocol: A microarray of fresh-frozen DMBA/TPA-induced squamous cell carcinoma samples, surgically removed from mouse back skin, was cryo-sectioned with a 10µm thickness (Leica, CM3050S) and placed on the capture area of a spatial gene expression slide (10x Genomics, 1000188), resulting in 4 different tumors on a single capture area. Samples were stored in -80°C before processing. The workflow of Mirzazadeh et al. (2023) for RNA-Rescue Spatial Transcriptomics (RRST) was followed to overcome low RNA integrity number (RIN) score. The samples were fixed with 4% PFA and hematoxylin and eosin (H&E) staining was performed. The spatial libraries were then generated from the probe hybridization step (10x Genomics, 1000365) according to Visium Spatial Gene Expression Reagent Kits for FFPE (10x Genomics, User Guide CG000407 Rev D, 1000361). The resulting libraries were sequenced by the Genomics Facility Basel. The sequencing was performed in a paired-end manner with dual indexing (10x Genomics, 1000251) on a NovaSeq 6000 (Illumina) with a S4 flow cell. Libraries were then sequenced with the following cycle settings: Read1 101 cycles, i7 10 cycles, i5 10 cycles, Read2 101 cycles.

treatment_protocol: DMBA/TPA chemical carcinogenesis: Two-stage cutaneous chemical carcinogenesis was performed as previously described by Filler et al. (2007). In brief: the back of 6-8 week old animals were shaved with electric hair clippers and treated once with 400 nmol 7,12-dimethylbenz[a]anthracene (DMBA)(Sigma-Aldrich D3254) per 100 µl (dissolved in acetone) application as an initiator mutagen. After a two-week rest period, 40 nmol 2-o-tetradecanoylphorbol-13-acetate (TPA)(Sigma-Aldrich 79346) dissolved in 100 µl 100% Ethanol was applied twice a week. Tumor formations were monitored regularly by visual inspection and mice were euthanized after 12 weeks of treatment or when tumors reached a cumulative length of 2cm.

tag: tissue

tag: strain


title: MMRF_1735

city: St. Louis

first: Jessica

last: Silva-Fisher

address: 425 Euclid

institute: Washington University in St. Louis

data_processing:
The assembled data was integrated with supplementary contigs, including the complete repeating unit of ribosomal DNA, cancer-related viruses, and data from the external RNA Control Consortium.
For gene and transcript models, Ensemble version 74 was used, along with additional annotations provided in the GTF file (https://github.com/tgen/MMRF_CoMMpass.git).
TopHat 2.0.11.Linux_x86_84 was used for generating the transcriptome and Salmon was utilized to quantify expression levels
Picard Tools suite, with the 'SAMtools faidx' function, was applied to create an index for the reference genome
For sequence alignment, Bowtie, Bowtie 2, and Salmon (p – 12 and type - Quasi), were used, followed by STAR 2.3.12
BAM files were subsequently generated using SAMtools v.1.19
Quality control checks on the RNA BAM files using Picard RNA metrics and BamTools Ig Counts
Assembly: GRCh37 reference genome (hs37d5 version)
Supplementary files format and content: Comma Separated Values text file with normalized counts for each sample

source_name: Bone marrow aspirates

extract_protocol:
RNA isolation
TSMRU - Illumina TruSeq mRNA Unstranded Library Prep

value: Bone marrow aspirates

tag: experimental group

value: Non - Responders


title: mCherry_negative_tumor_WTA

description: DMBA/TPA-induced skin tumor mCherry-negative cells scRNA-seq WTA

library_source: transcriptomic single cell

molecule: polyA RNA

extract_protocol:
Tumors and 1-2 mm of surrounding tissue from DMBA/TPA treated mice were excised and finely minced with a scalpel. Tumor pieces were submerged in 5 mL prewarmed DMEM with 0.25% Trypsin/EDTA and 3 U/mL DNAse I at 37°C on an orbital shaker with rigorous resuspension every 5 minutes for a total of 30 minutes. Reaction was quenched with 1 ml FBS and strained through a 70 µM mesh, pelleted at 1400rpm for 10 minutes with excess PBS and resuspended in 0.5 mL FACS buffer before mCherry-negative sorting. Single-Cell capture was performed with BD Rhapsody using Rhapsody Enhanced Cartridge Kit (BD Biosciences 664887).
Reverse transcription from beads and sequencing library production was carried out according to manufacturer's instructions (BD Biosciences, Doc ID: 210967 Rev. 1.0). Whole Transcriptome Amplification Analysis (WTAs) from BD Rhapsody Cartridges were sequenced in full SP flowcells at 1.8nM concentration with 10% PhiX, dual indexing i5 - 8bp, i7 - 8bp and Read1 64 cycles.


title: primary cortical neurons, KO+H2BE biol rep 3


title: SUM159_attached_doxorubicin_rep1

city: Athens

first: Spyros

last: Foutadakis

address: Soranou

country: Greece

institute: Biomedical Research Foundation Academy of Athens

data_processing:
The quality of the sequencing reads was assessed with FastQC
Reads were mapped to the human genome with HISAT2
Gene counts were calculated with HTSeq count
Bigwig files were prepared with the bamcoverage command from Deeptools
Assembly: hg19
Supplementary files format and content: bigwig files
Supplementary files format and content: text files containg gene counts for each replicate

growth_protocol: SUM159 cells were cultured in Ham's medium  supplemented with 5% fetal bovine serum, 1% penicillin/streptomycin, 5 μg/mL insulin and 1 μg/mL hydrocortisone in a humidified atmosphere of 5% CO2 at 37 °C.

extract_protocol:
Cells were collected and lysed with TRI reagent to isolate total RNA
The NEBNext Ultra II Directional RNA Library Prep Kit was used to prepare libraries following the manual

treatment_protocol: SUM159 attached cells were made resistant to doxorubicin by long term treatment with escalating doses

tag: cell line

value: Wt

value: doxorubicin


title: MMRF_1664


title: Sample_18_guides

description: sgRNA dial-out PCR amplification, P60 skin, mCherry CROP-seq, replicate 8. Same underlying biological sample as for Sample_18_WTA

library_source: other

data_processing:
The raw sequencing data comprising the BCL files were demultiplexed into FASTQ files using the Illumina bcl2fastq program v2.20 with default options allowing one mismatch of the sample barcode sequences. The PCR dialouts data was processed by custom in-house python scripts (refer to manuscript for details, and to scripts 'sgRNA_umi_counts.py' and 'sgRNA_umi_counts_annotation.py' in repository https://github.com/sendoellab/single-cell_CRISPR). Briefly, from the input FASTQ files we extract three 9 nucleotide (nt) long cell barcodes out of possible 97 barcodes at specific locations from the Read 1 ('*_R1_001.fastq.gz' files) allowing one mismatch per barcode (Hamming distance of 1). 8 nt UMI sequences are also obtained from the Read 1 as given in the BD manual. For valid barcodes, we count the presence of sgRNAs in Read 2 ('*_R2_001.fastq.gz' files) by an exact match of the 20 nt sgRNA sequence. Next, we deduplicate the UMI counts per cell to obtain the final sgRNA UMI counts per cell. If multiple sgRNAs are detected in one cell, we assign the cell to a specific sgRNA if the sgRNA UMI count is higher than the quantile 0.99.
Supplementary files format and content: *_umi_counts_anno.csv.gz compressed files contain a comma-separated (csv) table with the following 6 columns: 1) 'barcode' (the 3x9nt variable regions of the BD Rhapsody cell barcodes), 2) 'sgRNA' (the label of the single guide assigned to that cell), 3) 'counts' (the sgRNA UMI counts per cell), 4) 'q99' (the quantile 0.99 in that cell), 5 'id' (the unique cell index number corresponding to the cell barcode, matching those reported in the WTA *_MolsPerCell.csv.gz files), 6) 'sgrna_detected' (the number of guides detected).
Library strategy: dial-out nested PCR for sgRNA amplification

molecule: other

extract_protocol:
P60 back skin was harvested with surgical scissors and placed dermis-facing up on a styrofoam tray with pins. Fat and subcutis were scraped off with a scalpel. Fat-free skin was then washed, dermis-side facing down in 1xPBS. Skin was then placed in the same orientation in 0.5% Trypsin-EDTA (10X, Gibco; 15400054) and incubated at 37°C on an orbital shaker for 25 (females) or 50 min (males). Using a glass microscopy slide, skin was secured to the bottom of the dish and scraped again with a scalpel until it started to break. Excess cold PBS with 2% chelexed FBS (FBS(-)) was added to neutralize trypsin. Cell suspension was strained on ice first through a 70 µm filter, washed with an additional 15 mL of cold 1X PBS with 2% FBS(-) and then filtered through a 40 µm filter. Cell suspension was spun down at 400 x g for 10 min and resuspended in FACS buffer before sorting mCherry positive cells on a BD Aria II.
The sgRNA containing region has been amplified from BD Rhapsody beads in a separate nested PCR reaction. In PCR1, we used 100 µl KAPA HiFi HotStart ReadyMix (Roche; 07958935001), 6 µl forward primer (5? ACACGACGCTCTTCCGATCT 3?, 10 ?M), 6 µl reverse primer (5?TCTTGTGGAAAGGACGA 3? 10 ?M), 12 ?l Bead RT/PCR Enhancer reagent from BD Biosciences and 72 ?l nuclease free water for a total volume of 200 ?l. Rhapsody bead from each separate cartridge preparation were resuspended in PCR mix and aliquoted in 4x 50 ?l reactions and quickly moved into a pre-heated PCR machine without allowing beads to settle with the following conditions: Initial denaturation at 95°C for 5 minutes, followed by 25 cycles of denaturation 95°C - 30s, annealing 53°C - 30 s, extension 72°C - 20 s, and a final extension of 10 min at 72°C. PCR1 product was pooled, beads were removed magnetically and amplicons were cleaned up with Agencourt AMPure XP beads (Beckman Coulter; A63881) according to manufacturer?s instructions before the second PCR. For PCR 2, 3 ?l of cleaned up PCR1 was used as template along with 2 ?l forward primer (5? ACACGACGCTCTTCCGATCT 3?, 10 ?M), 2 ?l reverse primer (CAGACGTGTGCTCTTCCGATCTCTTGTGGAAAGGACGAAACA*C*C*G 3?, 10 ?M), 18 ?l nuclease free-water, along with 25 ?l of KAPA HiFi HotStart ReadyMix for a total volume of 50 ?l. PCR2 was performed under following conditions: Initial denaturation at 95°C for 3 minutes, followed by 10 cycles of denaturation 95°C - 30 s, annealing 60°C - 3 min, extension 72°C - 60 s, and a final extension of 5 min at 72°C. PCR2 product was cleaned up with Agencourt AMPure XP beads according to manufacturer?s instructions before the indexing PCR. Indexing PCR was performed according to BD Biosciences ?mRNA Targeted Library Preparation? protocol(Doc ID: 210968 Rev. 3.0).

value: 60 days post natal


title: primary cortical neurons, KO+H2BE biol rep 2


title: primary cortical neurons, KO+I39V biol rep 1


title: MMRF_1656


title: Sample_17_guides

description: sgRNA dial-out PCR amplification, P60 skin, mCherry CROP-seq, replicate 7. Same underlying biological sample as for Sample_17_WTA


title: MMRF_1637


title: primary cortical neurons, KO+H2BE biol rep 1


title: S325E mutant, CD3/CD28 stimulated, biol rep 3

city: Los Angeles

first: Richard

last: Ainsworth

address: 8700 Beverly Blvd

institute: Cedars-Sinai Medical Center

description: mRNA J325E-16_3

data_processing:
Reads aligned to mouse genome GRCh38.p13 using STAR version=2.7.9a
Assembly: GRCh38.p13 genome assembly
Supplementary files format and content: count_matrix: counts matrix

source_name: Peripheral blood

growth_protocol: Jurkat E6.1 derived cell lines were cultured in complete RPMI 1640 medium with 10% fetal bovine serum (FBS) and supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 2.5 mg/mL D-glucose, 10 mM HEPES, 100 units/mL penicillin, and 100 μg/mL streptomycin under 5% CO2 at 37 °C

extract_protocol:
RNA samples were extracted by using RNeasy Micro kit (Qiagen)
library was prepared with NEBNext Ultra II Directional RNA library prep kit for Illumina, with 500ng input

treatment_protocol: Cells were starved and stimulated with or without antibodies against human CD3/CD28 for 16h

value: Peripheral blood

value: Jurkat E6.1

value: T lymphoblast

value: CD3/CD28 stimulated


title: S325E mutant, CD3/CD28 stimulated, biol rep 2

description: mRNA J325E-16_2


title: MMRF_1539


title: S325E mutant, CD3/CD28 stimulated, biol rep 1

description: mRNA J325E-16_1


title: Sample_16_guides

description: sgRNA dial-out PCR amplification, P60 skin, mCherry CROP-seq, replicate 6. Same underlying biological sample as for Sample_16_WTA

value: mouse epidermis


title: primary cortical neurons, KO+H2B biol rep 3


title: Sample_15_guides

description: sgRNA dial-out PCR amplification, P60 skin, mCherry CROP-seq, replicate 5. Same underlying biological sample as for Sample_15_WTA

value: skin cells


title: MMRF_1496


title: MMRF_1502


title: S325E conterpart WT, CD3/CD28 stimulated, biol rep 3

description: mRNA JWT-E-16_3


title: primary cortical neurons, KO+H2B biol rep 2


title: Sample_14_guides

description: sgRNA dial-out PCR amplification, P60 skin, mCherry CROP-seq, replicate 4. Same underlying biological sample as for Sample_14_WTA

value: JAX #026179, CD1 wildtype x Tg(B6J.129(Cg)-Gt(ROSA)26Sortm1.1(CAG-cas9*,-EGFP)Fezh/J


title: primary cortical neurons, KO+H2B biol rep 1


title: Sample_13_guides

description: sgRNA dial-out PCR amplification, P60 skin, mCherry CROP-seq, replicate 3. Same underlying biological sample as for Sample_13_WTA


title: S325E conterpart WT, CD3/CD28 stimulated, biol rep 2

description: mRNA JWT-E-16_2


title: S325E conterpart WT, CD3/CD28 stimulated, biol rep 1

description: mRNA JWT-E-16_1


title: Sample_12_guides

description: sgRNA dial-out PCR amplification, P60 skin, mCherry CROP-seq, replicate 2. Same underlying biological sample as for Sample_12_WTA


title: primary cortical neurons, KO+GFP biol rep 3

value: GFP


title: MMRF_1452


title: S325A mutant, CD3/CD28 stimulated, biol rep 3

description: mRNA J325A-16_3


title: primary cortical neurons, KO+GFP biol rep 1


title: Sample_11_guides

description: sgRNA dial-out PCR amplification, P60 skin, mCherry CROP-seq, replicate 1. Same underlying biological sample as for Sample_11_WTA


title: MMRF_1415


title: S325A mutant, CD3/CD28 stimulated, biol rep 2

description: mRNA J325A-16_2


title: MMRF_1293


title: S325A mutant, CD3/CD28 stimulated, biol rep 1

description: mRNA J325A-16_1


title: Sample_10_guides

description: sgRNA dial-out PCR amplification, P4 skin, mCherry CROP-seq, replicate 7. Same underlying biological sample as for Sample_10_WTA

extract_protocol:
P4 back skin was surgically removed and scraped for fat, the back skin was washed once in cold PBS and then placed in dispase (Corning; 354235) for 35 min at 37°C, epidermis facing up, on an orbital shaker. Next, epidermis was separated from dermis with fine forceps, torn into smaller pieces and placed in 4 mL 0.25% Trypsin-EDTA (1X, Gibco; 25200056) for 15 min at 37°C with orbital shaking. Epidermis was washed with cold PBS and pipetted vigorously to achieve a single cell suspension. Suspension was then afterwards filtered through a 70 µm and a 40 µm strainer (Corning; 431750, 431751) consecutively, then centrifuged for 10 min at 1400rpm and resuspended in FACS buffer (PBS + 2 % FBS(-) + DAPI) before sorting mCherry positive cells on a BD Aria II.
The sgRNA containing region has been amplified from BD Rhapsody beads in a separate nested PCR reaction. In PCR1, we used 100 µl KAPA HiFi HotStart ReadyMix (Roche; 07958935001), 6 µl forward primer (5? ACACGACGCTCTTCCGATCT 3?, 10 ?M), 6 µl reverse primer (5?TCTTGTGGAAAGGACGA 3? 10 ?M), 12 ?l Bead RT/PCR Enhancer reagent from BD Biosciences and 72 ?l nuclease free water for a total volume of 200 ?l. Rhapsody bead from each separate cartridge preparation were resuspended in PCR mix and aliquoted in 4x 50 ?l reactions and quickly moved into a pre-heated PCR machine without allowing beads to settle with the following conditions: Initial denaturation at 95°C for 5 minutes, followed by 25 cycles of denaturation 95°C - 30s, annealing 53°C - 30 s, extension 72°C - 20 s, and a final extension of 10 min at 72°C. PCR1 product was pooled, beads were removed magnetically and amplicons were cleaned up with Agencourt AMPure XP beads (Beckman Coulter; A63881) according to manufacturer?s instructions before the second PCR. For PCR 2, 3 ?l of cleaned up PCR1 was used as template along with 2 ?l forward primer (5? ACACGACGCTCTTCCGATCT 3?, 10 ?M), 2 ?l reverse primer (CAGACGTGTGCTCTTCCGATCTCTTGTGGAAAGGACGAAACA*C*C*G 3?, 10 ?M), 18 ?l nuclease free-water, along with 25 ?l of KAPA HiFi HotStart ReadyMix for a total volume of 50 ?l. PCR2 was performed under following conditions: Initial denaturation at 95°C for 3 minutes, followed by 10 cycles of denaturation 95°C - 30 s, annealing 60°C - 3 min, extension 72°C - 60 s, and a final extension of 5 min at 72°C. PCR2 product was cleaned up with Agencourt AMPure XP beads according to manufacturer?s instructions before the indexing PCR. Indexing PCR was performed according to BD Biosciences ?mRNA Targeted Library Preparation? protocol(Doc ID: 210968 Rev. 3.0).

value: 4 days post natal


title: primary cortical neurons, KO biol rep 8

value: primary cortical neurons


title: MMRF_1261


title: primary cortical neurons, KO biol rep 7


title: S325A counterpart WT, CD3/CD28 stimulated, biol rep 3

description: mRNA JWT-A-16_3

value: WT


title: wd-1,treated,48h,3

city: Nanchang

first: shaochun

address: Jiaoqiao town

institute: Jiangxi Agricultural University

data_processing:
Fastp( version 0.19.7 ) was used to quality control the raw data to obtain clean reads
Assembly: the indica reference genome (Oryza_indica.ASM465v1.dna.toplevel. fa),the  Japonica reference genome(Oryza sativa Japonica IRGSP-1.0)
Supplementary files format and content: tab-delimited text files include RPKM values for each Sample

organism: Oryza sativa Japonica Group

source_name: leaf

extract_protocol:
Total RNA was isolated according to the protocol of Trizol Reagent kit and purification (Invitrogen)
The experimental samples were constructed and sequenced by cDNA library of Beijing Novogene using Novaseq 6000 platform.

value: leaf

value: MESOPHYLL cell

value: knockdown

value: 48h with Guy11


title: primary cortical neurons, KO biol rep 6


title: S325A counterpart WT, CD3/CD28 stimulated, biol rep 2

description: mRNA JWT-A-16_2


title: MMRF_1157

value: Bone marrow aspirates

value: Non - Responders


title: Sample_8_guides

description: sgRNA dial-out PCR amplification, P4 skin, mCherry CROP-seq, replicate 5. Same underlying biological sample as for Sample_8_WTA


title: primary cortical neurons, KO+GFP biol rep 2


title: S325A counterpart WT, CD3/CD28 stimulated, biol rep 1

description: mRNA JWT-A-16_1

value: Peripheral blood


title: Sample_7_guides

description: sgRNA dial-out PCR amplification, P4 skin, mCherry CROP-seq, replicate 4. Same underlying biological sample as for Sample_7_WTA


title: primary cortical neurons, KO biol rep 5


title: MMRF_1068


title: PBMC9, PBMC

city: Birmingham

first: Jason

middle: Dale

last: Turner

address: Queen Elizabeth Hospital

department: Rheumatology research group

country: United Kingdom

institute: University of Birmingham

data_processing:
Counts generated using Subread v2.0.1
Assembly: hg38
Supplementary files format and content: Subread counts table as tab delimited table

source_name: Minor labial salivary gland

extract_protocol:
RNA extraction from the microdissected samples was completed as per the protocols provided using the RNeasy FFPE kit (Qiagen)
NEBNext rRNA Depletion Kit v2 and NEBNext Ultra™ II Directional RNA Library Prep Kit for Illumina® using NEBNext® Multiplex Oligos for Illumina (Unique Dual Index UMI Adaptors RNA Set 1)

value: Minor labial salivary gland

value: Whole minor slaivary gland regions

tag: group

value: PBMC

tag: batch

value: Two


title: MMRF_1289


title: S325E mutant, Nonstimulated, biol rep 3

description: mRNA J325E-0_3

value: Jurkat E6.1

value: T lymphoblast


title: Sample_5_guides

description: sgRNA dial-out PCR amplification, P4 skin, mCherry CROP-seq, replicate 2. Same underlying biological sample as for Sample_5_WTA

value: mouse epidermis

value: skin cells

value: JAX #026179, CD1 wildtype x Tg(B6J.129(Cg)-Gt(ROSA)26Sortm1.1(CAG-cas9*,-EGFP)Fezh/J


title: S325E mutant, Nonstimulated, biol rep 2

description: mRNA J325E-0_2


title: primary cortical neurons, KO biol rep 4

value: primary cortical neurons


title: Sample_9_guides

description: sgRNA dial-out PCR amplification, P4 skin, mCherry CROP-seq, replicate 6. Same underlying biological sample as for Sample_9_WTA

value: 4 days post natal


title: Sample_6_guides

description: sgRNA dial-out PCR amplification, P4 skin, mCherry CROP-seq, replicate 3. Same underlying biological sample as for Sample_6_WTA


title: primary cortical neurons, KO biol rep 3


title: Sample_4_guides

description: sgRNA dial-out PCR amplification, P4 skin, mCherry CROP-seq, replicate 1. Same underlying biological sample as for Sample_4_WTA


title: PBMC8, PBMC


title: S325E mutant, Nonstimulated, biol rep 1

description: mRNA J325E-0_1


title: primary cortical neurons, KO biol rep 1


title: PBMC7, PBMC


title: S325E counterpart WT, Nonstimulated, biol rep 3

description: mRNA JWT-E-0_3

value: WT


title: Sample_17_WTA

description: scRNA-seq WTA of P60 skin, mCherry CROP-seq, replicate 7. Same underlying biological sample as for Sample_17_guides

data_processing:
The raw sequencing data comprising the BCL files were demultiplexed using the Illumina bcl2fastq program v2.20 with default options allowing one mismatch of the sample barcode sequences. The resulting FASTQ files were processed using the BD Rhapsody™ Whole Transcriptome Analysis (WTA) Pipeline (v1.8-1.9.1) hosted at the Seven Bridge’s cloud platform (https://www.sevenbridges.com/). We utilized a custom mouse reference genome based on the Gencode version 25 (mm10) combined with our 500 sgRNA sequences. Since the BD pipeline uses STAR aligner in the backend, the custom STAR reference genome was generated by the genomeGenerate command in STAR (version 2.5.2b). The Refined Putative Cell Calling option was disabled while running the pipeline. Unique Molecular Identifier (UMI) count errors that are single-base substitution errors were identified and adjusted to the parent UMI barcode in the pipeline using RSEC (Recursive Substitution Error Correction) method, developed by BD Biosciences. The output consisted of thus corrected UMI counts, and was used for further downstream analysis.
Assembly: mm10
Supplementary files format and content: *_Expression_Data.st.gz compressed files contain a tab-separated (tsv)  expression sparse matrix, in which each row records counts for cell-gene combinations that have non-zero RSEC molecule counts. Columns are the following: 1) Cell_Index: unique cell index number. 2) Gene: gene symbols. 3) RSEC_Reads: Number of reads after the RSEC molecular identifier adjustment algorithm. 4) Raw_Molecules: Number of UMIs before molecular identifier adjustment algorithms. 5) RSEC_Adjusted_Molecules: Number of UMIs after RSEC molecular identifier adjustment algorithm.  *.h5 files are HDF5 files containing RSEC molecule counts with only the final subset of cells used in our publication.

extract_protocol:
P60 back skin was harvested with surgical scissors and placed dermis-facing up on a styrofoam tray with pins. Fat and subcutis were scraped off with a scalpel. Fat-free skin was then washed, dermis-side facing down in 1xPBS. Skin was then placed in the same orientation in 0.5% Trypsin-EDTA (10X, Gibco; 15400054) and incubated at 37°C on an orbital shaker for 25 (females) or 50 min (males). Using a glass microscopy slide, skin was secured to the bottom of the dish and scraped again with a scalpel until it started to break. Excess cold PBS with 2% chelexed FBS (FBS(-)) was added to neutralize trypsin. Cell suspension was strained on ice first through a 70 µm filter, washed with an additional 15 mL of cold 1X PBS with 2% FBS(-) and then filtered through a 40 µm filter. Cell suspension was spun down at 400 x g for 10 min and resuspended in FACS buffer before sorting mCherry positive cells on a BD Aria II.
mCherry-positive cells were sorted on a BD FACSAria using a 70µm nozzle and processed for single-cell capture with a BD Rhapsody Single-Cell Analysis System. For P60 we loaded 8 cartridges (Cat No 633733) with an average of 64625 cells. Reverse transcription from beads and sequencing library production was carried out according to manufacturer's instructions (BD Biosciences, Doc ID: 210967 Rev. 1.0). Whole Transcriptome Amplification Analysis (WTAs) from BD Rhapsody Cartridges were sequenced in full SP flowcells at 1.8nM concentration with 20% PhiX and the following read configuration: Read1 60, I1 8, Read2 62 cycles.

value: 60 days post natal


title: S325E counterpart WT, Nonstimulated, biol rep 1

description: mRNA JWT-E-0_1


title: PBMC6, PBMC


title: primary cortical neurons, WT biol rep 4


title: PBMC3, PBMC


title: primary cortical neurons, WT biol rep 1


title: S325A mutant, Nonstimulated, biol rep 2

description: mRNA J325A-0_2


title: primary cortical neurons, WT biol rep 5


title: S325E counterpart WT, Nonstimulated, biol rep 2

description: mRNA JWT-E-0_2


title: primary cortical neurons, KO biol rep 2


title: Sample_16_WTA

description: scRNA-seq WTA of P60 skin, mCherry CROP-seq, replicate 6. Same underlying biological sample as for Sample_16_guides


title: Sample_15_WTA

description: scRNA-seq WTA of P60 skin, mCherry CROP-seq, replicate 5. Same underlying biological sample as for Sample_15_guides


title: S325A mutant, Nonstimulated, biol rep 3

description: mRNA J325A-0_3


title: PBMC4, PBMC


title: PBMC12, PBMC


title: Sample_18_WTA

description: scRNA-seq WTA of P60 skin, mCherry CROP-seq, replicate 8. Same underlying biological sample as for Sample_18_guides


title: primary cortical neurons, WT biol rep 2


title: primary cortical neurons, WT biol rep 3


title: KO biol rep 3 ATAC-Seq

data_processing:
Fastq files were aligned by Bowtie2
bam files from the two sequencing runs were merged for each biological replicate and 50 million reads were subset for each biol rep using SAMTools for downstream analysis
NGSplot was used to generate metaplots
deepTools was used to generate bigwig files
MACS3 was used for peak-calling and DiffBind was used to identify differential peaks
Assembly: mm10
Supplementary files format and content: bigwig files generated by deepTools from bam files aligned to bowtie2

tag: developmental stage


title: Sample_14_WTA

description: scRNA-seq WTA of P60 skin, mCherry CROP-seq, replicate 4. Same underlying biological sample as for Sample_14_guides


title: Sample_13_WTA

description: scRNA-seq WTA of P60 skin, mCherry CROP-seq, replicate 3. Same underlying biological sample as for Sample_13_guides


title: S325A mutant, Nonstimulated, biol rep 1

description: mRNA J325A-0_1


title: PBMC2, PBMC


title: PBMC13, PBMC


title: Sample_10_WTA

description: scRNA-seq WTA of P4 skin, mCherry CROP-seq, replicate 7. Same underlying biological sample as for Sample_10_guides

extract_protocol:
P4 back skin was surgically removed and scraped for fat, the back skin was washed once in cold PBS and then placed in dispase (Corning; 354235) for 35 min at 37°C, epidermis facing up, on an orbital shaker. Next, epidermis was separated from dermis with fine forceps, torn into smaller pieces and placed in 4 mL 0.25% Trypsin-EDTA (1X, Gibco; 25200056) for 15 min at 37°C with orbital shaking. Epidermis was washed with cold PBS and pipetted vigorously to achieve a single cell suspension. Suspension was then afterwards filtered through a 70 µm and a 40 µm strainer (Corning; 431750, 431751) consecutively, then centrifuged for 10 min at 1400rpm and resuspended in FACS buffer (PBS + 2 % FBS(-) + DAPI) before sorting mCherry positive cells on a BD Aria II.
mCherry-positive cells were sorted on a BD FACSAria using a 70µm nozzle and processed for single-cell capture with a BD Rhapsody Single-Cell Analysis System. For P4 we loaded 8 separate cartridges (Cat No 633733) with an average of 50037 cells. Reverse transcription from beads and sequencing library production was carried out according to manufacturer's instructions (BD Biosciences, Doc ID: 210967 Rev. 1.0). Whole Transcriptome Amplification Analysis (WTAs) from BD Rhapsody Cartridges were sequenced in full SP flowcells at 1.8nM concentration with 20% PhiX and the following read configuration: Read1 60, I1 8, Read2 62 cycles.


title: KO biol rep 2 ATAC-Seq


title: Sample_12_WTA

description: scRNA-seq WTA of P60 skin, mCherry CROP-seq, replicate 2. Same underlying biological sample as for Sample_12_guides


title: KO biol rep 4 ATAC-Seq


title: PBMC5, PBMC


title: Sample_11_WTA

description: scRNA-seq WTA of P60 skin, mCherry CROP-seq, replicate 1. Same underlying biological sample as for Sample_11_guides


title: Sample_8_WTA

description: scRNA-seq WTA of P4 skin, mCherry CROP-seq, replicate 5. Same underlying biological sample as for Sample_8_guides


title: PBMC11, PBMC


title: S325A counterpart WT, Nonstimulated, biol rep 1

description: mRNA JWT-A-0_1


title: S325A counterpart WT, Nonstimulated, biol rep 2

description: mRNA JWT-A-0_2


title: WT biol rep 3 ATAC-Seq


title: S325A counterpart WT, Nonstimulated, biol rep 3

description: mRNA JWT-A-0_3


title: Sample_9_WTA

description: scRNA-seq WTA of P4 skin, mCherry CROP-seq, replicate 6. Same underlying biological sample as for Sample_9_guides


title: PBMC1, PBMC

value: Three


title: Sample_7_WTA

description: scRNA-seq WTA of P4 skin, mCherry CROP-seq, replicate 4. Same underlying biological sample as for Sample_7_guides


title: TON2, Tonsil

value: Tonsil

value: One


title: HeLa, gammaH2AX, siDDX3X, CUT&Tag, rep3

city: Nanjing

first: Hongzhan

last: Xu

address: 163 Xianlin Road

institute: Nanjing university

data_processing:
Basecalls were performed using bcl2fastq (version 2.19) for NovaSeq output.
Raw FASTQ reads were trimmed by fastp (version 0.12.4). Reads were aligned to reference of hg38 along with the spike-in sequence by bowtie2 (version 2.4.4) with the options --end-to-end --very-sensitive --no-mixed --no-discordant.
Reads from spike-in and those not were separated by samtools (version 1.9) with option -b -L. Resulted BAM files were filtered with a  MAPQ score cutoff of 20 using samtools (version 1.9) and deduplicated using picard (version 2.27.1).
BAM files of different replicates and treatments were merged to identify a unified set of peaks for cross-sample comparison by MACS2 (version 2.2.7.1). Blacklisted regions were removed by intersecting peaks with the ENCODE blacklist bed file using Bedtools (version 2.30.0).
Data was normalized by spike-in scale-factor and library-size with bedtools genomecov (version 2.30.0) comparison and visualization.
Assembly: hg38
Supplementary files format and content: bigWig
Library strategy: CUT&Tag

source_name: HeLa

growth_protocol: HeLa cells were cultured in MEM media supplemented with 10% fetal bovine serum, 100 units/mL penicillin, and 100 μg/mL streptomycin at 37 °C with 5% CO2.

extract_protocol:
DNA was purified with minElute PCR kit (Qiagen, 28004). DNA was size-selected firstly by 0.55× DNA clean beads to remove the fragment above 600 bp and secondly by 2× DNA clean beads to get the DNA fragment between 100 bp to 600 bp.
To generate the sequencing libraries, DNA was mixed with barcoded i5 primer and barcoded i7 primer and amplified with high-fidelity master mix (NEB, M0541S). Finally, libraries were cleaned by 1.3× DNA clean beads.

treatment_protocol: For the CUT&Tag of DNMT1 protein, HeLa cells were treated with 10 uM BQQ or DMSO for 4 hours before experiment. For the CUT&Tag of DDX3X protein, HeLa cells were transfected with DDX3X-Flag plasmid for 48 hours before experiment. For the CUT&Tag of gammaH2AX protein, HeLa cells were transfected with DDX3X siRNA or scrambled siRNA for 48 hours before experiment. The antibodies used in this experiment included: Anti-DNMT1 (Active Motif, 39204, 1:100 dilution), Anti-Flag (Abclonal, AE092, 1:100 dilution), Anti-gammaH2AX (Abcam, ab303656, 1:500 dilution), Goat anti-rabbit-IgG antibody (Beyotime, A0468, 1:400 dilution) and Goat anti-mouse-IgG antibody (Beyotime, A0428, 1:400 dilution).

value: HeLa

value: Human cervical cell

tag: antibody

value: gammaH2AX (Abcam, ab303656)


title: LF1, Large_foci

value: Minor labial salivary gland

value: Whole minor slaivary gland regions


title: HeLa, gammaH2AX, scrambled siRNA, CUT&Tag, rep2

value: Scrambled siRNA


title: Sample_4_WTA

description: scRNA-seq WTA of P4 skin, mCherry CROP-seq, replicate 1. Same underlying biological sample as for Sample_4_guides


title: WT biol rep 2 ATAC-Seq


title: HeLa, gammaH2AX, siDDX3X, CUT&Tag, rep1


title: HeLa, gammaH2AX, siDDX3X, CUT&Tag, rep2


title: Sample_5_WTA

description: scRNA-seq WTA of P4 skin, mCherry CROP-seq, replicate 2. Same underlying biological sample as for Sample_5_guides


title: PBMC10, PBMC

value: PBMC

value: Two


title: TON3, Tonsil


title: H2BE-KO biol rep 3

data_processing:
Fastq files were aligned by Bowtie2
bam files from the two sequencing runs were merged for each biological replicate and 6 million reads were subset for each biol rep using SAMTools for downstream analysis
NGSplot was used to generate metaplots
deepTools was used to generate bigwig files
MACS3 was used for peak-calling
Assembly: mm10
Supplementary files format and content: bigwig files generated by deepTools from bam files aligned to bowtie2
Library strategy: CUT&Tag

extract_protocol:
At 12DIV, primary cortical neurons were collected in wash buffer (Kaya-Okur et al. 2019) and CUT&Tag was performed with anti-H2BE antibody (Millipore ABE1384)
Transposition reaction mix from Nextera Library Prep kit was used (Tagment DNA Enzyme 1 and Tagment DNA Buffer)
Nextera primer sets with NEBNext Master Mix was used to generate libraries with AMPure beads used for cleanup


title: KO biol rep 1 ATAC-Seq


title: TON1, Tonsil


title: H2BE-KO biol rep 2


title: WT biol rep 1 ATAC-Seq


title: HeLa, gammaH2AX, scrambled siRNA, CUT&Tag, rep3


title: Sample_6_WTA

description: scRNA-seq WTA of P4 skin, mCherry CROP-seq, replicate 3. Same underlying biological sample as for Sample_6_guides

value: mouse epidermis

value: skin cells


title: HeLa, gammaH2AX, scrambled siRNA, CUT&Tag, rep1


title: HeLa, DDX3X, CUT&Tag, rep3

value: Flag (Abclonal, AE092)

value: Ovexpression Flag-DDX3X


title: WT biol rep 3

value: primary cortical neurons

value: WT


title: GC15, GC

value: GC


title: H2BE-KO biol rep 1


title: GC14, GC


title: T113 shRNA Renilla

city: CAMBRIDGE

first: Giulia

last: Biffi

state: Cambridgeshire

address: Cancer Research UK, Robinson Way

institute: University of Cambridge

data_processing:
Estimated transcript counts were counted using Salmon (version 1.4.0)
DEA was performed in R using DESeq package (V2) with default parameters.
GSEA was performed using genes ranked by their p-values using the GSEA program (Broad Institute)
Assembly: GRCm38 (mm10)
Supplementary files format and content: Raw count data in tab-delimited text format.

source_name: Mouse Pancreatic Tumor Organoid

growth_protocol: Organoids were grown in Matrigel

extract_protocol:
The RNA form the organoids was extracted using Trizol reagent and the RNeasy Plus Mini Kit (Qiagen).
mRNA library preparations (Illumina) were performed using 30 µL of 10 ng/mL (300 ng) per sample

treatment_protocol: 3 KPC organoid lines (T91, T113, T118) were infected with virus for shRNA targeting the Renilla locus or for shRNAs targeting Scrib (#5523)

value: Mouse Pancreatic Tumor Organoid

value: T113 Renilla

value: PDAC KPF/FC organoid

value: KPF/FC

value: shRNA Renilla

tag: passage

tag: Sex

value: Male


title: HeLa, DDX3X, CUT&Tag, rep2

value: HeLa

value: Human cervical cell


title: EPI_INF2, Epithelium_inf

value: Minor labial salivary gland

value: Whole minor slaivary gland regions

value: One


title: T118 shRNA Scrib #5523

value: shRNA Scrib #5523


title: HeLa, DDX3X, CUT&Tag, rep1

value: Flag (Abclonal, AE092)


title: GC5, GC


title: T118 shRNA Renilla

value: T118 Renilla


title: WT biol rep 1

value: primary cortical neurons


title: WT biol rep 2

value: WT


title: T91 shRNA Scrib #5523

value: Female


title: GC6, GC

value: GC


title: T113 shRNA Scrib #5523

value: Mouse Pancreatic Tumor Organoid


title: output C

city: Barcelona

first: Mariano

last: Martín

address: c/ Baldiri Reixac 10-12

country: Spain

institute: IBEC

description:
from plasmid library
MT_MM_TSM_BB_BL_processed_data.xlsx (sheet NNKreplication_set)

data_processing:
Illumina NextSeq500 v2.5 sequencer
FastQ files from paired-end sequencing of each library library before (‘input’) and after selection (‘output’) were processed using the DiMSum pipeline (https://github.com/lehner-lab/DiMSum)
Assembly: no genome used for assembling - Random Library
Supplementary files format and content: excel book (.xlsx). Processed sequencing data. Each sequence has nucleator status (1 o 0); nucleation score; associated error (sigma); mean input counts

organism: Saccharomyces cerevisiae

source_name: yeast cell

extract_protocol:
Input and output pellets were thawed and resuspended in 1.5 ml extraction buffer (2% Triton-X, 1% SDS, 100 mM NaCl, 10 mM Tris pH 8, 1 mM EDTA pH 8), and underwent two cycles of freezing and thawing in an ethanol-dry ice bath (10 min) and at 62˚C (10 min). Samples were then vortexed together with 1.5 ml of phenol:chloroform:isoamyl 25:24:1 and 1.5 g of glass beads (Sigma). The aqueous phase was recovered by centrifugation and mixed again with 1.5 ml phenol:chloroform:isoamyl 25:24:1. DNA precipitation was performed by adding 1:10 V of 3M NaOAc and 2.2 V of 100% cold ethanol to the aqueous phase and incubating the samples at -20°C for 1 hr. After a centrifugation step, pellets were dried overnight at RT. Pellets were resuspended in 900ul resuspension buffer (10 mM Tris pH 8, 1 mM EDTA pH 8) and treated with 7.5 ml RNase A (Thermo Scientific) for 30 min at 37˚C. The DNA was finally purified using 30ul of silica beads (QIAEX II Gel Extraction Kit, Qiagen), washed and eluted in 22 ul of elution buffer. Plasmid concentrations were measured by quantitative PCR with SYBR green (Merck) and primers annealing to the origin of replication site of the PCUP1-Sup35N plasmid at 58 °C for 40 cycles (TSO_05 and TSO_06, Supplementary Data Primers).
The library for high-throughput sequencing was prepared in a two-step PCR (Q5 high-fidelity DNA polymerase, NEB). In PCR1, 160 million of molecules were amplified for 15 cycles at 68ºC with frame-shifted primers with homology to Illumina sequencing primers (primers TSO_7 to TSO_20, Supplementary Data Primers). The products were purified with ExoSAP treatment (Affymetrix) and by column purification (MinElute PCR Purification Kit, Qiagen). They were then amplified for 10 cycles in PCR2 with Illumina-indexed primers (primers TSO_21 to TSO_54, Supplementary Data Primers).
Libraries of random sequences were synthesized by Integrated DNA Technologies (IDT) as ultramers of 20 NNK codons (60 nucleotides). A library containing 600 sequences selected from the previous four random libraries was synthesized as an oligopool by IDT for validation purposes (Supplemetary Data). In both cases, sequences were flanked by constant regions of 25 nt upstream and 21 nt downstream for cloning. The NNK ultramers and the validation oligopool were extended in a 1-cycle PCR (Q5 high-fidelity DNA polymerase, NEB) with primers TSO_2 and TSO_65 (Supplementary Data Primers). The resulting products were treated with 2ul/tube of ExoSAP (ExoSAP-IT, Applied Biosystems) for 30 minutes at 37 °C and 20 minutes at 80 °C and purified through a MinElute column (Qiagen). In parallel, the PCUP1-Sup35N plasmid was linearized by PCR (Q5 high-fidelity DNA polymerase, NEB; primers TSO_3 and TSO_4, Supplementary Data Primers). The products were purified from a 1% agarose gel (QIAquick Gel Extraction Kit, Qiagen) and ligated by Gibson with 3 h of incubation at 50°C (Supplementary Data Table 3 Gibson) followed by dialysis for 3 h on a membrane filter (MF-Millipore 0.025 μm membrane, Merck) and vacuum concentration. The resulting libraries were transformed into 10-beta Electrocompetent E. coli (NEB), by electroporation with 2.0 kV, 200 Ω, 25 μF (BioRad GenePulser machine). Cells were recovered in SOC medium for 30 min and grown overnight in 50 ml of LB ampicillin medium. A small amount of cells was also plated in LB ampicillin plates to assess transformation efficiency. Total transformants were estimated (Supplementary Data Table 4 transformants in Bacteria), 50 ml of overnight culture were harvested to purify each library with a midi prep (Plasmid MIDI Kit, Qiagen).

treatment_protocol: Cells were thawed from −80 °C in 50 ml plasmid selection medium at OD = 0.05 and grown until exponential for 15 h. At this stage, cells were harvested and resuspended in 300 ml protein induction medium (-URA, 2% glucose, 100 μM Cu2SO4) at OD = 0.1. After 24 h the 250 ml input pellets were collected, and cells were plated on -ADE-URA selection medium in 145-cm2 plates (Nunc, Thermo Scientific). Plates were incubated at 30 °C for 7 days. Finally, colonies were scraped off the plates with PBS 1x and harvested by centrifugation to collect the output pellets. The number of colonies collected for each experiment is reported in Supplementary Data Table 1. Both input and output pellets were stored at −20 °C before DNA extraction. For each random library experiment, one input samples and three technical replicates of the output pellet were processed for sequencing. Selection experiments for validation were instead performed for three biological replicates, following the same steps as above. Three input and three output samples were processed for sequencing.

value: yeast cell

value: [psi-pin-] (MATa ade1-14 his3 leu2-3,112 lys2 trp1 ura3-52)

tag: mol type

value: PCR amplicon


title: EPI_INF1, Epithelium_inf


title: HeLa, DNMT1, BQQ4h, CUT&Tag, rep1

value: DNMT1 (Active Motif, 39204)

value: BQQ 4h


title: T91 shRNA Renilla

value: T91 Renilla

value: PDAC KPF/FC organoid

value: KPF/FC

value: shRNA Renilla


title: output B


title: GC13, GC

value: Minor labial salivary gland

value: Whole minor slaivary gland regions

value: One


title: Microglia, Vehicle, biol rep 2

city: Bath

first: Robert

middle: J

last: Williams

address: University Of Bath, The Avenue, Claverton Down

institute: University of Bath

description: 40M reads per sample

data_processing:
Data preprocessing performed by Novogene UK, including filtering of raw reads for adapter contamination and low-quality reads
Sequence alignment was performed with HISAT2 to map clean reads to the reference mouse genome (GRCm38.p6)
Assembly: GRCm38.p6
Supplementary files format and content: comma-separated values (.csv) file including unnormalised gene count data for each sample

source_name: Primary Microglia

extract_protocol:
Microglia RNA was extracted using the RNeasy mini kit (Qiagen) following the manufacturer’s instructions, including on column DNase digestion
Directional mRNA library preparation (PolyA enrichment)

value: Primary Microglia

value: Vehicle

tag: time


title: Microglia, EGCG, biol rep 2

description: 40M reads per sample

value: 1uM EGCG


title: output A


title: HeLa, DNMT1, DMSO, CUT&Tag, rep1

value: HeLa

value: Human cervical cell

value: DMSO


title: GC4, GC

value: GC


title: Microglia, Vehicle, biol rep 1

description: 40M reads per sample


title: HeLa, DNMT1, DMSO, CUT&Tag, rep2


title: GC2, GC


title: HeLa, DNMT1, BQQ4h, CUT&Tag, rep2


title: input B

value: yeast cell

value: [psi-pin-] (MATa ade1-14 his3 leu2-3,112 lys2 trp1 ura3-52)

value: PCR amplicon


title: Microglia, EGCG, biol rep 1

description: 40M reads per sample


title: input A


title: output3 NNK4

description:
from plasmid library
MT_MM_TSM_BB_BL_processed_data.xlsx (sheet NNK4ValidationSet)


title: GC3, GC


title: Scrib null (KPF/+CSF/F) #23 [TB20831]

data_processing:
Estimated transcript counts were counted using Salmon (version 1.4.0)
DEA was performed in R using DESeq package (V2) with default parameters.
GSEA was performed using genes ranked by their p-values using the GSEA program (Broad Institute)
Genome_build: GRCm38 (mm10)
Supplementary_files_format_and_content: Raw count data in tab-delimited text format.

source_name: ko tumor

extract_protocol:
Primary tumors were frozen in dry ice and stored at -80C. The RNA form the tumors was extracted using the RNeasy Plus Mini Kit (Qiagen).
mRNA library preparations (Illumina) were performed using 30 µL of 10 ng/mL (300 ng) per sample

value: KPC

value: pancreatic tumor

tag: genotype/variation

value: Scrib null (KPF/+CSF/F)


title: output1 NNK4

tag: cell type


title: GC12, GC


title: Scrib null (KPF/+CSF/F) #18 [TB20825]


title: Scrib null (KPF/+CSF/F) #15 [TB19972]


title: output2 NNK4

tag: genotype


title: GC1, GC


title: Scrib null (KPF/+CSF/F) #14 [TB19971]


title: input C


title: SF2, Small_foci


title: input NNK4


title: output2 NNK3

description:
from plasmid library
MT_MM_TSM_BB_BL_processed_data.xlsx (sheet NNK3)


title: SF1, Small_foci


title: GC9, GC


title: output1 NNK3


title: output3 NNK3


title: input NNK3


title: GC10, GC


title: EPI_DUCT2, Epithelium_duct


title: Scrib null (KPF/+CSF/F) #7 [TB19962]


title: GC8, GC


title: Scrib WT (KPF/+C) #3 [TB20455]

source_name: wt tumor

value: Scrib WT (KPF/+C)


title: Scrib WT (KPF/+C) #6 [TB20655]


title: genomic DNA from ID 0015 for 'b' visit

type: genomic

city: Exeter

first: Emma

last: Walker

address: RILD Barrack Rd

institute: University of Exeter

description:
0015b
200397870043_R06C02

hyb_protocol: standard Illumina protocol

scan_protocol: illumina iScan

data_processing:
waterMelon
wateRmelon::dasen() normalised Average Beta

label: Cy3, Cy5

source_name: peripheral whole blood

label_protocol: KCL samples were profiled using the Illumina Infinium HumanMethylation450 BeadChip and UMC samples were profiled using the Illumina Infinium HumanMethylationEPIC BeadChip

extract_protocol: Approximately 500ng of blood-derived DNA from each sample was treated with sodium bisulfite in duplicate, using the EZ-96 DNA methylation kit (Zymo Research, CA, USA)

tag: visit

value: b

tag: institute

value: KCL

tag: age

value: M

tag: bcell

tag: gran

tag: mono

tag: nk

tag: smokingscore


title: EPI_DUCT1, Epithelium_duct

tag: tissue

value: Minor labial salivary gland

value: Whole minor slaivary gland regions

tag: group

tag: batch

value: One


title: output3 NNK2

description:
from plasmid library
MT_MM_TSM_BB_BL_processed_data.xlsx (sheet NNK2)


title: Scrib WT (KPF/+C) #2 [TB19734]

tag: strain


title: Scrib WT (KPF/+C) #9 [TB22018]


title: genomic DNA from ID 0013 for 'b' visit

description:
0013b
200397870043_R04C02

tag: Sex


title: output1 NNK2

tag: mol type


title: genomic DNA from ID 0015 for 'a' visit

description:
0015a
200397870043_R05C02

value: a


title: output2 NNK2


title: MS, Patient 140

city: Aarhus

first: Mikkel

last: Carstensen

address: Palle Juul-Jensens Boulevard 5

country: Denmark

institute: Aarhus University

description: RRMS + Ulcerative colitis

data_processing:
Mapping was carried out with STAR (v2.6.0b) against the mm10 mus musculus primary assembly from Ensembl (parameters: --outFilterMismatchNmax 1 --outSAMmultNmax 1 --outMultimapperOrder Random --outFilterMultimapNmax 1)
The SAM files were converted to BAM files and sorted by coordinate using samtools (v1.7)
BigWigs files were generated using Deeptools (v3.1.3)
Differentially expressed genes were obtained using DESeq2 (v1.18.1)
Assembly: hg19
Supplementary files format and content: Supplementary files include RNA-seq data in bigWig format (hg19 version of the human genome) and a gene expression analysis carried out with DeSeq2. The gene expression analysis compare patients with highest expression of CBX5 (upper quartile) or lowest expression of CBX5 (lower quartile) to the symptomatic controls.
Supplementary files format and content: Tab-delimited text files including differential gene expression made by DESeq2

source_name: Monocytes

extract_protocol:
Total RNA was extracted from the monocytes by Trizol LS (Thermo Fisher Scientific, ref. 10296028), according to the manufacturer's protocol
Total RNA library preparation and sequencing were performed on DNase-treated RNA samples by Novogene Co., Ltd, as a lncRNA sequencing service, including lncRNA stranded library preparation with rRNA depletion using the Ribo-Zero kit

treatment_protocol: Monocytes were isolated from patient PBMCs by a Pan Monocyte Isolation Kit (Miltenyi Biotech, ref. 130-096-537), according to the kit protocol, allowing the negative selection of untouched monocytes

tag: disease

value: MS

value: Monocytes


title: LF2, Large_foci

tag: cell type


title: MS, Patient 141

description:
SPMS + Psoriasis
DESeq2_MS_Low_CBX5_expression_vs_MS_High_CBX5_expression.txt
DESeq2_MS_Low_CBX5_expression_vs_Symptomatic_control.txt


title: EPI_NORM1, Epithelium_norm


title: genomic DNA from ID 0007 for 'b' visit

description:
0007b
200397870043_R02C02

value: F


title: GC7, GC

value: GC


title: Samp47, Large_foci

value: Three


title: output2 NNK1

description:
from plasmid library
MT_MM_TSM_BB_BL_processed_data.xlsx (sheet NNK1)

value: yeast cell

tag: genotype

value: [psi-pin-] (MATa ade1-14 his3 leu2-3,112 lys2 trp1 ura3-52)

value: PCR amplicon


title: genomic DNA from ID 0044 for 'd' visit

description:
0044d
200397870043_R06C01

tag: visit

value: d


title: output1 NNK1


title: input NNK2


title: MS, Patient 134

description:
RIS
DESeq2_MS_High_CBX5_expression_vs_Symptomatic_control.txt
DESeq2_MS_Low_CBX5_expression_vs_MS_High_CBX5_expression.txt


title: MS, Patient 138

description: RRMS


title: EPI_NORM2, Epithelium_norm


title: genomic DNA from ID 0007 for 'a' visit

description:
0007a
200397870043_R01C02

tag: institute


title: MS, Patient 135

description:
RRMS + Psoriasis
DESeq2_MS_High_CBX5_expression_vs_Symptomatic_control.txt
DESeq2_MS_Low_CBX5_expression_vs_MS_High_CBX5_expression.txt


title: MS, Patient 139

description:
RRMS + Crohns
DESeq2_MS_Low_CBX5_expression_vs_MS_High_CBX5_expression.txt
DESeq2_MS_Low_CBX5_expression_vs_Symptomatic_control.txt


title: genomic DNA from ID 0013 for 'a' visit

description:
0013a
200397870043_R03C02

tag: age


title: output3 NNK1


title: Samp46, healthy tissue

value: healthy tissue


title: input NNK1


title: genomic DNA from ID 0044 for 'b' visit

description:
0044b
200397870043_R05C01

value: b

tag: bcell


title: TOR1A.IR_R3

city: Boston

first: Yanxin

state: MA

address: 185 Cambridge Street

institute: Massachusetts General Hospital/ Harvard Medical School

data_processing:
Transcriptome mapping was performed with STAR (PMID:23104886) using the Ensembl annotation of hg19 reference genome. Read counts for individual genes were produced using HTSeq (PMID:25260700).
Read counts for individual genes were produced using HTSeq (PMID:25260700). Differential expression analysis was performed using the EdgeR package (PMID:19910308) after normalizing read counts and including only those genes with CPM > 1 for at least one out of all samples.
Assembly: hg19
Supplementary files format and content: txt

growth_protocol: Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) high glucose (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific), 100 units/mL penicillin, and 100 μg/mL streptomycin (Thermo Fisher Scientific). IMR90 cells were cultured in a 3% oxygen incubator.

extract_protocol:
mRNA was extracted using RNeasy Mini Kit (Qiagen), with a DNase I (Qiagen) digestion step to minimize genomic DNA contamination.
RNA-seq libraries were prepared from total RNA using polyA selection followed by the NEBNext Ultra II Directional RNA library kit workflow (New England Biolabs).

treatment_protocol: Cells were stably infected with pLentiCRISPRv2 vector encoding sgPRM1 (control), sgALIX, or sgTOR1A, and were induced to senescence by IR (20 Gy) and were harvested at Day 14 post IR.

tag: cell line

value: fibroblast cell

value: TOR1A KO

tag: treatment

value: Cells were induced to senescence by IR (20 Gy) and harvested at Day 14 post IR


title: MS, Patient 130

description: SPMS


title: TOR1A.IR_R2


title: MS, Patient 136

description: RRMS


title: genomic DNA from ID 0054 for 'e' visit

description:
0054e
200397870043_R03C01

value: e

value: KCL

value: M

tag: gran

tag: mono

tag: nk


title: Samp42, Large_foci

tag: tissue

value: Minor labial salivary gland

value: Whole minor slaivary gland regions

tag: group


title: MS, Patient 129

description:
RRMS
DESeq2_MS_Low_CBX5_expression_vs_MS_High_CBX5_expression.txt
DESeq2_MS_Low_CBX5_expression_vs_Symptomatic_control.txt

value: MS

value: Monocytes


title: genomic DNA from ID 0054 for 'a' visit

description:
0054a
200397870043_R01C01

value: a

tag: smokingscore


title: TOR1A.IR_R1


title: MS, Patient 128

description:
PPMS + Crohns
DESeq2_MS_Low_CBX5_expression_vs_MS_High_CBX5_expression.txt
DESeq2_MS_Low_CBX5_expression_vs_Symptomatic_control.txt


title: ALIX.IR_R3

value: ALIX KO


title: Samp38, Large_foci

tag: batch

value: Two


title: Samp37, GC

value: GC

value: Three


title: MS, Patient 127

description: RRMS + Psoriasis


title: ALIX.IR_R2


title: Samp36, GC


title: ALIX.IR_R1


title: genomic DNA from ID 0051 for 'c' visit

description:
0051c
200397870101_R04C02

value: c

tag: Sex


title: Samp35, healthy tissue

value: healthy tissue


title: MS, Patient 125

description: RRMS

tag: disease


title: PRM.IR_R3

tag: cell type

value: fibroblast cell

value: PRM1 KO


title: MS, Patient 86

description: RIS


title: genomic DNA from ID 0051 for 'a' visit

description:
0051a
200397870101_R03C02


title: Samp45, healthy tissue


title: MS, Patient 85


title: Samp34, Small_foci


title: Samp39, healthy tissue


title: genomic DNA from ID 0048 for 'e' visit

description:
0048e
200397870101_R02C02

value: F


title: PRM.IR_R2

tag: genotype

value: Cells were induced to senescence by IR (20 Gy) and harvested at Day 14 post IR


title: genomic DNA from ID 0048 for 'd' visit

description:
0048d
200397870101_R01C02

tag: visit

value: d

value: KCL


title: genomic DNA from ID 0051 for 'e' visit

description:
0051e
200397870101_R06C02

value: e

tag: institute

value: M


title: Samp33, GC

value: Minor labial salivary gland

value: Whole minor slaivary gland regions


title: Symptomatic Control, Patient 81

description:
DESeq2_MS_High_CBX5_expression_vs_Symptomatic_control.txt
DESeq2_MS_Low_CBX5_expression_vs_Symptomatic_control.txt

value: Symptomatic Control

value: Monocytes


title: genomic DNA from ID 0051 for 'd' visit

description:
0051d
200397870101_R05C02

tag: age


title: Symptomatic Control, Patient 80


title: Samp31, healthy tissue

value: Two


title: genomic DNA from ID 0048 for 'b' visit

description:
0048b
200397870101_R06C01

value: b

tag: bcell


title: genomic DNA from ID 0048 for 'a' visit

description:
0048a
200397870101_R05C01

value: a


title: Symptomatic Control, Patient 79


title: Samp29, Large_foci

value: Three


title: STAG1/2 WT HAP1 ChIP

city: London

first: LMS

last: Bioinformatics Core

address: Hammersmith Hospital Campus, Du Cane Road

department: Bioinformatics Core

institute: MRC London Institute of Medical Sciences

data_processing:
Reads were trimmed using TrimGalore v.0.6.0
Reads were mapped to hg19 using Bowtie v2.3.4
bigWig files were generated with DeepTools v.3.1.3 (minimum mapping quality of 15, bin length of 10 bp, extending reads to 200 bp, and reads per kilobase per million reads normalization)
Assembly: hg19
Supplementary files format and content: bigWig files

extract_protocol:
Immunoprecipitation using a mouse anti-RAD21 anitbody was performed on WT and STAG1/2 mutant HAP1 cells as described in 10.1038/s41586-019-1910-z
DNA was sheared using Biorupter Pico (Diagenode),6cycles of 15-s on and 90-s off
Library preparation was performed using a KAPA Library preparation kit using the manufacturer’s protocol

tag: cell line

value: WT

tag: chip antibody

value: anti-RAD21 (Millipore #05-908)


title: STAG1/2 MT HAP1 ChIP

value: STAG1-W337A STAG2-W334A


title: genomic DNA from ID 0045 for 'e' visit

description:
0045e
200397870101_R04C01

tag: gran


title: Samp26, GC

value: GC


title: MS, Patient 73

description:
CIS + Graves
DESeq2_MS_High_CBX5_expression_vs_Symptomatic_control.txt
DESeq2_MS_Low_CBX5_expression_vs_MS_High_CBX5_expression.txt

value: MS


title: Samp27, Small_foci


title: Symptomatic Control, Patient 75


title: MS, Patient 69

description:
CIS + Psoriasis
DESeq2_MS_High_CBX5_expression_vs_Symptomatic_control.txt
DESeq2_MS_Low_CBX5_expression_vs_MS_High_CBX5_expression.txt


title: Samp25, healthy tissue

value: healthy tissue


title: genomic DNA from ID 0045 for 'd' visit

description:
0045d
200397870101_R03C01

value: d

value: KCL

tag: mono


title: genomic DNA from ID 0045 for 'a' visit

description:
0045a
200397870101_R01C01

value: M

tag: nk


title: Samp20, nerve-like

tag: tissue

value: Minor labial salivary gland

value: nerve-like


title: PRM.IR_R1

value: fibroblast cell

value: PRM1 KO

tag: treatment

value: Cells were induced to senescence by IR (20 Gy) and harvested at Day 14 post IR


title: MS, Patient 68

description: RIS

value: Monocytes


title: Samp17, Large_foci

value: Whole minor slaivary gland regions

tag: group

value: Two


title: genomic DNA from ID 0043 for 'e' visit

description:
0043e
200397870076_R06C02

value: e

tag: smokingscore


title: Samp16, Thick sections

value: Thick sections

tag: batch

value: Three


title: Symptomatic Control, Patient 63

tag: disease

value: Symptomatic Control


title: genomic DNA from ID 0043 for 'd' visit

description:
0043d
200397870076_R05C02

tag: Sex


title: Samp15, Thick sections

tag: cell type


title: genomic DNA from ID 0043 for 'c' visit

description:
0043c
200397870076_R04C02

tag: visit

value: c


title: MS, Patient 61

description:
RRMS + IDDM
DESeq2_MS_High_CBX5_expression_vs_Symptomatic_control.txt
DESeq2_MS_Low_CBX5_expression_vs_MS_High_CBX5_expression.txt

value: MS


title: genomic DNA from ID 0043 for 'a' visit

description:
0043a
200397870076_R03C02

value: a

tag: institute


title: Symptomatic Control, Patient 62


title: MS, Patient 76

description:
CIS
DESeq2_MS_High_CBX5_expression_vs_Symptomatic_control.txt
DESeq2_MS_Low_CBX5_expression_vs_MS_High_CBX5_expression.txt


title: genomic DNA from ID 0042 for 'e' visit

description:
0042e
200397870076_R02C02

value: KCL

tag: age


title: Samp13, Thick sections


title: genomic DNA from ID 0045 for 'b' visit

description:
0045b
200397870101_R02C01

value: b

value: M

tag: bcell


title: Symptomatic Control, Patient 58


title: 22RV1-piR-hsa-4447944-3

city: SHA TIN

first: Qiang

last: PENG

state: HONG KONG

address: 30-32 NGAN SHING ST

country: Hong Kong

institute: The Chinese University of HongKong

data_processing:
The sequencing reads were aligned to the human genome (hg38) using STAR 2.7.1a and gene expression was quantified by RSEM v1.3.0
Differentially expressed genes (DEGs) were determined with R 4.1.1 using the package DESeq2 1.34.0
The log2 fold change (L2FC) values were shrunk with the DESeq2 function lfcShrink (type="normal") to control for variance of L2FC estimates for genes with low read counts.
Assembly: hg38
Supplementary files format and content: TPM value for each sample

growth_protocol: Prostate cancer cells were cultured in RPMI-1640 medium supplemented with 10% FBS, and incubated at 37 ℃ with 5% CO2.

extract_protocol:
The total RNA of cells was extracted using miRNeasy Mini Kit (Qiagen, #217004) according to the manufacturer’s instructions.
Preparation of RNA library  was conducted by Novogene Co., LTD

treatment_protocol: Prostate cancer cells were cultured under androgen-deprived medium with 10%  charcoal-stripped fetal bovine serum (CS-FBS).

tag: cell line

value: prostatecancer cells

tag: genotype

value: piR-hsa-4447944 overexpression

value: androgen deprivation


title: Samp11, Thick sections

value: Minor labial salivary gland


title: genomic DNA from ID 0031 for 'd' visit

description:
0031d
200397870076_R01C02

value: d

tag: gran


title: genomic DNA from ID 0042 for 'b' visit

description:
0042b
200397870076_R06C01

tag: mono


title: Samp10, Thick sections

value: Whole minor slaivary gland regions


title: 22RV1-piR-hsa-4447944-1


title: Samp9, Thick sections


title: 22RV1-piR-hsa-4447944-2


title: Samp8, Thick sections


title: genomic DNA from ID 0038 for 'e' visit

description:
0038e
200397870076_R04C01

tag: nk


title: 22RV1-piR-Ctrl-3

value: control


title: genomic DNA from ID 0042 for 'a' visit

description:
0042a
200397870076_R05C01


title: 22RV1-piR-Ctrl-2


title: Samp7, Thick sections

tag: tissue

value: Two


title: genomic DNA from ID 0038 for 'b' visit

description:
0038b
200397870076_R02C01


title: Samp12, Thick sections

value: Thick sections


title: 22RV1-piR-Ctrl-1

tag: treatment


title: Samp5, Thick sections

tag: group


title: LNCaP-piR-hsa-4447944-3

source_name: LNCaP

value: LNCaP


title: GC11, GC

value: GC

value: One


title: Samp4, Thick sections

value: Three


title: LNCaP-piR-hsa-4447944-1


title: LNCaP-piR-hsa-4447944-2


title: genomic DNA from ID 0037 for 'd' visit

description:
0037d
200397870081_R05C02

value: F

tag: smokingscore


title: genomic DNA from ID 0037 for 'e' visit

description:
0037e
200397870081_R06C02

value: e

value: KCL


title: LNCaP-piR-Ctrl-3


title: genomic DNA from ID 0037 for 'c' visit

description:
0037c
200397870081_R04C02

value: c


title: genomic DNA from ID 0037 for 'a' visit

description:
0037a
200397870081_R03C02

value: a


title: LNCaP-piR-Ctrl-2

value: prostatecancer cells


title: Samp3, Thick sections

tag: batch


title: genomic DNA from ID 0038 for 'd' visit

description:
0038d
200397870076_R03C01

value: M


title: Samp2, Thick sections

value: Minor labial salivary gland


title: F/T Replicate #2

city: Gainesville

first: Holger

middle: Andreas

last: Russ

state: FL

address: 1275 Center Drive, Rm. #J545

department: Pharmacology and Therapeutics

institute: University of Florida

data_processing:
Demultiplexing, barcode processing, gene counting, and aggregation were performed with Cell Ranger Count v7.1.0 on the 10X genomics cloud analysis platform
Assembly: Human (GRCh38) v3.0.0
Supplementary files format and content: Filtered matrix files, Cellranger summary CSV, 10X Genomics Loupe browser file

source_name: Pancreas

extract_protocol:
sBC Clusters were single-celled via a 0.05% trypsin incubation for 12 minutes at 37 degrees celsisus. Following this, the single cell solution was quenched with 2% FBS PBS and a cell count was acquired via a Countess III (Cat # AMQAX2000). Following this, the single-cell solution was immediately used in the 10X Genomics Chromium Next GEM Single Cell 5' v2 (Dual Index)  scRNAseq protocol.
Library construction was performed via the 10X Genomics Chromium Next GEM Single Cell 5' v2 (Dual Index) scRNAseq protocol.

value: Pancreas

value: Mel1 INS GFP hESC

value: Stem cell derived beta cell

value: Frozen/Thawed


title: 47627_B2-IL1b

city: Arcadia

first: Mark

last: Boldin

state: California

address: 1500 Duarte Road

institute: City of Hope Beckman Research Institute

data_processing:
Reads were mapped to GRCh38 (hg38).dna.chromosome with STAR (2.6.0a) to generate counts
edgeR (v.3.30.3) was used to perform differential expression analysis
Assembly: GRCh38 (hg38).dna.chromosome

source_name: Colorectal carcinoma

growth_protocol: HCT116 cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS) and antibiotics in humidified atmosphere with 5% CO2 at 37C

extract_protocol:
miRNeasy kits
The KAPA mRNA HyperPrep kit was used for sequencing library preparation
Ribosomal RNA removal method and stranded RNA-seq library

treatment_protocol: Cells were treated with 100ng/ml hIL1b for 4 hours

value: Colorectal carcinoma

value: Epithelial cells

value: NDUFA4 KO


title: Control Replicate #2


title: Samp6, Thick sections

value: Whole minor slaivary gland regions

value: Thick sections


title: genomic DNA from ID 0031 for 'e' visit

description:
0031e
200397870081_R02C02


title: F/T Replicate #1


title: LNCaP-piR-Ctrl-1

value: LNCaP

value: control

value: androgen deprivation


title: 47626_B2


title: Control Replicate #1


title: genomic DNA from ID 0038 for 'a' visit

description:
0038a
200397870076_R01C01

value: KCL


title: 47625_A2-IL1b


title: genomic DNA from ID 0031 for 'a' visit

description:
0031a
200397870081_R05C01

value: a


title: genomic DNA from ID 0042 for 'd' visit

description:
0042d
200397870081_R01C02

value: d


title: MAC+ET-1 treated HG-SOC cells rep3

city: Torino

first: Ivan

last: Molineris

state: TO

address: Via Nizza 52

department: Life Science and System Biology

country: Italy

institute: University of Turin

data_processing:
Sequencing reads were aligned to human reference genome (version GRCh38.p10) using STAR v2.5.3a [PMID:23104886] (with parameters --outFilterMismatchNmax 999 --outFilterMismatchNoverLmax 0.04) and providing a list of known splice sites extracted from GENCODE comprehensive annotation (version 27)
Read that align on ribosomal gene or ribosomal repeated sequences in the genome are removed
Gene expression levels were quantified with featureCounts v1.6.3 [PMID:24227677] (options: -t exon -g gene_name) using GENCODE gene annotation (version 27 basic). Multi-mapped reads were excluded from quantification.
Assembly: GRCh38.p10
Supplementary files format and content: raw gene count

source_name: HG-SOC

growth_protocol: Patient-derived high-grade serous ovarian cancer (HG-SOC) primary cells were isolated from the ascitic fluid of a HG-SOC patient through Ficoll-Histopaque 1077 (Sigma-Aldrich, St. Louis, Missouri,USA). Interface cells were washed in culture medium, and 5×106 viable cells were seeded in 75-cm2 culture flasks, in RPMI 1640 (Gibco, Grovemont Cir, Gaithersburg, USA) containing 1% penicillin streptomycin and 10% fetal bovine serum. All experiments were conducted between the first and second in vitro passage. The purity of primary cultures was assessed by immunophenotyping with a panel of monoclonal Abs (including WT1, keratin 7, calretinin and OCT-125) recognizing ovarian tumor-associated antigens by the alkaline phosphatase-peroxidase-antiperoxidase method. The early passage HG-SOC patient-derived cell line is named PMOV10 where PM stands for Preclinical Models, OV stands for ovarian serous cancer, and # is the order in which the cell line was established

extract_protocol:
Total RNA was isolated using the RNeasy Quick Start kit (Qiagen), according to the manufacturer’s protocol. Quantity and quality of the starting RNA were checked by NanoDrop (Thermo Scientific).
Before starting with library prep, RNA quality was determined running samples on Tapestation 4100 (Agilent). Only RNA samples with RIN above 7 were used in the next steps. To generate the NGS libraries, we exploited the TruSeq Stranded mRNA (Illumina) kit, starting from 100ng of total RNA. Libraries were barcoded, pooled, and sequenced on an Illumina NextSeq 500 sequencing system, in order to obtain for each sample ~ 20-30M single-end reads, 100 nt long.

treatment_protocol: Patient-derived HG-SOC cells (PMOV10) were seeded 1x106 cells/75-cm2 culture flask and incubated at 37°C for 24 hours, then were starved for 24 hours and stimulated with endothelin-1 (ET-1, 100 nM), or with macitentan (MAC, 1µM), alone or in combination with ET-1, or with acetic acid (1% in H20) as vehicle (CTR) for 24 hours.

value: HG-SOC

value: Patient derived HG-SOC primary cells isolated from the ascitic fluid


title: genomic DNA from ID 0031 for 'b' visit

description:
0031b
200397870081_R06C01

value: b

value: M


title: MAC+ET-1 treated HG-SOC cells rep2


title: 47624_A2


title: 47623_83-IL1b-2

value: C15ORF48 OE


title: MAC+ET-1 treated HG-SOC cells rep1


title: genomic DNA from ID 0030 for 'e' visit

description:
0030e
200397870081_R04C01

value: e


title: 47622_83-IL1b-1

value: Colorectal carcinoma


title: genomic DNA from ID 0030 for 'b' visit

description:
0030b
200397870081_R02C01


title: MAC treated HG-SOC cells rep3

value: MAC


title: genomic DNA from ID 0030 for 'd' visit

description:
0030d
200397870081_R03C01


title: 47621_83-2

value: Epithelial cells


title: MAC treated HG-SOC cells rep2


title: genomic DNA from ID 0030 for 'a' visit

description:
0030a
200397870081_R01C01


title: 47619_PCW-IL1b-2

value: WT


title: MAC treated HG-SOC cells rep1


title: 47620_83-1


title: genomic DNA from ID 0035 for 'e' visit

description:
0035e
3998567096_R06C02


title: genomic DNA from ID 0035 for 'b' visit

description:
0035b
3998567096_R04C02


title: 47618_PCW-IL1b-1


title: ET-1 stimulated HG-SOC cells rep3


title: genomic DNA from ID 0035 for 'd' visit

description:
0035d
3998567096_R05C02


title: genomic DNA from ID 0035 for 'a' visit

description:
0035a
3998567096_R03C02


title: TAF7L Wild type 4

city: Kansas City

first: Ayelen

last: Moreno-Irusta

state: Kansas

address: 3901 Rainbow Blvd., 3050

institute: University of Kansas Medical Center

description: Testis from TAF7L Wild type rat at postnatal day 60

data_processing:
Following generation of sequencing images, the pixel-level raw darta collection, image analysis, and base-calling were performed by Real Time Analysis software (Illumina)
Base call files were converted to qseq files by BCL converter (Illumina), and qseq files were converted to fastq files for downstream analysis.
Reads from fastq files were mapped to the rat reference genome (Rnor_5.0.7) using CLC Bio Genomics Workbench 7.0 (CLC Bio, Aarhus, Denmark)
Assembly: Rnor5
Supplementary files format and content: excel file containing reads per kilobase per million reads (RPKM).

organism: Rattus norvegicus

source_name: Testis

extract_protocol:
Testes were removed, flash frozen on dry ice, and RNA was harvested using Trizol reagent according to the manufacturer's recommendations.
Libraries were prepared from poly-A containing RNAs according to protocols provided by the TruSeq RNA sample preparation kits (Illumina).

tag: strain

value: Holtzman Sprague-Dawley

value: Post natal day 60

value: TAF7L Wild type


title: TAF7L Wild type 3


title: 47617_PCW-2


title: ET-1 stimulated HG-SOC cells rep1


title: genomic DNA from ID 0026 for 'e' visit

description:
0026e
3998567096_R02C02

tag: Sex


title: TAF7L Wild type 2


title: 47616_PCW-1

tag: cell type


title: Untreated HG-SOC cells rep3


title: HepG2 cells/ shContrl#3

city: Chengdu

first: Yida

last: Wang

address: No.24, South Section of First Ring Road, Wuhou District

institute: Sichuan University

data_processing:
trimmomatic
Hisat2
samtools
stringtie
Assembly: hg18
Supplementary files format and content: FPKM values for each Sample

growth_protocol: HepG2 cells were cultivated in DMEM with 10% FBS and maintained at 37°C in a 5% CO2 environment.

extract_protocol:
Cell lysis was carried out using TRIzol reagent, followed by total RNA extraction.
RNA libraries for RNA-seq were prepared using NEBNext® UltraTM RNA Library Prep Kit for Illumina®(NEB,USA) following manufacturer's protocols.

treatment_protocol: The target plasmid and packaging plasmids were co-transfected into 293T cells using Lipofectamine 3000 (Thermo Fisher, USA) for virus packaging. After 48 hours of transfection, the supernatant was collected, filtered, and employed for the infection experiments.


title: Untreated HG-SOC cells rep2


title: genomic DNA from ID 0026 for 'b' visit

description:
0026b
3998567096_R01C02

tag: visit


title: HepG2 cells/ shContrl#2


title: Untreated HG-SOC cells rep1


title: TAF7L Knockout 4

description: Testis from TAF7L Knockout rat at postnatal day 60

value: TAF7L Null


title: TAF7L Wild type 1


title: NES, EV, H3K27me3 CUT&RUN, Replicate 2

city: San Francisco

first: Bjoern

last: Schwer

state: CA

institute: University of California, San Francisco

data_processing:
For data analysis, we used the nf-core framework CUT&RUN analysis pipeline (v3.0; https://github.com/nf-core/cutandrun/tree/3.0)19,20. In brief, adapter-trimmed reads (TrimGalore v0.6.6) were mapped to the human (hg38) and spike-in (S. cerevisiae, sacCer3) genome reference assemblies by Bowtie2 (v2.4.4). Duplicates were marked by Picard (v2.27.4). Reads were normalized against spike-in. CUT&RUN peaks were called by SEACR (v1.3) using stringent settings, and consensus peaks were identified as peaks that were present in all replicates per condition.
Assembly: hg38
Supplementary files format and content: bigwig files are replicate-merged and BPM-normalized
Supplementary files format and content: consensus.peaks.awk.bed files contain SEACR consensus peaks.

source_name: Neuroepithelial stem cells

growth_protocol: For human neuroepithelial stem (NES) cells: NES cells were generated from human iPS cells (WTC10). NES cells were cultured on poly-L-ornithine/laminin-coated wells in NES cell medium (DMEM/F-12 with GlutaMAX, 1x N2 supplement, 0.05x B27 without vitamin A supplement, 1.6 g/L Glucose, 20 ng/mL EGF, 20 ng/mL FGF2) in a humidified 37 C incubator with 5% CO2. NES cells were fed daily, passaged every three to four days using TrypLE Express, and plated at 500,000-600,000 cells per well of a 6-well plate in 2 mL of NES cell medium.

extract_protocol:
Preparation of CUT&RUN libraries was done with the CUT&RUN assay kit (Cell Signaling Technology). Briefly, 250,000 NES cells per sample were fixed with 0.1% formaldehyde in PBS for 2 min and quenched with 1 M glycine for 5 min at room temperature. Cells were washed in 1x wash buffer containing spermidine and protease inhibitor cocktail, and incubated in antibody binding buffer containing spermidine, proteinase inhibitor cocktail, digitonin, and 10 µl of activated concanavalin A beads per sample for 16-18 h at 4 C. Per CUT&RUN reaction, 0.2 µg of H3K27me3 antibody (clone C36B11, #9733, Cell Signaling Technology) or 0.96 µg of V5 antibody (clone D3H8Q, #13202, Cell Signaling Technology) was used. pAG-MNase in 1x wash buffer containing spermidine, protease inhibitor cocktail, and digitonin was bound to the antibody for 1 h at 4 C. pAG-MNase was activated by adding 3 µL calcium chloride per sample and incubated for 30 min at 4 C. Digestion was stopped by adding 1x stop buffer (digitonin solution, RNase A, and 50 pg S. cerevisiae spike-in DNA (Cell Signaling Technology) for normalization) and samples were incubated for 10 min at 37 C. Samples were subjected to SDS and proteinase K treatment for 2 h at 65 C. Zymo DNA Clean and Concentrator (D4034) was used to clean up the samples.
NEBNext Ultra II DNA Library Prep Kit for Illumina (E7645L) was used for library preparation. Library clean-up and size selection was done by using SPRI beads (103778-488, Bulldog Bio). Libraries were quantified and analyzed by Qubit and Tapestation 4200 (Agilent) analysis and sequenced for 38 cycles in paired-end mode on a NextSeq 500 (Illumina).

value: Neuroepithelial stem cells

tag: antibody

value: H3K27me3, clone C36B11, #9733, Cell Signaling Technology

value: EV


title: genomic DNA from ID 0026 for 'a' visit

description:
0026a
3998567096_R06C01

value: a

tag: institute

value: KCL

tag: age


title: genomic DNA from ID 0009 for 'e' visit

description:
0009e
3998567096_R05C01

tag: bcell


title: ET-1 stimulated HG-SOC cells rep2

value: HG-SOC

value: Patient derived HG-SOC primary cells isolated from the ascitic fluid


title: NES, EV, H3K27me3 CUT&RUN, Replicate 1

tag: genotype


title: genomic DNA from ID 0009 for 'd' visit

description:
0009d
3998567096_R04C01

value: d

value: M

tag: gran


title: HepG2 cells/ shSLC7A11-AS1#3

tag: cell line


title: TAF7L Knockout 3


title: HepG2 cells/ shSLC7A11-AS1#2


title: TAF7L Knockout 2


title: TAF7L Knockout 1


title: genomic DNA from ID 0008 for 'd' visit

description:
0008d
3998567096_R02C01

tag: mono

tag: nk


title: PRDM6-MYCN NES-derived tumor, 94867

data_processing:
Raw RNA-seq data was pre-processed using HTStream (https://github.com/s4hts/HTStream). Pre-processed reads were aligned using STAR (v2.7.10, GRCh38).
Assembly: hg38
Supplementary files format and content: Counts table in txt or csv file format

source_name: Tumor

extract_protocol:
Total RNA was extracted from cultured cells or flash frozen tumor tissue using RNA extraction kit (Zymo Research). RNA quality was checked using Agilent TapeStation 4200.
Total RNA was subjected to polyA enrichment and library preparation via the Illumina TruSeq stranded mRNA kit

tag: tissue

value: Tumor


title: HepG2 cells/ shSLC7A11-AS1#1


title: SW480_Reg_Rep1

first: Darshan

last: Shimoga Chandrashekar

state: Alabama

address: 619 19th Street South, WP P220D

department: Pathology

institute: University of Alabama at Birmingham

data_processing:
The raw sequence reads obtained as FASTQ files were trimmed using Trim Galore (v0.4.1) [http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/] to remove adapter sequences and low quality reads.
After performing the quality control analysis using FastQC (v0.11.5) [http://www.bioinformatics.babraham.ac.uk/projects/fastqc/], the trimmed reads were mapped to human genome (GRCh38/hg38) using hisat2.
The aligned reads were sorted via samtools (Version: 1.3.1). HTSeq-count (version 0.6.0) was used to obtain raw read counts for each annotated human gene. Differential expression analysis was carried out using DESeq19 considering “blind” method for estimating dispersion and “fit-only” sharing mode.  Genes with absolute fold change of 2 or more and p-value <0.05 were considered as differentially expressed.
Assembly: hg38
Supplementary files format and content: tab delimited text file include normalized read counts for each sample from DESeq.

extract_protocol:
RNA was extracted from using Trizol reagent following standard manufacturer’s instructions.
RNA integrity and quantification was  assessed using the Agilent TapeStation instrument and High Senstivity RNA ScreenTape and Qubit RNA assay kit all RIN score were higher than 9. Poly A enrichment from total RNA was performed, and library were prepared using Agilent Technologies: Poly A enrichment from total RNA, cDNA Synthesis (Cat #5500-0135), cDNA library prep, (cat# 5500-0140 and cat# 5190-6444). Final library were run on Illumina Nextseq 550 sequencer USING Illumina NextSeq 500/550 High Output Kit, as 75-bp single-end reads

treatment_protocol: SW480 cells were treated at 500nM concentration of JAK/HDACi  and regorafenib as single drugs, and their combination. Dmso was used as control.

tag: treatment

value: Reg


title: SW480_JH_Rep1

value: JH


title: SW480_Reg_Rep2


title: SW480_JH_Rep2


title: genomic DNA from ID 0009 for 'a' visit

description:
0009a
3998567096_R01C01

tag: smokingscore


title: corpus callosum, SHR_C1, rep1

city: Hangzhou

first: Rui

last: Xue

address: 88# Jiefang Road

department: Department of Neurology

institute: the Second Affiliated Hospital of Zhejiang University, School of Medicine

data_processing:
Reads obtained from the sequencing machines includes raw reads containing adapters or low quality bases which will affect the following assembly and analysis. Thus, to get high quality clean reads, reads were further filtered by Cutadapt (https://cutadapt.readthedocs.io/en/stable/,version:cutadapt-1.9). We aligned reads of all samples to the research species reference genome using HISAT2 (https://daehwankimlab.github.io/hisat2/,version:hisat2-2.2.1) package, which initially remove a portion of the reads based on quality information accompanying each read and then maps the reads to the reference genome.
The mapped reads of each sample were assembled using StringTie (http://ccb.jhu.edu/software/stringtie/,version:stringtie-2.1.6) with default parameters. Then, all transcriptomes from all samples were merged to reconstruct a comprehensive transcriptome using gffcompare software(http://ccb.jhu.edu/software/stringtie/gffcompare.shtml,version:gffcompare-0.9.8). After the final transcriptome was generated, StringTie and ballgown (http://www.bioconductor.org/packages/release/bioc/html/ballgown.html) were used to estimate the expression levels of all transcripts and perform expression abundance for mRNAs by calculating FPKM (fragment per kilobase of transcript per million mapped reads) value.
Genes differential expression analysis was performed by DESeq2 software between two different groups (and by edgeR between two samples). The genes with the parameter of false discovery rate (FDR) below 0.05 and absolute fold change ≥ 2 were considered differentially expressed genes. Differentially expressed genes were then subjected to enrichment analysis of GO functions and KEGG pathways.
Assembly: ftp://ftp.ensembl.org/pub/release-101/fasta/rattus_norvegicus/dna/
Supplementary files format and content: tab-delimited text files include FPKM values for each Sample

source_name: corpus callosum

extract_protocol:
Total RNA was extracted using Trizol reagent (thermofisher, 15596018) following the manufacturer's procedure. The total RNA quantity and purity were analysis of Bioanalyzer 2100 and RNA 6000 Nano LabChip Kit (Agilent, CA, USA, 5067-1511) , high-quality RNA samples with RIN number > 7.0 were used to construct sequencing library.
mRNA was purified from total RNA (5ug) using Dynabeads Oligo (dT) (Thermo Fisher, CA, USA) with two rounds of purification. Following purification, the mRNA was fragmented into short fragments using divalent cations under elevated temperature (Magnesium RNA Fragmentation Module (NEB, cat.e6150, USA) under 94℃ 5-7min). Then the cleaved RNA fragments were reverse-transcribed to create the cDNA by SuperScript™ II Reverse Transcriptase (Invitrogen, cat. 1896649, USA), which were next used to synthesise U-labeled second-stranded DNAs with E. coli DNA polymerase I (NEB, cat.m0209, USA), RNase H (NEB, cat.m0297, USA) and dUTP Solution (Thermo Fisher, cat.R0133, USA). An A-base was then added to the blunt ends of each strand, preparing them for ligation to the indexed adapters. Each adapter contained a T-base overhang for ligating the adapter to the A-tailed fragmented DNA. Dual-index adapters were ligated to the fragments, and size selection was performed with AMPureXP beads. After the heat-labile UDG enzyme (NEB, cat.m0280, USA) treatment of the U-labeled second-stranded DNAs, the ligated products were amplified with PCR by the following conditions: initial denaturation at 95℃ for 3 min; 8 cycles of denaturation at 98℃ for 15 sec, annealing at 60℃ for 15 sec, and extension at 72℃ for 30 sec; and then final extension at 72℃ for 5 min. The average insert size for the final cDNA librarys were 300±50 bp. At last, we performed the 2×150bp paired-end sequencing (PE150) on an Illumina NovaseqTM 6000 following the vendor's recommended protocol.

value: corpus callosum

value: Spontaneously hypertensive rats (SHR)


title: HepG2 cells/ shContrl#1

value: WT


title: corpus callosum, SHR_C2, rep2


title: genomic DNA from ID 0027 for 'b' visit

description:
0027b
3998567015_R04C02

value: b

value: F


title: PRDM6-MYCN NES-derived tumor, 94836


title: SW480_DMSO_Rep2

city: Birmingham

value: DMSO


title: genomic DNA from ID 0027 for 'd' visit

description:
0027d
3998567015_R05C02


title: genomic DNA from ID 0009 for 'c' visit

description:
0009c
3998567096_R03C01

value: c


title: corpus callosum, SHR_P3, rep3


title: corpus callosum, SHR_C3, rep3


title: corpus callosum, SHR_P2, rep2


title: MYCN NES-derived tumor, 94863

value: MYCN


title: genomic DNA from ID 0027 for 'a' visit

description:
0027a
3998567015_R03C02

value: a


title: MYCN NES-derived tumor, 94861


title: SW480_Combo_Rep2

value: JH+Reg


title: corpus callosum, SHR3, rep3

value: No treatment


title: corpus callosum, SHR_P1, rep1


title: corpus callosum, SHR2, rep2


title: genomic DNA from ID 0027 for 'e' visit

description:
0027e
3998567015_R06C02

value: e

value: KCL


title: PRDM6 NES-derived tumor, 94958


title: OPM2, KRAS A146T, replicate 3

city: Würzburg

first: Larissa

middle: Anita

last: Haertle

state: Bavaria

address: Josef-Schneider-Str. 2, Haus C11

department: Medizinische Klinik und Poliklinik II

country: Germany

institute: Universitätsklinikum Würzburg (UWK)

data_processing:
Sequencing quality was evaluated via FastQC.
RNA-sequencing reads were mapped to the human genome using STAR.
The mapped reads were quantified by featureCounts (v. 1.6.3) from the Subread package
Assembly: GRCh38.p12
Supplementary files format and content: tab-delimited files including raw counts for each Sample
Supplementary files format and content: tab-delimited files including normalized counts (TPM) for each Sample

source_name: cell line

growth_protocol: Cells were cultured in RPMI-1640 medium supplemented with 10% FBS, 1 mM sodium pyruvate, 2 mM glutamine, 100 U/ml penicillin, and 100 g/ml streptomycin and kept at 5% CO2 and 37°C. Cultures were regularly split (every 3-4 days) and routinely checked for mycoplasma negativity. OPM2 cells are semi-adherent and were scratched with a cell scraper before each splitting.

extract_protocol:
For the RNA-Seq analysis, the OPM2 KRAS WT and the OPM2-derived KRAS A146T and G12A cells were seeded in triplicates at a density of 100,000 cells/ml. Three days after seeding, two million cells were pelleted and stored at -80°C. The RNeasy Mini kit (Qiagen) was used to extract the RNA and the quality was checked with the 2100 Bioanalyzer and the RNA 6000 Nano kit (Agilent). The RIN was ~10 for all samples.
Libraries were prepared from 1000ng of total RNA using the TruSeq stranded mRNA kit (Illumina). Quantification was conducted with the Qubit 3.0 Fluorometer (ThermoFisher) and the 2100 Bioanalyzer with the DNA 1000 kit (Agilent). Pooled libraries were sequenced (single-end) on the NextSeq 500 (Illumina) using the High Output Kit v2.5 (75 Cycles).

value: cell line

value: Multiple Myeloma

value: KRAS A146T

value: no teatment


title: OPM2, KRAS A146T, replicate 2


title: PRDM6-MYCN NES-derived tumor, 94840

value: Tumor


title: MYCN NES-derived tumor, 94973


title: genomic DNA from ID 0024 for 'd' visit

description:
0024d
3998567015_R01C02

value: d

value: M


title: genomic DNA from ID 0024 for 'e' visit

description:
0024e
3998567015_R02C02


title: OPM2, KRAS A146T, replicate 1


title: corpus callosum, SHR1, rep1

value: corpus callosum

value: Spontaneously hypertensive rats (SHR)


title: PRDM6 NES-derived tumor, 94957


title: genomic DNA from ID 0024 for 'a' visit

description:
0024a
3998567015_R05C01


title: SW480_DMSO_Rep1

value: DMSO


title: PRDM6 NES-derived tumor, 94856


title: genomic DNA from ID 0024 for 'b' visit

description:
0024b
3998567015_R06C01

value: b


title: SW480_Combo_Rep1


title: corpus callosum, WKY3, rep3

value: Wistar Kyoto rats (WKY),


title: OPM2, KRAS G12A, replicate 3

value: KRAS G12A


title: genomic DNA from ID 0023 for 'e' visit

description:
0023e
3998567015_R04C01


title: NES, PRDM6, Replicate 3

value: Neuroepithelial stem cells


title: OPM2, KRAS G12A, replicate 2


title: corpus callosum, WKY1, rep1

value: No treatment


title: genomic DNA from ID 0023 for 'a' visit

description:
0023a
3998567015_R01C01

value: a


title: NES, PRDM6, Replicate 2


title: genomic DNA from ID 0023 for 'd' visit

description:
0023d
3998567015_R03C01

value: KCL


title: OPM2, KRAS wildtype, replicate 3

value: cell line

value: wildtype


title: genomic DNA from ID 0023 for 'b' visit

description:
0023b
3998567015_R02C01


title: genomic DNA from ID 0018 for 'e' visit

description:
0018e
3998567059_R06C02

value: e

value: F


title: NES, EV, Replicate 3

value: EV


title: NES, PRDM6, Replicate 1


title: NES, EV, Replicate 2


title: Nucleus pulposus cells from LSI-E #1, RNA-seq

city: Wuhan

first: Cao

last: Yang

state: Hubei

address: Jiefang average

institute: Wuhan Union Hospital

description:
LSI_E_1
Sham_LSI_LSI_E_RPKM.txt

data_processing:
Illumina Casava 1.7 software was used for basecalling.
Sequenced reads were qualified using FastQC and masked for low-complexity or low-quality sequence, then mapped to Homo_sapiens.GRCH38.94 whole genome and Rattus_norvegicus.Rnor_6.0.
Read Per Kilobase of exon per Megabase of library size (RPKM)  were calculated using a procotol from Feng, Jianxing et al. Bioinformatics (Oxford, England) vol. 28,21 (2012): 2782-8. In short, exons from all isoforms of a gene were merged to create one meta-transcript. The number of reads falling of the meta-transcript and by the size of the library.
Assembly: Homo_sapiens.GRCh38.94; Rattus_norvegicus.Rnor_6.0
Supplementary files format and content: IVDD_Normal_RPKM.txt
Supplementary files format and content: Sham_LSI_LSI_E_RPKM.txt

source_name: Nucleus pulposus tissue from LSI-E

growth_protocol: Nucleus pulposus cells were isolated from human or rat lumbar nucleus pulposus tissues

extract_protocol:
Total RNA was isolated from samples using TRIzol reagent. Illumina TruSeq RNA Sample Pre Kit (Cat#FC-122-1001) was used with 1 μg of total RNA for the construction of sequencing libraries.
Libraries were prepared according to instructions of Illumina HiSeq X ten accompanying the Ovation RNA-Seq System V2 (M01206 v9). Briefly, RNA was elutated according to the manuscription user guide. Then RNA was fragmented using fragment buffer. cDNA was synthed using dNTPs, DNA polymeraseI and RNaseH. Then cDNA was purified using AMPure XP beads. PCR Was used to amplify with Illumina primers for 15 cycles and library fragments of ~200 bp (insert plus adaptor and PCR primer sequences) were band isolated from an agarose gel. The purified RNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Illumina HiSeq X ten following the manufacturer's protocols.

treatment_protocol: Nucleus pulposus tissues were digested with 0.4% type II collagenase for 4-6 hours.

value: Nucleus pulposus tissue from LSI-E

value: Nucleus pulposus cells

value: LSI-E


title: OPM2, KRAS wildtype, replicate 2

value: Multiple Myeloma

value: no teatment


title: Nucleus pulposus cells from LSI-E #2, RNA-seq

description:
LSI_E_2
Sham_LSI_LSI_E_RPKM.txt


title: NES, EV, Replicate 1


title: corpus callosum, WKY2, rep2

value: corpus callosum