title: runx1 homozygous [ko3]

description:
Single-cell RNA sequencing, 10x Genomics
seurat_integrated_clustered.rds

data_processing:
The libraries were sequenced on NovaSeq platforms. BCL files were analyzed with the Cell Ranger pipeline (version 3.1.0). The demultiplexed FASTQ data was aligned to zebrafish Ensembl GRCz11 reference and generated the single cell count matrix. Gene-level counts were imported into R using the Seurat suite version 3.0.
Count matrices from the NextSeq and NovaSeq platforms were merged together prior to quality control and filtering. For each cell that was present in both datasets, gene-level count values were summed to retain a final count matrix of 21,727 features across 35,178 cells. Low quality cells were filtered out by keeping cellular barcodes with the following metrics: (1) >= 500 UMIs per cell; (2) >= 250 genes per cell; (3) >= 0.75 novelty score, calculated as log10(genes detected) / log10(UMI counts per cell); and (4) <= 15 percentage of reads mapping to mitochondrial genes.
Genome_build: Zebrafish Ensembl GRCz11 reference
Supplementary_files_format_and_content: Count matrices from the NovaSeq platform.

source_name: unsorted whole kidney marrow cells

growth_protocol: Freshly isolated kidney marrow cells from euthanized adult zebrafish at 8 months.

extract_protocol:
After euthanasia, the adult zebrafish were subjected to cardiac bleeding to remove most mature erythrocytes from the marrow. The zebrafish were then dissected at room temperature and the dissected marrow was placed into 500ul of 0.9x PBS with 2% FBS and 1 USP units/mL heparin, placed on ice. The marrow samples were then mechanically dissociated by repeated pipetting into single cell suspension and passed through a 40-μm nylon mesh. All 8 samples were washed by spinning down at 350 x g for 5 min at 4C. The cell pellets were collected in 300ul of 1x DPBS with 0.04% BSA (Gemini Bio-Products), then washed again after spinning down at 350 x g for 5 minutes at 4°C and counted after resuspension in DPBS/BSA to target 6,000 cells per reaction.
We followed the 10x Genomics sample preparation protocol and the 10x Genomics single cell RNA sequencing v3.1 library preparation.

treatment_protocol: No additional treatment for unsorted cells.

value: 8 months

value: runx1 homozygous


title: rpoN mutant 3

status: Public on Dec 02 2021

type: SRA

city: Urbana

first: Frank

last: Zhao

state: IL

address: 1201 W. Gregory Dr.

department: Crop Sciences

country: USA

institute: University of Illinois

library_source: transcriptomic

data_processing:
Fastq files were generated and demultiplexed with with the bcl2fastq v1.8.4 Conversion Software (Illumina)
Sequenced reads were trimmed for adaptor sequence, then mapped to Erwinia amylovora Ea1189  whole genome using bowtie v0.12.7 with parameters -t --sam -v 2 -B 1 -3 20 --best
The number of reads mapped to CDS of Erwinia amylovora Ea1189  were counted using bedtools 2.17.0
Normalization and statistical analysis were carried out using the R package DESeq2 as described in Love et al. Genome biology, 2014.
Genome_build: bowtieNC_013961
Supplementary_files_format_and_content: tab-delimited text files include read counts for each sample

molecule: total RNA

organism: Erwinia amylovora

source_name: bacterial cells

growth_protocol: Overnight cultures of wild-type and mutant strain grown in LB were harvested and washed twice with hrp-inducing minimal media.

extract_protocol:
Bacterial cells were harvested, and RNA was isolated using Qiagen RNeasy bacteria Mini kit
The stranded RNAseq libraries were prepared with Illumina's 'TruSeq Stranded RNA Sample Prep kit'

treatment_protocol: Bacterial cells were adjusted to 2 x 10^8 cfu/ml in 5 mL of hrp-inducing minimal media and incubated for 6 h at 18 °C.

tag: strain

tag: genotype

value: deltarpoN


title: runx1 heterozygous [het3]

city: Boston

first: Leonard

last: Zon

state: MA

address: 1 Blackfan Circle

department: Oncology/Hematology

institute: Boston Children's Hospital

organism: Danio rerio

tag: cell type

value: unsorted whole kidney marrow cells

tag: sorting

value: unsorted

tag: age

value: runx1 heterozygous


title: runx1 homozygous [ko1]


title: rpoN mutant 2


title: runx1 homozygous [ko2]


title: rpoN mutant 1


title: control 1

data_processing:
The libraries were sequenced on NextSeq and NovaSeq platforms. BCL files were analyzed with the Cell Ranger pipeline (version 3.1.0). The demultiplexed FASTQ data was aligned to zebrafish Ensembl GRCz11 reference and generated the single cell count matrix. Gene-level counts were imported into R using the Seurat suite version 3.0
Count matrices from the NextSeq and NovaSeq platforms were merged together prior to quality control and filtering. For each cell that was present in both datasets, gene-level count values were summed to retain a final count matrix of 35,227 features across 39,424 cells. Low quality cells were filtered out by keeping cellular barcodes with the following metrics: (1) >= 500 UMIs per cell; (2) >= 200 genes per cell; (3) >= 0.7 novelty score, calculated as log10(genes detected) / log10(UMI counts per cell); and (4) <= 10 percentage of reads mapping to mitochondrial genes.
Genome_build: Zebrafish Ensembl GRCz11 reference
Supplementary_files_format_and_content: FASTQ files and merged count matrices from the NextSeq and NovaSeq platforms

source_name: control

growth_protocol: Freshly isolated kidney marrow cells from euthanized adult zebrafish at 8 months

extract_protocol:
After euthanasia, the adult zebrafish were subjected to cardiac bleeding to remove most mature erythrocytes from the marrow. The zebrafish were then dissected at room temperature and the dissected marrow was placed into 500ul of 0.9x PBS with 2% FBS and 1 USP units/mL heparin, placed on ice. The marrow samples were then mechanically dissociated by repeated pipetting into single cell suspension and passed through a 40-μm nylon mesh. Part of the mutant marrow cell preparation was used for sorting the dominant color marrow cells on BD Aria 2 into 500ul 1x PBS. All 8 samples were washed by spinning down at 350 x g for 5 min at 4C. The cell pellelts were collected in 300ul of 1x DPBS with 0.04% BSA (Gemini Bio-Products), then washed again after spinning down at 350 x g for 5 minutes at 4°C and counted after resuspension in DPBS/BSA to target 10,000 cells per reaction.
We followed the 10x genomics sample preparation protocol and the 10x genomics single cell RNA sequencing V2 library preparation
single-cell RNA sequencing, 10x genomics

treatment_protocol: No additional treatment for unsorted cells; dominant cell samples were sorted into PBS over 30 minutes per sample

value: 8 months old

tag: genotype/variation

value: 2 control guide RNAs and cas9 mRNA


title: Wildtype 3

value: wild-type


title: runx1 heterozygous [het4]


title: runx1 heterozygous [het1]


title: WT-1 (Sample 25)

status: Public on Dec 03 2021

first: Hui

last: Pan

address: 1 Joslin Place

department: Bioinformatics and Biostatistics Core

institute: Joslin Diabetes Center

data_processing:
The reads are trimmed for adapters and filtered by sequencing Phred quality (>= Q15) using fastp.
Adapter-trimmed reads are aligned to the human transcriptome (Ensembl version 102) using kallisto.
Transcript counts are converted  to gene counts using tximport.
Genome_build: human transcriptome (Ensembl version 102)
Supplementary_files_format_and_content: count_table.csv

organism: Homo sapiens

source_name: Endoc-bH1 cell line

growth_protocol: EndoC-bH1 cells were grown in conditioned media collected from HEK293 stable OE cells (media change everyday for 10 days).

extract_protocol:
Total RNA is isolated using Qiagen Kit.
TruSeq Stranded mRNA seq, NovaSeq 6000, 100PE, ~50M reads (25PR)

treatment_protocol: Conditioned media collected from EV, WT-CEL, MUT-CEL stable OE HEK293 cell line by culturing HEK293 cells in EndoC-bH1 media for 3 hours, filtered, and protein levels were normalized.

tag: cell line

value: Endoc-bH1 cell line

value: Pancreatic beta cells

tag: treatment

value: Treated with HEK293-WT OE conditioned media for 10 days

tag: batch


title: runx1 heterozygous [het2]


title: Wildtype 1


title: CD45+ immune cells from 2.5-month-old mice: sample3

city: Los angeles

first: Gaoyan

last: Li

state: CA

address: 1929 beloit ave, apt315

institute: UCLA

data_processing:
The 10X Genomics Cell Ranger version 3.0.2 single-cell software was used to perform sample demultiplexing, sequence alignment to mouse genome mm10, filtering, and unique molecular identifier (UMI) counting to generate gene count matrices.
Data analysis was performed in R using Seurat v3 package.
Genome_build: mm10 for mouse cells and hg19 for human cells
Supplementary_files_format_and_content: Feature-barcode matrix. Each matrix is stored in the Market Exchange Format (MEX) for sparse matrices. It also contains gzipped TSV files with feature and barcode sequences corresponding to row and column indices.

organism: Mus musculus

source_name: epididymal white adipose tissue

extract_protocol: Mouse epididymal adipose tissue (eWAT) was minced and transferred to a 50 mL Falcon tube containing 10 mL digestion buffer (100mM HEPES pH 7.4; 120mM NaCl; 50mM KCl; 5mM Glucose; 1mM CaCl2, 1.5% BSA, and 1 mg/mL collagenase D). The tissue was incubated in a 37℃ shaking water bath for 30 min. Digestion was stopped by adding 35 mL 2% FBS in PBS. The solution was passed through 100 µm and then 40 µm strainers, and centrifuged at 400 g for 5 min. The supernatant was aspirated, and SVF pellet was collected. Lin- SVFs was obtained by Multicolor Flow Cytometry (MACS) (Stemcell Technologies, Cat# 17656). To obtain Lin- SVFs, anti-mouse-CD45-PE, anti-mouse-CD31-PE, and anti-mouse-Ter119-PE (1:200, BioLegend) were used.  To obtain immune cells from eWAT, only anti-mouse-CD45-PE was used and isolated by MACS. Human peripancreatic white adipose tissue was minced and transferred to a 50 mL Falcon tube containing 10 mL digestion buffer (1 × PBS, and 10 mg/mL collagenase D). The tissue was incubated in a 37℃ shaking water bath for 45 min. Digestion was stopped by adding 35 mL 2% FBS in PBS. The solution was passed through a 250 µm mesh (Spectrum), and then 100 µm and 40 µm strainers, and centrifuged at 600 g for 5 min. The supernatant was aspirated, and SVF pellet was collected. To obtain Lin- SVF, cells were incubated with anti-human-CD45-PE, anti-human-CD31-PE, and anti-human-CD235a-PE (1:200, BioLegend), and isolated by MACS (Stemcell Technologies, Cat# 17654). To obtain immune cells, only anti-human-CD45-PE (1:200, BioLegend) was used and isolated by MACS.

tag: tissue

value: epididymal white adipose tissue

value: CD45+ immune cells

tag: cell population

value: immune cells

value: 2.5 months

tag: Sex

value: M


title: runx1 homozygous [ko4]

value: 8 months


title: CD45+ immune cells from 2.5-month-old mice: sample2


title: fb-B-4

city: Nanning

first: xing

last: lin

address: Shuangyong Road

country: China

institute: Guangxi medical university

data_processing:
We applied DESeq2 algorithm to filter the differentially expressed genes, after the significant analysis and FDR analysis under the following criteria: i) |log2FC| > 1 ; ii) Pvalue < 0.05.
Genome_build: mm9

organism: Rattus norvegicus

source_name: rat treated with SCL rep2

growth_protocol: rats in model group  were feed with high-fat diet for 8 weeks to induce  non-alcoholic fatty liver diseace, and normal group were given normal diet

extract_protocol:
Total RNA was extracted from liver tissues with Trizol reagent
NEBNext UltraTM RNA Library Prep Kit（NEB #E7490）,AMPure XP system

treatment_protocol: rats of SCL group were given SCL

value: male

value: SD

value: non-alcoholic fatty liver

value: treated with Smilax china L.(SCL)


title: fb-B-7

source_name: rat treated with SCL rep3


title: fb-M-6

source_name: rat model rep3

value: model group (high-fat diets for 8 weeks)


title: fb-M-5

source_name: rat model rep2


title: CD45+ immune cells from 2.5-month-old mice: sample1


title: fb-M2-1

source_name: rat model rep1


title: CD45+ immune cells from 12-month-old mice: sample 2

value: 12 months


title: CD45+ immune cells from 12-month-old mice: sample3


title: fb-N-3

source_name: rat wt rep3

value: normal control liver

value: normal group


title: CD45+ immune cells from 12-month-old mice: sample 1


title: fb-N-1

source_name: rat wt rep1


title: fb-N-2

source_name: rat wt rep2


title: Lin- SVF cells from 2.5-month-old mice: sample3

value: Lin- SVF cells

value: non-immune cells


title: Lin- SVF cells from 2.5-month-old mice: sample1


title: Lin- SVF cells from 2.5-month-old mice: sample2


title: Wildtype 2

value: wild-type


title: KO_SSt_rep2

city: Zunyi

first: Jiao

state: Guizhou

address: No.6 West Xuefu Road

institute: Zunyi Medical University

description: processed data file: KO_genes_fpkm_expression.txt

data_processing:
Novaseq 6000 software used for basecalling.
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence, then mapped to mm10 whole genome using bowtie v0.12.2 with parameters -q -p 4 -e 100 -y -a -m 10 --best --strata
Reads Per Kilobase of exon per Megabase of library size (RPKM) were calculated.
Genome_build: mm10
Supplementary_files_format_and_content: tab-delimited text files include FPKM values for each Sample.

extract_protocol:
RNA was harvested using Trizol reagent.
RNA libraries were prepared for sequencing using standard protocols

treatment_protocol: MSCs isolated from WT and miR-337-/- rats and cultured. At passage 3, MSCs were treated by CMS or FBS for 24h after serum starved for 8h.

tag: strain background

value: Bone marrow

value: mesenchymal stem cell

value: serum starvation for 24h


title: KO_FBS_rep3

value: FBS for 24h


title: KO_SSt_rep1


title: Lin- SVF cells from 12-month-old mice: sample2

value: epididymal white adipose tissue

value: M


title: fb-B-2

source_name: rat treated with SCL rep1

value: male


title: KO_FBS_rep2

value: SD


title: KO_FBS_rep1


title: Primary Visual Cortex (V1)

city: San Francisco

first: Victoria

last: Cheung

address: 1550 4th Street

institute: University of California, San Francisco

description: 10x seq output

data_processing:
Cellranger pipeline was used to assign UMI
Scanpy was used for QC check
Scanpy was used for analyzing the data via cluster analysis and generation of gene expression plots
Genome_build: mm10 from 10x Genomics
Supplementary_files_format_and_content: Matrix table with raw gene counts for every gene and every sample
Supplementary_files_format_and_content: Matrix table with normalized gene counts for every gene and every sample

source_name: brain

extract_protocol:
tissue were microdissected and either flash froze for single nuclei isolation or taken straight to single cell isolation protocol.
RNA libraries were prepared for sequencing using standard Illumina protocols

value: brain

value: adult 8-12 weeks

value: wild type

tag: brain region

value: Primary Visual Cortex (V1)


title: KO_CMS_rep3

value: 10% cyclic mechanical stretch for 24h


title: KO_CMS_rep1


title: WT_SSt_rep2

description: processed data file: WT_genes_fpkm_expression.txt


title: WT_FBS_rep3


title: WT_SSt_rep1


title: GEX_Meningeals cells of 2 PBS & 2 LPS mice

city: Marseille

first: Rejane

last: Rua

state: PACA

address: Centre d'Immunologie de Marseille Luminy Parc Scientifique et technologique de Luminy 163 Avenue de Luminy Case 906

country: France

institute: CIML

description:
Meningeal cells were extracted as described in the Mononuclear cell isolation and Flow cytometry sections above (apart from sacrifice, which was performed using an overdose of Ketamine/Xylasine) and stained with Hoescht 33342 (10µg/ml, Sigma) and Draq7 (0.3µM, ThermoFischer). The 4 samples were differentially labelled using 4 Hashtags (TotalSeq-A, clone M1/42; 30-F11, Biolegend) at 0.1mg/ml for 15min at 4°C. After a wash, nucleated cells were isolated from 2 PBS or 2 LPS-injected mice (7 weeks-old males) at day 1 post-injection using a FACS Aria III SORP (BD) using a 100 µm nozzle. 52 000 cells of each sample were pooled, centrifuged at 350g for 3 minutes, and 35 000 cells of the pool were loaded on a Chromium Single Cell Instrument (10x Genomics) to generate single-cell gel beads-in-emulsion (GEM). GEM and scRNA-libraries were prepared using the Single Cell 3’v3 Gel Bead and Library Kit (10X Genomics, No. 1000092) and the Chromium i7 Multiplex kit (10x Genomics, No 120262) according to the manufacturer’s instructions. Briefly, GEM reverse-transcription incubation was performed in a 96-deep-well reaction module at 53 °C for 45 min, 85 °C for 5 min and ending at 4 °C. Next, GEMs were broken and complememtary DNA (cDNA) was cleaned up with DynaBeads MyOne Silane Beads (Thermo Fisher Scientific, No. 37002D) and SPRIselect Reagent Kit (Beckman Coulter, No. B23317). Fulllength, barcoded cDNA was PCR amplified with a 96-deep-well reaction module at 98 °C for 3 min, 11 cycles at 98 °C for 15 s, 63 °C for 20 s and 72 °C for 1 min, one cycle at 72 °C for 1 min and ending at 4 °C. Following cleaning up with the SPRIselect Reagent Kit and enzymatic fragmentation, library construction to generate Illumina-ready sequencing libraries was performed by the addition of R1 (read 1 primer), P5, P7, i7 sample index and R2 (read 2 primer) via endrepair, A-tailing, adapter ligation, post-ligation SPRIselect cleanup/size selection and sample index PCR. After generation of the gene expression and HTO libraries, the cDNA content of post-sample index PCR samples was analyzed using the 2100 BioAnalyzer (Agilent) and the samples were sequenced by the TGML platform (TAGC, Marseille). Sequencing libraries (molar ratio 92/8 for gene expression/HTO) were loaded on a NextSeq500 Illumina flow cell High output (75 cycles) with sequencing settings following the recommendations of 10x Genomics (read 1:28 cycles; read 2: 55 cycles; index i7: 8 cycles; index i5: no cycles; 1.8 pM loading concentration).
S003890_S1_L001

data_processing:
Raw fastq files from gene expression libraries were processed using Cell Ranger software, with alignment on the mm10 reference genome.
Genome_build: mm10
Supplementary_files_format_and_content: tsv and mtx files containing cell barcodes, features and count matrix for mRNA and HTO libraries, csv files containing gene UMI counts, csv files containing additional cell-specific metadata (QC values, index sorting parameters) for each filtered cell identified by a UniqueCellID.

molecule: polyA RNA

source_name: Meninge

extract_protocol:
Hundred μl of LPS (Sigma, 0.1 mg/ml)  were injected intraveinously via the retro-orbital vein.
Anesthetized mice received an intracardiac perfusion with PBS to remove contaminating erythrocytes. Leukocytes were isolated from the meninges using forceps to gently separate them from the underside of skull cap (the same method used to prepare meningeal whole mounts above). This was followed by enzymatic digestion in RPMI containing 2.5 mg/ml collagenase D (Roche) + 0.1 mg/ml DNase (Roche) for 30 min at 37 °C with gentle shaking every 10 min. Leukocytes were also isolated from the brain and the spleen as described. Following digestion, supernatants were isolated, washed, and finally passed through a 35-μm pore cell strainer. After this step, meningeal cells were used for staining. Cells from the brain, however, were resuspended in 40% Percoll (GE Healthcare) gradient in PBS1X and centrifuged to remove myelin and debris from the preparation. Afterwards, brain leukocytes were stained comparably to meningeal cells. Spleen samples were similarly digested. After straining, spleen leukocytes were treated with RBC1X to eliminate red blood cells, then washed and 1:20 of total cells were isolated for cell staining.

tag: cell types

value: Macrophages, Stromal, Mast cell, Lymphatics, B cell, ILCs, Osteoblasts, Neutrophils, Monocytes, NK, NKT, Schwann, Smooth muscle, DC, Neurons, Endothelium


title: WT_FBS_rep2


title: WT_CMS_rep3


title: WT_CMS_rep1


title: WT_CMS_rep2


title: KO_SSt_rep3


title: Lin- SVF cells from 12-month-old mice: sample3

value: Lin- SVF cells

value: 12 months


title: Organoid_day_60_Dex_Veh_rep4

city: Munich

first: Elisabeth B

last: Binder

address: Kraepelinstr. 2-10

country: Germany

institute: Max Planck Institute of Psychiatry

description:
MUC4910
Single-cell RNA sequencing (Wafergen iCell8).
Each GEO Sample includes DEX-treated organoids and VEH-treated organoids.

data_processing:
The initial quality check was performed using FastQC before demultiplexing the cells by their barcode with Je multiplexer version 1.0.6 requiring a perfect match of the sequence. Subsequent adaptor trimming was performed using cutadapt version 1.11.
For read alignment, the STAR module of the Cell Ranger 2.1.1 release with the corresponding reference index of GRCh38 version 1.2.0 was used, both provided by 10x Genomics.
Only uniquely aligned reads with a minimum overlap of 31 bp to the transcriptome were regarded for gene quantification with the featureCounts package version 1.6.4.
We converted Ensembl Gene IDs to Gene Symbols using the R package BED with database version UCB-Human 2019.10.11.
Genome_build: GRCh38
Supplementary_files_format_and_content: *.csv: Comma-separated text files with raw gene counts per sample.
Supplementary_files_format_and_content: *barcodes.tsv: Tab-delimited text files include valid barcodes per sample. These files make demultiplexing of the raw data files possible.

growth_protocol: Brain organoids were grown using an unpatterend protocol (Lancaster et al. 2014).

extract_protocol:
Single cells were dissociated using StemPro Accutase Cell Dissociation Reagent (Life Technologies), filtered through 30uM and 20uM filters (Miltenyi Biotec) and cleaned of debris using a Percoll (Sigma, P1644) gradient. Single cells were resuspended in ice-cold Phosphate-Buffered Saline (PBS) supplemented with 0.04% Bovine Serum Albumin and prepared for single-cell separation. All experiments were performed in quadruplicate at all three time points, with a paired design of a treatment (Dex) or control (Veh) condition per each experiment/iCell8 chip, for a total of 12 chips, using the iCELL8 Single-Cell System (Wafergen, Takara Bio) according to the manufacturer’s recommendations. Briefly, cells were stained with Propidium Iodide (for dead cells) and DAPI (for live cells) on ice for 5-10 minutes and dispensed in the loading plate. Following microfluidic separation, iCell8 chips were imaged using the built-in fluorescence microscope and snap-frozen and stored at -80C until library preparation.
Single-cell sequencing was performed according to the manufacturer’s guidelines using the iCell8 Chip and Reagent Kit using in-chip RT-PCR amplification chemistry (Wafergen, Takara Bio). On each chip, 2 samples were loaded in parallel (treatment and vehicle condition) and subsequenctly, based on fluorescence labeling, only wells containing live single cells were selected for library preparation.
All libraries were prepared using the Nextera XT DNA Library Preparation Kit and Nextera XT Index Kit (Illumina). Sequencing was performed on an Illumina HiSeq4000 (Illumina, San Diego, CA), 1 lane per chip, sequencing paired-end with 26nt/100nt configuration.

treatment_protocol: Treatment with Dexamethasone (100nM) or vehicle (DMSO) for 12 hours in culture.

value: brain organoid

value: 60 days

value: mixed sample (demultiplexed)

tag: replicate


title: Organoid_day_60_Dex_Veh_rep3

description:
MUC4909
Single-cell RNA sequencing (Wafergen iCell8).
Each GEO Sample includes DEX-treated organoids and VEH-treated organoids.


title: Organoid_day_90_Dex_Veh_rep2

description:
MUC4912
Single-cell RNA sequencing (Wafergen iCell8).
Each GEO Sample includes DEX-treated organoids and VEH-treated organoids.

value: 90 days


title: Organoid_day_90_Dex_Veh_rep4

description:
MUC4914
Single-cell RNA sequencing (Wafergen iCell8).
Each GEO Sample includes DEX-treated organoids and VEH-treated organoids.


title: Lin- SVF cells from 12-month-old mice: sample1

value: non-immune cells


title: Organoid_day_60_Dex_Veh_rep2

description:
MUC4908
Single-cell RNA sequencing (Wafergen iCell8).
Each GEO Sample includes DEX-treated organoids and VEH-treated organoids.


title: Organoid_day_60_Dex_Veh_rep1

description:
MUC4907
Single-cell RNA sequencing (Wafergen iCell8).
Each GEO Sample includes DEX-treated organoids and VEH-treated organoids.


title: Organoid_day_90_Dex_Veh_rep1

description:
MUC4911
Single-cell RNA sequencing (Wafergen iCell8).
Each GEO Sample includes DEX-treated organoids and VEH-treated organoids.


title: SUVH456-G0

city: Freising

first: Rashmi

middle: Rekha

last: Hazarika

address: Liesel Beckmann-Str-2

institute: Technical University of Munich

library_source: genomic

data_processing:
Run MethylStar pipeline (Shahryary et al., 2020)
Methylation state calls were obtained using METHimpute (Taudt et al., 2018)
Only cytosines with a maximum posterior probability of at least 0.99 were retained
Genome_build: A. thaliana
Supplementary_files_format_and_content: tab-delimited text files for methylome generated using METHimpute

molecule: genomic DNA

organism: Arabidopsis thaliana

source_name: leaf tissue

growth_protocol: MA_cmt3, and MA_suvh4/5/6 seeds were planted and grown under a 16-h light/8-h-dark cycle in growth chambers.

extract_protocol:
Samples were harvested from young above ground tissue. Tissue was flash frozen in liquid nitrogen and DNA was isolated using a Qiagen Plant DNeasy kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions.
MethylC-seq libraries were prepared according to the protocol described in Urich et al.2015. Libraries were sequenced to 150 bp per read.

tag: line

tag: generation


title: Organoid_day_30_Dex_Veh_rep2

description:
MUC4511
Single-cell RNA sequencing (Wafergen iCell8).
Each GEO Sample includes DEX-treated organoids and VEH-treated organoids.

value: 30 days


title: SUVH456-L8-G5

tag: genotype

tag: lineage


title: HTO_Meningeals cells of 2 PBS & 2 LPS mice

description:
Meningeal cells were extracted as described in the Mononuclear cell isolation and Flow cytometry sections above (apart from sacrifice, which was performed using an overdose of Ketamine/Xylasine) and stained with Hoescht 33342 (10µg/ml, Sigma) and Draq7 (0.3µM, ThermoFischer). The 4 samples were differentially labelled using 4 Hashtags (TotalSeq-A, clone M1/42; 30-F11, Biolegend) at 0.1mg/ml for 15min at 4°C. After a wash, nucleated cells were isolated from 2 PBS or 2 LPS-injected mice (7 weeks-old males) at day 1 post-injection using a FACS Aria III SORP (BD) using a 100 µm nozzle. 52 000 cells of each sample were pooled, centrifuged at 350g for 3 minutes, and 35 000 cells of the pool were loaded on a Chromium Single Cell Instrument (10x Genomics) to generate single-cell gel beads-in-emulsion (GEM). GEM and scRNA-libraries were prepared using the Single Cell 3’v3 Gel Bead and Library Kit (10X Genomics, No. 1000092) and the Chromium i7 Multiplex kit (10x Genomics, No 120262) according to the manufacturer’s instructions. Briefly, GEM reverse-transcription incubation was performed in a 96-deep-well reaction module at 53 °C for 45 min, 85 °C for 5 min and ending at 4 °C. Next, GEMs were broken and complememtary DNA (cDNA) was cleaned up with DynaBeads MyOne Silane Beads (Thermo Fisher Scientific, No. 37002D) and SPRIselect Reagent Kit (Beckman Coulter, No. B23317). Fulllength, barcoded cDNA was PCR amplified with a 96-deep-well reaction module at 98 °C for 3 min, 11 cycles at 98 °C for 15 s, 63 °C for 20 s and 72 °C for 1 min, one cycle at 72 °C for 1 min and ending at 4 °C. Following cleaning up with the SPRIselect Reagent Kit and enzymatic fragmentation, library construction to generate Illumina-ready sequencing libraries was performed by the addition of R1 (read 1 primer), P5, P7, i7 sample index and R2 (read 2 primer) via endrepair, A-tailing, adapter ligation, post-ligation SPRIselect cleanup/size selection and sample index PCR. After generation of the gene expression and HTO libraries, the cDNA content of post-sample index PCR samples was analyzed using the 2100 BioAnalyzer (Agilent) and the samples were sequenced by the TGML platform (TAGC, Marseille). Sequencing libraries (molar ratio 92/8 for gene expression/HTO) were loaded on a NextSeq500 Illumina flow cell High output (75 cycles) with sequencing settings following the recommendations of 10x Genomics (read 1:28 cycles; read 2: 55 cycles; index i7: 8 cycles; index i5: no cycles; 1.8 pM loading concentration).
S003891_2_Htlib


title: Organoid_day_30_Dex_Veh_rep3

description:
MUC4512
Single-cell RNA sequencing (Wafergen iCell8).
Each GEO Sample includes DEX-treated organoids and VEH-treated organoids.


title: WT_SSt_rep3

value: Bone marrow

value: mesenchymal stem cell

value: serum starvation for 24h


title: Organoid_day_30_Dex_Veh_rep1

description:
MUC4510
Single-cell RNA sequencing (Wafergen iCell8).
Each GEO Sample includes DEX-treated organoids and VEH-treated organoids.


title: KO_CMS_rep2

value: SD


title: SUVH456-L4-G13


title: SUVH456-L8-G16


title: Donor3-RIOK2 KO

first: Shrestha

last: Ghosh

state: Massachusetts

address: SM 752, Smith building, 1 Jimmy Fund Way

institute: Dana Farber Cancer Institute, Boston

data_processing:
The sequence reads were trimmed to remove possible adapter sequences and nucleotides with poor quality using Trimmomatic v.0.36. The trimmed reads were mapped to the Homo sapiens reference genome available on ENSEMBL using the STAR aligner v.2.5.2b to generate BAM files. Unique gene hit counts were calculated by using feature Counts from the Subread package v.1.5.2. Only unique reads that fell within exon regions were counted.
Genome_build: GRCh38.
Supplementary_files_format_and_content: tab-delimited text files include raw gene counts.

source_name: Primary human hematopoietic stem and progenitor cells

growth_protocol: Primary human HSPCs were cultured in expansion medium- StemSpan SFEM II medium supplemented with 1X CC100, 1% penicillin-streptomycin (P/S), 1% Glutamine and 10ng/ml TPO.

extract_protocol:
RNA was isolated from cells using the RNeasy Plus Micro kit (Qiagen) using manufacturer’s protocols. Genomic DNA (gDNA) was removed using gDNA eliminator spin columns, followed by isolation of total RNA using phenol-free RNeasy MinElute spin columns.
RNA sequencing libraries were prepared using the NEBNext Ultra RNA Library Prep Kit for Illumina following manufacturer’s instructions (NEB, Ipswich, MA, USA). Briefly, mRNAs were first enriched with Oligo(dT) beads. Enriched mRNAs were fragmented for 15 minutes at 94 °C. First strand and second strand cDNAs were subsequently synthesized. cDNA fragments were end repaired and adenylated at 3’ends, and universal adapters were ligated to cDNA fragments, followed by index addition and library enrichment by limited-cycle PCR. The sequencing libraries were validated on the Agilent TapeStation (Agilent Technologies, Palo Alto, CA, USA), and quantified by using Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA) as well as by quantitative PCR (KAPA Biosystems, Wilmington, MA, USA).
The sequencing libraries were clustered on 1 lane of a flowcell. After clustering, the flowcell was loaded on the Illumina HiSeq instrument (4000 or equivalent) according to manufacturer’s instructions. The samples were sequenced using a 2x150bp Paired End (PE) configuration. Image analysis and base calling were conducted by the HiSeq Control Software (HCS). Raw sequence data (.bcl files) generated from Illumina HiSeq was converted into fastq files and de-multiplexed using Illumina's bcl2fastq 2.17 software. One mismatch was allowed for index sequence identification.

treatment_protocol: After genome-editing, the cells were cultured in differentiation media containing IMDM reconstituted with 3% human AB serum, 2% human AB plasma, 1% P/S, 0.06% heparin solution, 1 ng/ml IL-3 , 10 ng/ml SCF, 200 ug/ml holo-transferrin and 3U/ml erythropoietin for 48 hours.

tag: individual

tag: cell type

value: RIOK2 KO


title: SUVH456-L4-G5


title: SUVH456-L8-G13


title: WT_FBS_rep1

tag: age

value: wild type

value: FBS for 24h


title: Donor2-RIOK2 KO

value: Primary human hematopoietic stem and progenitor cells


title: SUVH456-L4-G16


title: SUVH456-L8-G11


title: Donor1-RIOK2 KO


title: SUVH456-L4-G11


title: SUVH456-L4-G10


title: Organoid_day_90_Dex_Veh_rep3

description:
MUC4913
Single-cell RNA sequencing (Wafergen iCell8).
Each GEO Sample includes DEX-treated organoids and VEH-treated organoids.


title: SUVH456-L8-G9


title: Organoid_day_30_Dex_Veh_rep4

description:
MUC4513
Single-cell RNA sequencing (Wafergen iCell8).
Each GEO Sample includes DEX-treated organoids and VEH-treated organoids.


title: SUVH456-L8-G8


title: Donor3-RIOK2 KD

value: RIOK2 KD


title: SUVH456-L8-G3


title: SUVH456-L4-G8


title: Donor1-RIOK2 KD


title: MCF7 cells, shCTR9-Input-Rep1

city: Madison

first: NGAI TING

last: CHAN

state: WI

address: 4849 Sheboygan Ave

department: Oncology

institute: University of Wisconsin -- Madison

data_processing:
Basecalls performed using CASAVA version 1.8
ChIP sequencing reads that passed quality score were aligned to the human reference genome (hg38) using Bowtie2 (v2.4.1)
Only unique alignments that passed high mapping quality (MAPQ>30) were retained for downstream analysis. Duplicated reads and reads resides in hg38 blacklist regions (https://github.com/Boyle-Lab/Blacklist/tree/master/lists) were eliminated by sambamba tools (v0.7.1). Sequencing reads were also aligned to the Drosophila genome (dm6) and normalization factors (Rx factor) were calculated after removing multi-mapping and duplicated reads.
Bigwig files were generated at a resolution of 20bp using the bamCoverage utility from the deepTools (v3.4.3) suits and data were subsequently visualized as ChIP-Rx normalized tracks using IGV genome browser (v2.8.9).
Peaks were called using MACS2 (v2.1.4) with FDR<0.01 for narrow beaks and FDR<0.05 for broad peaks. Peaks intersection was performed by bedtools (v2.29.2) and bedops (v2.4.40). Overlapped peaks that shorter than sequencing tag (100bp) will be discarded.
Genome_build: hg38
Supplementary_files_format_and_content: bigwig  (a continous track representing the normalized ChIP-seq signal)

source_name: MCF7-shCTR9 + eGFP

growth_protocol: Cells are maintained in Dulbecco’s modified medium (DMEM) (Gibco) supplemented with 10% fetal bovine serum (FBS) (VWR) and 1% Penicillin-Streptomycin (P/S) (Gibco) and cultured at 37°C in a humidified atmosphere containing 5% CO2.

extract_protocol:
Lysates were clarified from sonicated nuclei and protein-DNA complexes were isolated with corresponding antibodies.
Libraries were prepared according to manufacturer’s protocol of  the Ovation Ultralow System V2 1-16 Kit (NuGEN Technologies).  Briefly, the DNA was end-repaired and ligated to Illumina sequencing adaptors. The ligated DNA was purified using Agencourt RNAClean XP beads (Beckman Coulter). A subsequent PCR amplification step (8-15 cycles) was performed to add linker sequence to the purified fragments for annealing to the Genome Analyzer flow-cell. Following PCR amplification, the library was separated on a 2% agarose gel (120 V, 1.5 hours) to select a narrow range of fragment sizes, and bands between 200-500 bp were excised. The library was purified from the excised agarose gel using the Qiagen MiniElute PCR Purification Kit following the manufacturer’s protocol.

tag: cell line

value: human luminal breast cancer cells

value: CTR9 KD with eGFP expression

tag: chip antibody

value: in regular DMEM medium


title: MCF7 cells, PRC2.1-OE-Input-Rep2

source_name: MCF7-shControl + PRC2.1-eGFP-OE

value: PRC2.1 -- MTF2&EPOP overexpression with eGFP reporter

tag: treatment


title: CMT3-L8-G16


title: CMT3-L8-G11


title: Donor2-RIOK2 KD


title: Donor3-Control

value: Control


title: CMT3-L10-G5


title: MCF7 cells, PRC2.1-OE-Input-Rep1


title: MCF7 cells, shCTR9-Input-Rep2


title: Donor1-Control


title: SUVH456-L4-G3


title: Donor2-Control


title: MCF7 cells, shPRC2.2-Input-Rep2

source_name: MCF7-shPRC2.2 + eGFP

value: PRC2.2 --JARID2&AEBP2 KD with eGFP expression


title: CMT3-L10-G1


title: CMT3-L8-G5


title: MCF7 cells, shPRC2.2-Input-Rep1


title: CMT3-L8-G1


title: Purified neurons 3

city: Chicago

first: Rajeshwar

last: Awatramani

address: 320 E. Superior Ave.

institute: Northwestern University

data_processing:
cellranger v3.0.2 was used for demultiplexing, alignment, and generating digital gene matrices for 10x single sequencing data. Sequences were aligned to GRCh38.
Genome_build: hg38
Supplementary_files_format_and_content: cellranger 3.0.2 generated gene barcode matrices files

source_name: Differentiated cells

extract_protocol:
For neural co-culture dissociation, cells were dissociated with TrypLE (Gibco) for 5 minutes 37º C. For purified neurons, cells were dissociated with 0.25% Trypsin-EDTA (Gibco) for 5 minutes 37º C. Dissociated cells were washed in PBS+10% FBS and stained with live/dead DAPI (0.5ug/ml) for 10min on ice. Cells were strained with a 40µ filter in flow buffer of PBS+2% FBS and live cells (neural co-culture) and live tdTomato+ cells (purified neurons) were sorted with FACS Aria for library preparation using Single Cell 3’ Reagent Kit v3 (10xGenomics, Chromium).
Samples for single-cell RNA-seq (scRNA-seq) was examined first for cell viability, density, and prep quality ensuring no clumps, cell debris, etc. Samples passing the QC process were loaded onto 10x Genomics Chromium for partitioning and encapsulation of single cells into nanoliter-sized GEMs (Gel beads-in-EMulsion). Each encapsulated cell was then lysed within its GEM. The total RNA released was reverse transcribed to cDNA with primers attached to the gel bead, each of which carries a unique 10x barcode for downstream cell separation. Subsequently the GEMs were broken, and all uniquely barcoded cDNAs were pooled and then PCR amplified to generate enough material for Illumina sequencing. This sequencing library construction process was carried out using the 10X Genomics Single Cell 3’ v3 Protocol. Sequencing of the 10x scRNA-seq libraries was performed on Illumina HiSeq 4000.
10x RNA seq


title: Purified neurons 2


title: PR 5

city: Houston

first: Anil

middle: K.

last: Sood

address: 1515 Holcombe Blvd

institute: MD Anderson Cancer Center

data_processing:
[count matrix output]  The Visium sequencing data were processed by spaceranger v1.1.0 with commands spaceranger mkfastq and spaceranger count. The count matrix output and image data were further analyzed by R package Seurat v4.0.1. Specifically, the matrices of each sample were loaded individually and the function SCTransform was used to perform data transformation
[remove batch effect]  To remove batch effects, the list of data from individual samples was processed by functions SelectIntegrationFeatures, PrepSCTIntegration, and FindIntegrationAnchors, and finally integrated into a single Seurat object by the function IntegrateData
[PCA and clusters]  The integrated data underwent principal component analysis, and 30 principal components were used for Uniform Manifold Approximation and Projection. Cluster-specific cell type scores were calculated by performing gene set variation analysis on the average expression matrix of clusters using R package GSVA v1.36.319. The pairwise distances between two clusters were calculated by median Euclidean distances of each spot pair on the slides in the two clusters.
[differential gene expression and LR analysis]  The differential gene expression analyses between two clusters were performed by the function FindMarkers(), and the resulting ranks were used for gene set enrichment analyses by R package fgsea v1.14.020. The ligand receptor interaction (SingleCellSignalR) scores were calculated by multiplying the average expression of the ligand and the receptor in the respective clusters and further normalized to [0,1], as performed in previous studies.
Genome_build: GRCh38
Supplementary_files_format_and_content: tab-delimited text files and matrix table with gene counts, image files

source_name: tumor tissue

growth_protocol: tissues were FFPE and sectioned

extract_protocol:
For spatial transcriptomics, we used the Visium Spatial Gene Expression Reagent Kit (10X Genomics). Tissue optimization was done using the Visium Spatial Tissue Optimization Slide & Reagent kit. The optimization, as well as the analysis, was performed using tissue at 10-µm thickness per the manufacturer’s protocol. The selected permeabilization time was 20 minutes. Bright-field images of the tissue were captured according to the requirements of the manufacturer, using a Nikon Eclipse Ni microscope and Nikon DS-Ri1 8-bit camera. The objective was a Nikon Plan Apo with 10× magnification and 0.45 numerical aperture. We used Nikon Elements software with a resolution of 0.91 µm/pixel. Camera settings were as follows: format 1280×1024 (fine), exposure 10.0 ms (±0.0 EV), analog gain 1.00.
The Visium Spatial Gene Expression Slide & Reagent kit was used for library construction. One section per patient (10 µm) was mounted into each capture area. Each capture area on a Visium for FFPE slide has an array containing capture probes that bind to RNA. The probe pairs are extended to incorporate complements of the spatial barcodes, and sequencing libraries are prepared. The libraries are then sequenced and data visualized to determine which genes are expressed and where, as well as in what quantity. A capture area is defined as a 6.5 × 6.5 mm area that contains 5,000 barcoded spots that are 55 µm in diameter and 100 µm center to center between spots, providing an average resolution of 1 to 10 cells (10X Genomics). All following steps (cDNA synthesis and amplification, library construction, and post-library construction quality control) were done according to the manufacturer’s protocol. The libraries were sequenced on a NovaSeq 6000 (Illumina).

treatment_protocol: the section undergoes hematoxylin and eosin staining

tag: tissue

value: high-grade serous ovarian tumor

tag: response to nact

value: Poor


title: Purified neurons 1


title: MCF7 cells, shControl-Input-Rep2

source_name: MCF7-shControl + eGFP

value: scramble control KD with eGFP expression


title: PR 6


title: MCF7 cells, shControl-Input-Rep1


title: PR 2


title: PR 3


title: Neural co-culture 3


title: PR 4


title: CMT3-L4-G5


title: Neural co-culture 1


title: Erythroleukemia cells (TF-1)- Input

description: Identifier: TF-1 (ATCC® CRL-2003™)

data_processing:
Sequencing depth was 2x150 Paired End (PE) configuration, a total of 30 million reads for each sample.
Image analysis and base calling were conducted by the HiSeq Control Software (HCS). Raw sequence data (.bcl files) generated from Illumina HiSeq was converted into fastq files and de-multiplexed using Illumina's bcl2fastq 2.17 software. One mis-match was allowed for index sequence identification.
Sequence reads were trimmed to remove possible adapter sequences and nucleotides with poor quality at 3’ end (error rate > 0.01) using CLC Genomics Server 9.0. Trimmed data was then aligned to reference genome for human genome GRCh38.
During the mapping, only specific alignment was allowed. After investigating the quality of the raw data, sequence reads were trimmed to remove possible adapter sequences and nucleotides with poor quality using Trimmomatic v.0.36. The trimmed reads were mapped to the Homo sapiens reference genome available on ENSEMBL using the STAR aligner v.2.5.2b.
BAM files were generated as a result of this step. Unique gene hit counts were calculated by using feature Counts from the Subread package v.1.5.2. Only unique reads that fell within exon regions were counted.
Genome_build: Homo Sapiens GRCh38
Supplementary_files_format_and_content: fastq files and .bam, .bam.bai files

source_name: Bone marrow-derived erythroblast cells

growth_protocol: TF-1 were cultured as per ATCC's instructions; cells were maintained in RPM1 media containing 10% FBA, 2ng/ml GM-CSF and penicillin-streptomycin. Typically 2-3 × 106 cells were maintained in a T75 flask as a suspension cell culture.

extract_protocol:
Lysates were clarified from sonicated nuclei and protein-DNA complexes were isolated with monoclonal antibodies against RIOK2.
Libraries were prepared according to Illumina's instructions accompanying the DNA Sample Kit. Briefly, DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (32 to 52 exo minus) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3’ end. After adapter ligation DNA was PCR amplified with Illumina primers for 15 cycles and library fragments of ~250 bp (insert plus adaptor and PCR primer sequences) were band isolated from an agarose gel. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Illumina HiSeq following the manufacturer's protocols.

treatment_protocol: No treatment was given to the cells, they were maintained at steady state before harvesting.

value: Erythroleukemia cells, TF-1

tag: antibody


title: MCF7 cells, shPRC2.2-SUZ12-Rep2

value: SUZ12 (Cell signaling technology, Cat# 3737, RRID:AB_2196850)


title: MCF7 cells, PRC2.1-OE-SUZ12-Rep1


title: MCF7 cells, PRC2.1-OE-SUZ12-Rep2


title: MCF7 cells, shCTR9-SUZ12-Rep1


title: Organoid_Line3_Veh_rep4

description:
HPS0076_16
Single-cell RNA sequencing (10x Genomics Chromium)

data_processing:
Count matrices were obtained from the fastq-files using Cell Ranger v3.0.2 with the transcriptome hg38_ensrel94.
For read alignment, the STAR module of the Cell Ranger 2.1.1 release with the corresponding reference index of GRCh38 version 1.2.0 was used, both provided by 10x Genomics.
We converted Ensembl Gene IDs to Gene Symbols using the R package BED with database version UCB-Human 2019.10.11.
Genome_build: GRCh38
Supplementary_files_format_and_content: barcodes.tsv: Cell Ranger output - barcodes files.
Supplementary_files_format_and_content: features.tsv: Cell Ranger output - features files.
Supplementary_files_format_and_content: matrix.mtx: Cell Ranger output - count matrix files.

source_name: Organoid

extract_protocol:
Single cells were dissociated using StemPro Accutase Cell Dissociation Reagent (Life Technologies), filtered through 30uM and 20uM filters (Miltenyi Biotec) and cleaned of debris using a Percoll (Sigma, P1644) gradient. Single cells were resuspended in ice-cold Phosphate-Buffered Saline (PBS) supplemented with 0.04% Bovine Serum Albumin and prepared for single-cell separation. All experiments were performed in quadruplicate using a pair-wise design with a treatment (Dex) or control (Veh) condition ran in parallel.
Single cells were run through the Chromium controller to form gel emulsion beads containing barcoded single cells and prepared into single-cell libraries using the Chromium Single Cell 3’ Reagent Kits v2 according to the manufacturer’s recommendations without any modifications (10X Genomics).
All libraries were assessed using a High Sensitivity DNA Analysis Kit for the 2100 Bioanalyzer (Agilent) and KAPA Library Quantification kit for Illumina (KAPA Biosystems). Sequencing of the 10x Genomics single-cell libraries was performed on an Illumina NovaSeq (Illumina, San Diego, CA), generating paired-end reads of 100 bp length.

value: brain organoid

value: 90 days

value: Veh

tag: timepoint

value: 12 hr


title: Neural co-culture 2

value: wild type


title: Organoid_Line3_Veh_rep3

description:
HPS0076_15
Single-cell RNA sequencing (10x Genomics Chromium)


title: Erythroleukemia cells (TF-1)- RIOK2 immunoprecipitated

description: Identifier: TF-1 (ATCC® CRL-2003™)

value: RIOK2 immunoprecipitated


title: ER 3

value: Excellent


title: ER 5


title: ER 4


title: ER 2


title: ER 1


title: MCF7 cells, shCTR9-SUZ12-Rep2

value: human luminal breast cancer cells

value: CTR9 KD with eGFP expression


title: MCF7 cells, shPRC2.2-SUZ12-Rep1

value: in regular DMEM medium


title: Organoid_Line3_Veh_rep1

description:
HPS0076_13
Single-cell RNA sequencing (10x Genomics Chromium)


title: Organoid_Line3_Dex_rep3

description:
HPS0076_11
Single-cell RNA sequencing (10x Genomics Chromium)

value: Dex


title: Organoid_Line3_Veh_rep2

description:
HPS0076_14
Single-cell RNA sequencing (10x Genomics Chromium)


title: Organoid_Line3_Dex_rep4

description:
HPS0076_12
Single-cell RNA sequencing (10x Genomics Chromium)


title: Organoid_Line3_Dex_rep2

description:
HPS0076_10
Single-cell RNA sequencing (10x Genomics Chromium)


title: Organoid_Line3_Dex_rep1

description:
HPS0076_9
Single-cell RNA sequencing (10x Genomics Chromium)


title: Primary human CD34+ RIOK2 KO rep2

data_processing:
Sequencing depth was 2x150 Paired End (PE) configuration, a total of 30 million reads for each sample.
Image analysis and base calling were conducted by the HiSeq Control Software (HCS). Raw sequence data (.bcl files) generated from Illumina HiSeq was converted into fastq files and de-multiplexed using Illumina's bcl2fastq 2.17 software. One mis-match was allowed for index sequence identification.
Sequence reads were trimmed to remove possible adapter sequences and nucleotides with poor quality at 3’ end (error rate > 0.01) using CLC Genomics Server 9.0. Trimmed data was then aligned to reference genome for human genome GRCh38.
During the mapping, only specific alignment was allowed. After investigating the quality of the raw data, sequence reads were trimmed to remove possible adapter sequences and nucleotides with poor quality using Trimmomatic v.0.36. The trimmed reads were mapped to the Homo sapiens reference genome available on ENSEMBL using the STAR aligner v.2.5.2b.
BAM files were generated as a result of this step. Unique gene hit counts were calculated by using feature Counts from the Subread package v.1.5.2.
Genome_build: Homo Sapiens hg19
Supplementary_files_format_and_content: .bam files

source_name: Primary human CD34+ hematopoietic stem and progenitor cells isolated from healthy donors

growth_protocol: Primary CD34+ hematopoietic stem and progenitor cells were expanded and differentiated, as previously described in Khajuria et al., CELL 2018.

extract_protocol:
Whole cell lysates were collected, chromatin extracted, followed by QC, library preparation and sequencing.
Libraries were prepared according to Illumina's instructions accompanying the DNA Sample Kit. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Illumina HiSeq following the manufacturer's protocols.

treatment_protocol: No treatment was given to the cells.

tag: differentiation

value: presence of RIOK2


title: Primary human CD34+ Control rep2

value: control


title: MCF7 cells, shControl-SUZ12-Rep1


title: MCF7 cells, shCTR9-H3K27me3-Rep2

value: H3K27me3 (Cell signaling technology, Cat# 9733, RRID:AB_916252)


title: Organoid_Line2_Veh_rep4

description:
FOK4_8
Single-cell RNA sequencing (10x Genomics Chromium)


title: Organoid_Line2_Veh_rep3

description:
FOK4_7
Single-cell RNA sequencing (10x Genomics Chromium)


title: Organoid_Line2_Veh_rep2

description:
FOK4_6
Single-cell RNA sequencing (10x Genomics Chromium)


title: Primary human CD34+ Control rep1


title: MCF7 cells, PRC2.1-OE-H3K27me3-Rep2

value: PRC2.1 -- MTF2&EPOP overexpression with eGFP reporter


title: Organoid_Line2_Dex_rep3

description:
FOK4_3
Single-cell RNA sequencing (10x Genomics Chromium)


title: MCF7 cells, shCTR9-H3K27me3-Rep1


title: PR 1

value: high-grade serous ovarian tumor

value: Poor


title: Organoid_Line2_Dex_rep2

description:
FOK4_2
Single-cell RNA sequencing (10x Genomics Chromium)

value: brain organoid


title: ChIP-Seq EPL_3_Ac

city: Poznan

first: Piotr

middle: A

last: Ziolkowski

address: Uniwersytetu Poznanskiego 6

department: Institute of Molecular Biology and Biotechnology

country: Poland

institute: Adam Mickiewicz University

data_processing:
Quality and adapter trimming as well as (RNA-Seq only) rRNA sequnce removal were performed using BBDuk version 38.57
Gene expression values (Araport11 annotations) were estimated from RNA-Seq data using Salmon version 0.13.1
ChIP-Seq reads were mapped to the reference genome (TAIR10) and spike-in genome (GRCm38) using BWA version 0.7.17
Normalized and smoothed genome coverage profiles were calculated using DANPOS 2.2.2
Genome_build: TAIR10
Supplementary_files_format_and_content: Tab-delimited text files containing expression values of genes (TPM values and raw estimated read counts)
Supplementary_files_format_and_content: Tab-delimited text file containing genome mapping statistics for ChIP-Seq samples, used to correct genome coverage values for plotting
Supplementary_files_format_and_content: Wig files storing normalized and smoothed genome coverage data calculated by DANPOS

source_name: Leaves

growth_protocol: WT and mutant plants were grown under LD conditions (16 h light and 8 h darkness), under 80-90 µmol/(m2s) of photosynthetically active radiation. Material was collected at the developmental stage of ca. 10 leaves about 5 hours after light onset.

extract_protocol:
RNA for each biological repeat was extracted from 110 mg of rosette leaves number 5-6 (from at least six plants) with TRI Reagent (Merck) and rounds of phenol-chloroform and chloroform extractions followed by isopropanol precipitation. RNA was treated with RQ1 DNase I (Promega), then extracted with phenol-chloroform and precipitated with ethanol. Chromatin was isolated as previously described (Sura et al. Plant Cell 2017), enriched with 2% chromatin isolated from mouse embryonic fibroblasts as spike-in control, and incubated with antibody (α-H3, Abcam ab1791; α-H4K5ac, Millipore 07327; α-H3K9ac Millipore 07352; α-HTA9, Agrisera AS10718; α-H2A.Zac, Diagenode C15410173 pAb-173-050) overnight at 4°C. Dynabeads Protein A (Thermo Fisher Scientific) were added to the samples and incubated for another 2 hours at 4°C. The slurry was washed twice with low salt buffer (with 150 mM NaCl), twice with high salt buffer (with 500 mM NaCl) and once with LiCl wash buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 0.25 M LiCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 1 mM PMSF and protease inhibitor cocktail). Chromatin was eluted through incubation in 1% SDS and 0.8 M NaHCO3 at 65°C and the cross-links were reversed by overnight incubation at 65°C. Following Proteinase K treatment and phenol/chloroform extraction, DNA was precipitated and DNA concentration was measured with Qubit dsDNA HS Kit (Thermo Fisher Scientific).
Pure RNA solution was sent to Macrogen Europe (South Korea), where libraries were performed using TruSeq Stranded Total RNA sample preparation with Ribo-Zero Plant. Libraries were sequenced on the Illumina HiSeq 4000, 2x100bp run. 10 ng (H3) or 2 ng (other antibodies) of ChIP-ed DNA was used to prepare DNA libraries with MicroPlex Library Preparation Kit v2 (Diagenode). The library preparation was carried out accoring to the manufacturer (Diagenode) protocol.

value: Leaves

value: epl

value: H2AZac (Diagenode C15410173)


title: Organoid_Line2_Dex_rep4

description:
FOK4_4
Single-cell RNA sequencing (10x Genomics Chromium)

value: 90 days

value: 12 hr


title: ER 6

value: Excellent


title: ChIP-Seq EPL_4_T

value: H2AZ (Agrisera AS10718)


title: MCF7 cells, PRC2.1-OE-H3K27me3-Rep1

value: human luminal breast cancer cells


title: ChIP-Seq EPL_2_T


title: MCF7 cells, shPRC2.2-H3K27me3-Rep2

value: PRC2.2 --JARID2&AEBP2 KD with eGFP expression

value: in regular DMEM medium


title: Patient 25 cell line sample

city: Little Rock

first: Analiz

last: Rodriguez

state: AR

address: 4301 W. Markham St Slot 507

department: College of Medicine, Department of Neurosurgery

institute: University of Arkansas for Medical Sciences

data_processing:
RNA-Seq data were uniformly processed using the Toil RNA-Seq pipeline version 3.2 developed by the UCSC Computational Genomics Lab as described in the expression section of the pipelines repository https://github.com/UCSC-Treehouse/pipelines. These steps include:
Adapters are removed with CutAdapt v1.9 (Martin, 2011)
Reads are aligned by STAR v 2.4.2a using indices generated from the human reference genome GRCh38 and the human gene models Gencode 23 (Dobin et al., 2013)
RSEM 1.2.25 is used to quantify gene expression (Li and Dewey, 2011)
Gene level expression is in TPM
Genome_build: GRCh38
Genome_build: Gencode 23
Supplementary_files_format_and_content: Tab separated column of genes with the expression level estimate for each gene.

source_name: glioblastoma cells

growth_protocol: For clonogenic assays, cells were cultured in minimum essential media (MEM) containing 10% fetal bovine serum (FBS) and 1% (v/v) antibiotic/antimitotic. For organoid mini-ring assays, cells were cultured in Neurocult NS-A complete media (Stemcell Technologies, # 05751, Vancouver, Canada) containing NS-A proliferation supplement, 20 ng/mL human recombinant epidermal growth factor (EGF) (Stemcell Technologies, # 78006), 10 ng/mL human recombinant basic fibroblast growth factor (bFGF) (Stemcell Technologies, #78003), 0.0002% heparin (Stemcell Technologies, #07980) and 1% (v/v) antibi-otic/antimitotic. Cells were incubated at 37°C in 5% CO2.

extract_protocol:
Total RNA was extracted from cell pellets lysed in 600ul RNA Lysis Buffer using the Quick-RNA Miniprep Kit (Zymo Research).
Illumina RNA library preparation and sequencing was performed by the Vincent J. Coates Genomics Sequencing Laboratory at UC Berkeley. Total RNA quantity and quality was measured on a NanoDrop One (Thermo Scientific) and a Bioanalyzer 2100 (Agilent Technologies). An input amount of 500ng high quality total RNA (RIN score > 8.0 for all samples) was used for library preparation. Libraries were created using the Kapa Biosystems library preparation kit with custom Unique Dual Indexes and analyzed on a Bioanalyzer 2100 (Agilent Technologies). Samples were pooled on a 150PE NovaSeq S4 flowcell and run on a NovaSeq platform with a throughput of 50M reads per sample.

tag: donor

value: Patient 25

tag: sample type

value: patient derived cell line

tag: Sex

value: F

tag: cancer predisposition

tag: diagnosis

value: glioma

tag: treehouse id

tag: tumor grade


title: Patient 17 cell line sample

value: Patient 17

value: M


title: MCF7 cells, shControl-SUZ12-Rep2

tag: cell type

tag: genotype

value: scramble control KD with eGFP expression

value: SUZ12 (Cell signaling technology, Cat# 3737, RRID:AB_2196850)


title: ChIP-Seq WT_3_Ac

value: Leaves

value: WT


title: CUT&RUN_input_293T

first: Daniel

last: Foltz

address: 303 E Superior St

data_processing:
Library strategy: CUT&RUN
Raw BCL output files were processed using bclfastq (Illumina, version 2.17.1.14).
For ChIP-Seq, trimmed reads were aligned to the human genome (UCSC hg19) with bowtie (Langmead & Salzberg, 2012; Langmead et al, 2019). peaks were called from GFP-IP bam files using input bam files as control with macs2 (Zhang et al, 2008). To compare GFP-UBR7 ChIP-seq signal to changes in ATAC-seq and CUT&RUN signals at non-redundant TSS, deepTools (Ramirez et al, 2016) bamCompare was used to calculate the log2 fold change of GFP-IP versus input from GFP-UBR7 293T cells. DeepTools plotHeatmap was used to visualize the log2 fold changes >0 centered on TSS as clustered in ATAC-seq analysis.
For ATAC-seq, fastq files were trimmed and analyzed with trim_galore (https://github.com/FelixKrueger/TrimGalore). Fastq files across lanes within replicate were merged, then aligned to the human genome (UCSC hg19) using bowtie2 (Langmead & Salzberg, 2012; Langmead et al., 2019) allowing up to one mismatch resulting in >50% aligned reads in all samples. multimapping reads were removed with samtools (option -q 5), and mitochondrial genome reads were removed with the sed command. Duplicate reads were removed with Picard (Picard toolkit, 2019). All bam files were downsampled to the number of reads in the smallest library, sorted, and indexed with samtools. Peaks were called using macs2 in broad mode (Tripodi et al, 2018; Zhang et al., 2008). Bedtools (Quinlan & Hall, 2010) was used to identify and merge (sum) the macs2 scores across intersecting peaks between biological replicates, then convert the resulting bed files to bam files, which were sorted and indexed with samtools. DeepTools (Ramirez et al., 2016) bamCompare was used to calculate the log2 fold change of UBR7KO versus parental 293T cells. Unique TSS were derived by first building a bed file using the “chrom” and “txn start” columns of NCBI RefSeq GRCh37/hg19 file downloaded from UCSC Table Browser (http://genome.ucsc.edu/cgi-bin/hgTables) followed by a column of values (“txn start” +1). Bedtools merge was used to collapse redundant TSS. DeepTools plotHeatmap was used to visualize the log2 fold changed centered on non-redundant TSS using unsupervised kmeans clustering to divide the data into 4 clusters. A bed file of this sorted clustering was used to plot heatmaps of GFP-UBR7 ChIP-seq and H3K4me3 CUT&RUN data at the same sites.
For RNA-Seq, trimmed reads were aligned to the human genome (UCSC hg19) with TopHat (version 2.1.0) (Trapnell et al, 2009) with gene annotations sourced from Ensembl release 72 assigned using Python package HTSeq 0.6.1 (Anders et al, 2015). Differentially expressed reads between all three UBR7KO versus all three parental 293T samples were identified using the exact test in EdgeR (McCarthy et al, 2012; Robinson et al, 2010).
For CUT&RUN, Raw sequencing data was demultiplexed using bcl2fastq2 v2.19.1. Reads were trimmed for adaptor sequences using trimmomatic v0.36 and then aligned to concatenated hg38/Genome Reference Consortium Human Reference 38 Patch 15 (GRCh38.p15) and dm6/BDGP Release 6 + ISO1 MT assemblies of the human and Drosophila melanogaster genomes, respectively, using bowtie2 v2.3.1 with options --local --very-sensitive-local --no-unal --no-mixed --no-discordant --phred33. Only reads with a MAPQ ≥ 30 were retained using samtools v1.3.1. PCR duplicates were removed with picard MarkDuplicates v2.9.2. Coverage-based tracks were generated with deepTools bamCoverage with options --binSize 1 --normalizingUsing RPGC --effectiveGenomeSize 2800000000 --extendReads --samFlagInclude 64. CUT&RUN profiles were then calibrated by spike-in normalization using Drosophila chromatin as the internal reference or spike-in, analogous to that done for ChIP-seq (Orlando et al.), to determine the normalization coefficient or Occupancy Ratio (OR). Specifically, the OR was calculated as the product of IgG spike-in reads and immunocleaved (IC) human reads, divided by the product of IgG human reads and IC spike-in reads. Spike-in normalized tracks were generated with deepTools bamCoverage with options --binSize 1 --normalizeUsing RPGC --effectiveGenomeSize 2800000000 --extendReads --samFlagInclude 64 --scaleFactor OR. OR-normalized tracks were remapped to genome build hg19/GRCh37. BigWig files were converted to bedGraph using UCSC binary utils (http://hgdownload.soe.ucsc.edu/admin/exe/linux.x86_64/) bigWigToBedGraph . BedGraph files were remapped to hg19 using UCSC binary utils liftOver with options -positions -bedPlus=4 and hg38ToHg19.over.chain file. Resulting bedGraph files were sorted using LC_Collate=C sort with options -k1,1 -k2,2n. Overlapping coordinates were removed with UCSC binary utils bedRemoveOverlap. Resulting bedGraph files were converted to hg19 mapped tracks using UCSC binary utils bedGraphToBigWig. DeepTools bigWigCompare was used to calculate the log2 fold change of H3K4me3 using either the Millipore or Diagenode antibody (Supplementary Table S3) in UBR7KO versus parental 293T cells. DeepTools plotHeatmap was used to visualize the log2 fold changed centered on TSS as clustered in ATAC-seq analysis
Genome_build: hg19
Supplementary_files_format_and_content: Bed files contain lists of coordinates for mapping data in bigWig files, or conserved sum peaks from biological replicates ("interR1R2" for ATACseq). log2FC bigWig files contain tracks of comparisons generated with deepTools bamCompare, from bam files of IP v Input (ChIPseq) or UBR7KO v parental (ATACseq), or bigWigCompare, from bigWig files of UBR7KO v parental (CUT&RUN). Non-log2FC bigWig files contain tracks generated using bamCoverage. BroadPeak files contain peaks called from alignments using macs2. Tab-delimited text file contains normalized counts for each sample and log2FC differential gene expression.

growth_protocol: Cells were grown in DMEM with 10% fetal bovine serum and 1% Pen/Strep in a 37°C incubator with 5% CO2

extract_protocol:
The ProteinA-MNase (pA-MNase) was purified in E. coli cells as described previously using a protocol adapted from Schmidt et al, 2014. Parental and UBR7KO 293T cells or Drosophila Kc167 spike-in cells were harvested at RT and resuspended in Wash Buffer (20 mM HEPES-NaOH pH 7.5, 150 mM NaCl, 0.5 mM spermidine, 1x Roche protease inhibitor cocktail) and counted using a Vi-Cell Blu cell counter. For each antibody, 4e5 293T cells were mixed with 1e5 Kc167 cells, then incubated for 7m with 10uL of Concanavalin A coated magnetic beads (BioMag) in Binding Buffer (20 mM HEPES-KOH pH 7.9, 10 mM KCl, 1 mM CaCl2, 1 mM MnCl2). The bead-cell slurry was permeabilized in Wash Buffer supplemented with 0.005% digitonin (“Digitonin Buffer”) as determined by optimization, then incubated with anti-H3K4me3 antibody, or IgG as a control, (1:50) in Antibody Buffer (20 mM HEPES-NaOH pH 7.5, 150 mM NaCl, 0.5 mM spermidine, 0.005% digitonin, 2 mM EDTA, 0.1% BSA, 100 nM TSA, 0.1 unit/mL citrate synthase, 1 mM Oxaloacetic acid, 1x Roche protease inhibitor cocktail) for 2 h at RT with nutation. The beads were washed twice in Digitonin Buffer, then incucbated in 100uL Digintonin buffer with 700 ng/mL pA-MNase for 1 h at 4°C with nutation. The beads were washed twice in Digitonin Buffer, then resuspended in a low-salt Wash Buffer (20 mM HEPES-NaOH pH 7.5, 0.5 mM spermidine, 0.005% digitonin, 1x Roche protease inhibitor cocktail) and chilled to 0°C. The beads were then incubated with ice-cold Calcium Incubation Buffer (3.5 mM HEPES-NaOH pH 7.5, 10 mM CaCl2, 0.005% digitonin) for 30 m. The beads were washed with EGTA-Stop Buffer (170 mM NaCl, 20 mM EGTA, 0.005% digitonin, 50 µg/mL RNase A) for 30m at 37°C to release the chromatin fragments. To extract the DNA, 200ul of Oligo Binding Buffer (Zymo) was added to each sample and the total volume was then added to a Zymo-Spin DCC-5 column and centrifuged. The column was washed twice with DNA Wash buffer from the DNA Clean & Concentrator-5 Kit (Zymo). The DNA was eluted from the column in 15ul of DNA Elution Buffer and sample concentration was measured using a Qubit 3.0 fluorometer and Qubit dsDNA HS Assay Kit.
Illumina libraries were prepared using a NEBNext Ultra II Library Prep Kit for Illumina and NEBNext® Multiplex Oligos for Illumina® (Dual Index Primers Set 1) using a protocol adapted from the manufacturer. Modifications to the protocol are as follows: The End Prep heat inactivation step was conducted at 50 °C for 60 minutes. The NEBNext Adaptor was diluted 1:25 in 10 mM Tris-HCl, pH 8.0 with 10 mM NaCl. After Adaptor Ligation, a DNA cleanup using 1.75x volumes of Sera-Mag Select beads was conducted. The PCR cycle conditions were programmed as following: 1 cycle: 98° C for 30 seconds, 12 cycles: 98° C for 10 seconds, 65° C for 30 seconds, 1 cycle: 65° C for 5 minutes, Hold: 4° C. After PCR amplification, DNA cleanup was conducted twice with 1.2x volumes of Sera-Mag Select (Cytiva) beads. Quality control was conducted on the resulting 15ul elution using Agilent 4200 TapeStation D1000 ScreenTape to determine sample concentration and sample quality.

tag: cell line

value: Human embryonic kidney cells

tag: genotype (crispr-cas9)

value: parental


title: CUT&RUN_input_UBR7KO


title: MCF7 cells, shControl-H3K27me3-Rep2

tag: chip antibody

value: H3K27me3 (Cell signaling technology, Cat# 9733, RRID:AB_916252)

tag: treatment


title: Primary human CD34+ RIOK2 KO rep1

value: presence of RIOK2


title: Patient 23 cell line sample

value: Patient 23

tag: age


title: CUT&RUN_H3K4me3diagenode_293T

description:
log2FC_KOvWT_spikeInNorm_diag.bw
CR0308_spikeInNorm_hg19.bw


title: RNAseq_UBR7KO2_rep1

description:
UBR7KO.v.293T.611-207-227.b.htseq.edgeR.txt
AH05B1

data_processing:
Raw BCL output files were processed using bclfastq (Illumina, version 2.17.1.14).
For ChIP-Seq, trimmed reads were aligned to the human genome (UCSC hg19) with bowtie (Langmead & Salzberg, 2012; Langmead et al, 2019). peaks were called from GFP-IP bam files using input bam files as control with macs2 (Zhang et al, 2008). To compare GFP-UBR7 ChIP-seq signal to changes in ATAC-seq and CUT&RUN signals at non-redundant TSS, deepTools (Ramirez et al, 2016) bamCompare was used to calculate the log2 fold change of GFP-IP versus input from GFP-UBR7 293T cells. DeepTools plotHeatmap was used to visualize the log2 fold changes >0 centered on TSS as clustered in ATAC-seq analysis.
For ATAC-seq, fastq files were trimmed and analyzed with trim_galore (https://github.com/FelixKrueger/TrimGalore). Fastq files across lanes within replicate were merged, then aligned to the human genome (UCSC hg19) using bowtie2 (Langmead & Salzberg, 2012; Langmead et al., 2019) allowing up to one mismatch resulting in >50% aligned reads in all samples. multimapping reads were removed with samtools (option -q 5), and mitochondrial genome reads were removed with the sed command. Duplicate reads were removed with Picard (Picard toolkit, 2019). All bam files were downsampled to the number of reads in the smallest library, sorted, and indexed with samtools. Peaks were called using macs2 in broad mode (Tripodi et al, 2018; Zhang et al., 2008). Bedtools (Quinlan & Hall, 2010) was used to identify and merge (sum) the macs2 scores across intersecting peaks between biological replicates, then convert the resulting bed files to bam files, which were sorted and indexed with samtools. DeepTools (Ramirez et al., 2016) bamCompare was used to calculate the log2 fold change of UBR7KO versus parental 293T cells. Unique TSS were derived by first building a bed file using the “chrom” and “txn start” columns of NCBI RefSeq GRCh37/hg19 file downloaded from UCSC Table Browser (http://genome.ucsc.edu/cgi-bin/hgTables) followed by a column of values (“txn start” +1). Bedtools merge was used to collapse redundant TSS. DeepTools plotHeatmap was used to visualize the log2 fold changed centered on non-redundant TSS using unsupervised kmeans clustering to divide the data into 4 clusters. A bed file of this sorted clustering was used to plot heatmaps of GFP-UBR7 ChIP-seq and H3K4me3 CUT&RUN data at the same sites.
For RNA-Seq, trimmed reads were aligned to the human genome (UCSC hg19) with TopHat (version 2.1.0) (Trapnell et al, 2009) with gene annotations sourced from Ensembl release 72 assigned using Python package HTSeq 0.6.1 (Anders et al, 2015). Differentially expressed reads between all three UBR7KO versus all three parental 293T samples were identified using the exact test in EdgeR (McCarthy et al, 2012; Robinson et al, 2010).
For CUT&RUN, Raw sequencing data was demultiplexed using bcl2fastq2 v2.19.1. Reads were trimmed for adaptor sequences using trimmomatic v0.36 and then aligned to concatenated hg38/Genome Reference Consortium Human Reference 38 Patch 15 (GRCh38.p15) and dm6/BDGP Release 6 + ISO1 MT assemblies of the human and Drosophila melanogaster genomes, respectively, using bowtie2 v2.3.1 with options --local --very-sensitive-local --no-unal --no-mixed --no-discordant --phred33. Only reads with a MAPQ ≥ 30 were retained using samtools v1.3.1. PCR duplicates were removed with picard MarkDuplicates v2.9.2. Coverage-based tracks were generated with deepTools bamCoverage with options --binSize 1 --normalizingUsing RPGC --effectiveGenomeSize 2800000000 --extendReads --samFlagInclude 64. CUT&RUN profiles were then calibrated by spike-in normalization using Drosophila chromatin as the internal reference or spike-in, analogous to that done for ChIP-seq (Orlando et al.), to determine the normalization coefficient or Occupancy Ratio (OR). Specifically, the OR was calculated as the product of IgG spike-in reads and immunocleaved (IC) human reads, divided by the product of IgG human reads and IC spike-in reads. Spike-in normalized tracks were generated with deepTools bamCoverage with options --binSize 1 --normalizeUsing RPGC --effectiveGenomeSize 2800000000 --extendReads --samFlagInclude 64 --scaleFactor OR. OR-normalized tracks were remapped to genome build hg19/GRCh37. BigWig files were converted to bedGraph using UCSC binary utils (http://hgdownload.soe.ucsc.edu/admin/exe/linux.x86_64/) bigWigToBedGraph . BedGraph files were remapped to hg19 using UCSC binary utils liftOver with options -positions -bedPlus=4 and hg38ToHg19.over.chain file. Resulting bedGraph files were sorted using LC_Collate=C sort with options -k1,1 -k2,2n. Overlapping coordinates were removed with UCSC binary utils bedRemoveOverlap. Resulting bedGraph files were converted to hg19 mapped tracks using UCSC binary utils bedGraphToBigWig. DeepTools bigWigCompare was used to calculate the log2 fold change of H3K4me3 using either the Millipore or Diagenode antibody (Supplementary Table S3) in UBR7KO versus parental 293T cells. DeepTools plotHeatmap was used to visualize the log2 fold changed centered on TSS as clustered in ATAC-seq analysis
Genome_build: hg19
Supplementary_files_format_and_content: Bed files contain lists of coordinates for mapping data in bigWig files, or conserved sum peaks from biological replicates ("interR1R2" for ATACseq). log2FC bigWig files contain tracks of comparisons generated with deepTools bamCompare, from bam files of IP v Input (ChIPseq) or UBR7KO v parental (ATACseq), or bigWigCompare, from bigWig files of UBR7KO v parental (CUT&RUN). Non-log2FC bigWig files contain tracks generated using bamCoverage. BroadPeak files contain peaks called from alignments using macs2. Tab-delimited text file contains normalized counts for each sample and log2FC differential gene expression.

extract_protocol:
RNA was isolated from 1 million cells using the Qiagen RNeasy Mini kit. RNA quality was assessed at >9 on the Bioanlayzer 2100 using the Eukaryote Total RNA Nano kit (Agilent 5067-1511).
1ug of RNA was used as input for library preparation using the Illumina TruSeq LS protocol (Illumina 15031048).


title: CUT&RUN_H3K4me3millipore_293T

description:
log2FC_KOvWT_spikeInNorm.bw
CR0305_spikeInNorm_hg19.bw


title: ChIP-Seq WT_1_Ac

tag: tissue

value: H2AZac (Diagenode C15410173)


title: CUT&RUN_H3K4me3diagenode_UBR7KO

description:
log2FC_KOvWT_spikeInNorm_diag.bw
CR0314_spikeInNorm_hg19.bw


title: ChIP-Seq WT_1_T

value: H2AZ (Agrisera AS10718)


title: P1_NT

city: Minato-ku

first: Tomoko

last: Miyake

state: Tokyo

address: 7-1-19 Akasaka

department: Cosmetic R&D

country: Japan

institute: Takara Belmont Corp.

data_processing:
Illumina RtaVersion v3.3.3 software used for basecalling.
FASTQ files were imported into the Illumina BaseSpace Sequence Hub.
Adapter, Low-quality bases (score <30), and poly-A/T tail (length >10) trimmed from both ends of the reads using FASTQ Toolkit.
Reads shorter than 32 bases after trimming were removed. Quality check were performed using FASTQC.
Differential gene expression analysis by DESeq2 was performed using RNA-Seq Differential Expression.
Genome_build: hg19
Supplementary_files_format_and_content: CSV files contain comparison of differential gene expression between untreated and AGR-treated samples in fibroblasts, iPSCs, and keratinocytes respectively.

source_name: Keratinocytes

growth_protocol: NB1RGB fibroblasts were cultured with DMEM supplemented with 10% FBS and penicillin and streptomycin, C2 iPSCs derived NB1RGB fibroblasts were cultured with Nutristem XF/FF culture medium, P1 keratinocytes derived from C2 iPSCs were cultured with DKSFM supplemented with EGF and Y27632.

extract_protocol:
Total RNA was extracted using FastGene RNA premium kit (Nippon Genetics).
Illumina TruSeq stranded mRNA Library Kit was used for the construction of sequencing libraries. RNA libraries were prepared for sequencing using standard illumina protocols.

treatment_protocol: Each cells were treated with 50 μM AGR for 4 hours.

value: iPS cells-derived keratinocytes

tag: passage

value: P1 (after differentiation)

value: untreated


title: P1_AGR

value: treated with 50 mM AGR for 4 hours


title: Ventral Midbrain

description: 10x seq output

tag: strain

value: brain

value: adult 8-12 weeks

value: wild type

tag: brain region

value: Ventral Midbrain


title: CUT&RUN_H3K4me3millipore_UBR7KO

description:
log2FC_KOvWT_spikeInNorm.bw
CR0311_spikeInNorm_hg19.bw


title: Organoid_Line2_Veh_rep1

description:
FOK4_5
Single-cell RNA sequencing (10x Genomics Chromium)

value: brain organoid

value: 90 days

value: Veh

tag: timepoint

value: 12 hr

tag: line

tag: replicate


title: C2_ARG

source_name: iPSCs

value: Fibroblasts-derived iPSCs

value: P2-5 (after reprogramming)


title: ChIP-Seq EPL_2_Ac

value: Leaves

value: epl


title: Superior Colliculus (SC)

description: 10x seq output

source_name: brain

value: Superior Colliculus (SC)


title: RNA-Seq GLK_2

value: glk


title: RNAseq_293T_rep3

description:
UBR7KO.v.293T.611-207-227.b.htseq.edgeR.txt
AH293T

value: Human embryonic kidney cells

value: parental


title: Patient 1 cell line sample

tag: donor

value: Patient 1

tag: sample type

value: patient derived cell line

tag: Sex

value: M

tag: cancer predisposition

value: Li-Fraumeni syndrome

tag: diagnosis

value: glioma


title: Organoid_Line2_Dex_rep1

description:
FOK4_1
Single-cell RNA sequencing (10x Genomics Chromium)

value: brain organoid

value: Dex


title: MCF7 cells, shPRC2.2-H3K27me3-Rep1

tag: cell line

tag: cell type

value: human luminal breast cancer cells

tag: genotype

value: PRC2.2 --JARID2&AEBP2 KD with eGFP expression

value: H3K27me3 (Cell signaling technology, Cat# 9733, RRID:AB_916252)

value: in regular DMEM medium


title: C2_NT

value: Fibroblasts-derived iPSCs

value: P2-5 (after reprogramming)

tag: treatment

value: untreated


title: RNA-Seq GLK_1

value: Leaves

value: glk


title: ChIP-Seq WT_3_T

value: WT

tag: chip antibody

value: H2AZ (Agrisera AS10718)


title: NB1RGB_NT

source_name: Fibroblasts

value: Skin fibroblasts


title: RNAseq_293T_rep1

description:
UBR7KO.v.293T.611-207-227.b.htseq.edgeR.txt
293T_RNA_101515

value: Human embryonic kidney cells

tag: genotype (crispr-cas9)

value: parental


title: RNAseq_293T_rep2

description:
UBR7KO.v.293T.611-207-227.b.htseq.edgeR.txt
293T_1_20151203


title: RNAseq_UBR7KO1_rep2

description:
UBR7KO.v.293T.611-207-227.b.htseq.edgeR.txt
UBR7_KO_1_20151203


title: RNA-Seq GLK_3

tag: tissue


title: RNA-Seq EAF_3

value: eaf


title: Intestine treated (Ssrp1 KO)

city: Buffalo

first: Katerina

last: Gurova

state: NY

address: Elm and Carlton Str

department: Cell Stress Biology

institute: Roswell Park Cancer Institute

data_processing:
cellranger was used to generate the fastq files using mkfastq
cellranger was used to generate the barcode and gene expression count matrix using mouse mm10 reference genome
Seurat R package was used for downstream analysis.
Genome_build: mm10
Supplementary_files_format_and_content: gene expression count matrix

source_name: intestine

extract_protocol:
For bone marrow samples, mouse femurs were cleaned with alcohol, washed with ice-cold PBS containing antibiotics, and the metaphysis removed. The marrow was flushed with buffer (2 mM EDTA + 0.5% BSA in PBS) using a 23G syringe. The cells were gently mixed, filtered through a 40-mm cell strainer, and centrifuged at 300 g for 10 min. The pellets were resuspended in 0.04% BSA-PBS and counted using trypan blue. A total of 1 x 105 cells were used for the analysis (1000 cells/mL). For the intestinal samples, 2 cm of intestine above and below cecum, excluding cecum, were excised and flushed with ice-cold PBS containing gentamycin using a 20G needle. The intestine was placed in a dish with fresh PBS and cut open longitudinally. The walls were washed to remove any residual matter. The intestine was incubated in ice-cold PBS containing 10 mM EDTA (10 mL) at 4⁰C with gentle rocking for 30 min. The tissue was into ice-cold PBS and shaken with vigorous bursts to release the epithelial cells. The supernatant was centrifuged at 300 g for 5 min at 4⁰C. The pellet was washed once with ice-cold PBS, resuspended in PBS containing 1 mg/mL collagenase, and incubated at 37⁰C for 5 min. The tube was then placed immediately on ice, followed by the addition of 5% (v/v) FBS. The cell suspension was centrifuged, washed with PBS, and filtered through a 40-mm cell strainer. The cells were resuspended in 0.04 % BSA-PBS and counted using trypan blue. A total of 1 x 105 cells were used for the analysis (1000 cells/mL).
Single cell libraries are generated using the 10X Genomics Chromium Single Cell 3’ Reagent V3 Kit according to manufacturer’s protocol.  Cell suspensions are first assessed with Trypan Blue using a Countess FL automated cell counter (ThermoFisher), to determine concentration, viability and the absence of clumps and debris that could interfere with single cell capture.  Cells are loaded into the Chromium Controller (10X Genomics) where they are partitioned into nanoliter-scale Gel Beads-in-emulsion with a single barcode per cell.  Reverse transcription is performed and the resulting cDNA is amplified.  The full-length amplified cDNA is used to generate gene expression libraries by enzymatic fragmentation, end-repair, a-tailing, adapter ligation, and PCR to add Illumina compatible sequencing adapters.  The resulting libraries are evaluated on D1000 screentape using a TapeStation 4200 (Agilent Technologies), and quantitated using Kapa Biosystems qPCR quantitation kit for Illumina. They are then pooled, denatured, and diluted to 300pM with 1% PhiX control library added.
The resulting pool is then loaded into the appropriate NovaSeq Reagent cartridge and sequenced on a NovaSeq6000 following the manufacturer’s recommended protocol (Illumina Inc.).

treatment_protocol: vehicle or tamoxifen (2mg, IP, every day for 5 days)

value: Ssrp1 KO

tag: agent

value: tamoxifen


title: NB1RGB_AGR

tag: passage

value: treated with 50 mM AGR for 4 hours


title: Intestine control (WT)

value: vehicle


title: aHSC_FF_3

city: Bunkyo-ku

first: Yuta

last: Koui

address: 1-1-1 Yayoi

department: Institute for Quantitative Biosciences

institute: The University of Tokyo

data_processing:
Raw sequencing reads were cleaned and trimmed with cutadapt ver. 1.2.1 and PRINSEQ ver. 0.19.2.
The filtered reads were mapped to the human reference genome (Homo sapiens NCBI build 37.2 ) using TopHat ver 2.0.13
Cufflinks ver 2.2.1 was used to generate a transcriptome assembly and to calculate FPKMs.
Genome_build: Homo sapiens NCBI build 37.2
Supplementary_files_format_and_content: FPKMs for every gene and every sample

source_name: FF-1 hiPSC-derived aHSCs 3

extract_protocol:
Total RNA was extracted with RNeasy Mini Kit (Qiagen) according to the manufacture’s protocol.
Libraries were prepared using the TruSeq Stranded mRNA Library Prep Kit (Illumina).

value: activated hepatic stellate cell

tag: derived from


title: ATACseq_293T_rep2

description:
log2atacSum_refgeneMerge_k4.1_sort.bed
log2FC_bedIntersetMerge.bw
WTb_interR1R2.merged.bed
293T_2_5b_peaks.broadPeak

extract_protocol:
ATAC-seq was performed as previously described (Buenrostro et al, 2015). Briefly, transposition was performed from 5000 parental or UBR7KO 293T cells resuspended in 50 uL of ATAC buffer (25 uL 2x TD buffer, 2.5 uL TDE1 transposase, Nextera; 0.5uL Digitonin, Promega; 22uL nuclease-free water) for 30 m at 37C with 300 rpm agitation. Fragmented DNA was collected using a Qiagen MinElute PCR kit (Qiagen 28004).
Sequencing libraries were prepared by PCR and quality controlled as described (Buenrostro et al., 2015). Libraries were sequenced on the NextSeq 500 to generate 50 bp single-end reads.


title: Bone marrow treated (Ssrp1 KO)

source_name: bone marrow


title: RNA-Seq EPL_4

value: epl


title: ATACseq_UBR7KO_rep2

description:
log2atacSum_refgeneMerge_k4.1_sort.bed
log2FC_bedIntersetMerge.bw
UBR7KOb_interR1R2.merged.bed
AH05B1_2_6b_peaks.broadPeak


title: RNA-Seq EAF_2


title: RNA-Seq EPL_3


title: RNA-Seq EAF_1


title: RNAseq_UBR7KO1_rep1

description:
UBR7KO.v.293T.611-207-227.b.htseq.edgeR.txt
293_UBR7_KO_RNA_101515


title: ATACseq_UBR7KO_rep1

description:
log2atacSum_refgeneMerge_k4.1_sort.bed
log2FC_bedIntersetMerge.bw
UBR7KOb_interR1R2.merged.bed
AH05B1_2_4b_peaks.broadPeak


title: qHSC_FF_3

source_name: FF-1 hiPSC-derived qHSCs 3

value: quiescent hepatic stellate cell


title: RNA-Seq EPL_1


title: aHSC_FF_1

source_name: FF-1 hiPSC-derived aHSCs 1


title: Bone marrow control (WT)


title: aHSC_FF_2

source_name: FF-1 hiPSC-derived aHSCs 2


title: ChIPseq_GFPUBR7_input

description:
GFP-IP-GFP-UBR7-1.macsPeaks_peaks.bed
log2FCbam_GFPUBR7vInput.bw

extract_protocol:
2x15cm 90% confluent plates of GFPUBR7 293T cells were used for each ChIP-seq experiment. The cells were fixed with 1% paraformaldehyde in DMEM for 15 m at 37C, then quenced with  0.125M glycine (final concentration) for 5m 37C. The cells were washed with ice-cold PBS containing Protease inhibitors (cOmplete, Roche). Cells were sonicated with a Branson sonicator at  40% amplitude, 0.7s on 1.3s off, for 10 m. Samples were then spun at max speed for 10m at 40C. The supernatant was collected and 50uL retained as input sample. The lysate was pre-cleared with beads.  4ug anti-GFP antibody (Supplementary Table 3) was added and the samples were incubated overnight with rotation at 4C. The immune complex was collected by incubating with Protein-A Sepharose beads on a rotator at 4°C. The beads were washed 5x with washing buffer (50 mM HEPES pH 8.0, 1% NP-40, 0.7% DOC, 0.5 M LiCl, freshly added protease inhibitors) then 1x with TE buffer. Immunoprecipitated complexes were eluted twice in serial by resuspending beads with 100 μl elution buffer (50 mM Tris-HCl pH 8.0, 1 mM EDTA, 1% SDS) and incubating for 30 m at 65°C. The supernatant was collected and treated with proteinase K for 2h at 55°C, then purified DNA by phenol-chloroform extraction.
ChIPseq libraries were prepared with the KAPA HTP Library Preparation Kit (Illumina KK8234) using NEXTFLEX Adapters (Illumina 514104), according to manufacturer protocols, from 10ng of starting DNA. Libraries were size selected using SPRI beads (Beckman Coulter A63987). Libraries were assessed on an Agilent Bioanalyzer, and sequenced on a NextSeq500 (Illumina) to generate 50bp single end reads.

value: endogenously tagged GFPUBR7


title: ATACseq_293T_rep1

description:
log2atacSum_refgeneMerge_k4.1_sort.bed
log2FC_bedIntersetMerge.bw
WTb_interR1R2.merged.bed
293T_2_3b_peaks.broadPeak


title: qHSC_FF_1

source_name: FF-1 hiPSC-derived qHSCs 1


title: qHSC_FF_2

source_name: FF-1 hiPSC-derived qHSCs 2


title: ChIP-Seq epl1_4_H4K5ac

value: H4K5ac (Millipore 07327)


title: ChIPseq_GFPUBR7_GFPIP


title: RNA-Seq WT_4

value: Leaves


title: ChIP-Seq epl1_4_H3K9ac

value: H3K9ac (Millipore 07352)


title: aHSC_4M_3

source_name: TkDN4-M hiPSC-derived aHSCs 3


title: EWS single cells

city: San Antonio

first: Yidong

last: Chen

state: Texas

address: 8403 Floyd Curl Drive, MSC 7784

department: Population Health Sciences

institute: UT Health Science Center at San Antonio

data_processing:
Illumina Casava v1.8.2 software used for base-calling
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence,  merged into single fastq file and mapped to UCSC/hg19 human genome using STAR aligner (see supplementary notes)
gene count measurements were obtained using featureCounts (see supplementary notes)
Genome_build: UCSC/hg19
Supplementary_files_format_and_content: The processed data file "allSamples_Genes_featureCount.txt" is in tab-delimited tabular format.  The first column contains the gene symbols and the second and late columns contain the read counts of the gene using featureCounts
Supplementary_files_format_and_content: The processed data file "CellList_EWS_SC.txt" is in tab-delimited tabular format.  The first column contains the cell name, the second column is the batch number, the third column is the treatment/RNA-collection time, and the fourth column is the cluster ID (-1 are either dead cells or cells with fewer than 2000 reads sequenced)

source_name: Ewing Sarcoma cell line EW-8

growth_protocol: Ewing sarcoma cells (cell line: EW-8) were treated with siEWSR1/FLI1 and collected after 48h and 120h of transfection and cells in the control condition were collected while dividing exponentially after 48h of transfection with si-control.

extract_protocol:
Total RNA was extracted using Lysis Mix- Mix A Module 2(mRNA Seq) (Fluidigm) and Rnase inhibitor from SMARTer kit (Fluidigm).
RNA libraries were prepared using standard Illumina Nextera kit for HighSeq protocol

treatment_protocol: Control and knockdown cells were subjected to 50ng/μl of si-Control and si-EWSR1 in 1X RPMI+10%FBS. Cells were washed with 1x PBS and harvested at days 2, and 5 (n=200-300 cells for each time point/condition) for RNA extraction and cDNA synthesis

value: Ewing Sarcoma cell line

value: control and EWS/FLI1 knock-down cells Dormant 1 and Dormant2

tag: time point

value: control, 48h and 120h


title: aHSC_4M_2

source_name: TkDN4-M hiPSC-derived aHSCs 2


title: aHSC_4M_1

source_name: TkDN4-M hiPSC-derived aHSCs 1


title: H1836_SOX2_RD

city: Sioux Falls

first: Michael

last: Kareta

state: SD

address: 2301 East 60th Street North

department: Genetics & Genomics

institute: Sanford Research

data_processing:
Reads were quality trimmed using cutadapt 2.0 and python 3.7.1 to remove nucleotides with a quality score less than 20, while maintaining a minimum length of 30 nt and removing a poly-G sequence that remained from library preparation. The samples were aligned using Bowtie2 version 2.3.4 with the following parameters: --local --very-sensitive-local --no-unal --no-mixed --no-discordant --phred33 -I 10 -X 700. Output sam files were sorted and converted to bed files using samtools version 1.9 and bedtools version 2.28.0 using the following commands: samtools view -f 0x2 $name.bam | bamToBed -i stdin -bedpe | cut -f 1,2,6 > $name.bed. Aligned bed files were used for peak calling using macs2 version 2.1.2 keeping all duplicates. The top 99.5th percentile of peaks after sorting by q-values (including peaks with the same q-value at cutoff) were selected for further analysis.
Genome_build: hg38

source_name: SCLC H1836

growth_protocol: Cells were grown in RPMI suplemented with 10% bovine growth serum and penicillin/streptomycin.

extract_protocol:
Samples were prepared according the the protocol published by Janssens & Hennikoff. Janssens D, Henikoff, S. CUT&RUN: Targeted in situ genome-wide profiling with high efficiency for low cell numbers. protocols.io 2019. Available from: https://dx.doi.org/10.17504/protocols.io.zcpf2vn. using the CUTANATM pAG-MNase (EpiCypher), and concanavalin-A coated beads (BioMag Plus #86057). The optional high-calcium/low-salt conditions were included to prevent premature chromatin release after digestion.
NEBNext® Ultra™ II DNA Library Prep Kit for Illumina

value: human small cell lung cancer

tag: antibody

value: R&D Systems (AF2018)


title: H82_SOX2_EMD

source_name: SCLC H82

value: EMD/Millipore (17-656)


title: H82_SOX2_RD


title: ChIP-Seq epl1_3_H3K9ac

source_name: Leaves


title: ChIP-Seq epl1_4_H3

value: H3 (Abcam ab1971)


title: ChIP-Seq epl1_3_H4K5ac


title: qHSC_4M_3

source_name: TkDN4-M hiPSC-derived qHSCs 3


title: H29_SOX2_EMD

source_name: SCLC H29


title: qHSC_4M_1

source_name: TkDN4-M hiPSC-derived qHSCs 1


title: H29_SOX2_RD


title: ChIP-Seq epl1_2_H4K5ac


title: ChIP-Seq epl1_3_H3


title: qHSC_4M_2

source_name: TkDN4-M hiPSC-derived qHSCs 2


title: ChIP-Seq epl1_2_H3


title: ChIP-Seq WT_4_H4K5ac

value: WT


title: H1836_SOX2_EMD


title: ChIP-Seq WT_4_H3K9ac


title: Control 2

city: New Delhi

first: Rahul

last: Pal

state: Delhi

address: Aruna Asaf Ali Marg

country: India

institute: National Institute of Immunology

data_processing:
Raw reads are filtered based on length (read length should be greater than 50) and quality using Trimmomatic-0.39
Filtered reads are aligned using STAR-Aligner v 2.5.4b
HTSeq v0.11.2 is used to get read count of genes
Insert size and SD are calculated from BAM files using RSeQC v3.0.1
Genome_build: GRCh38
Supplementary_files_format_and_content: Tab deliminated files include gene name and their raw counts in each sample

source_name: Peripheral blood mononuclear cells_control

extract_protocol:
PBMCs were isolated from human blood using Ficoll (Amersham) gradient separation at 20°C, 400g for 40 min
The sequencing library was prepared by random fragmentation of the DNA or cDNA sample, followed by 5' and 3' adapter ligation. Alternatively, "tagmentation" combines the fragmentation and ligation reactions into a single step that greatly increases the efficiency of the library preparation process. Adapter-ligated fragments are then PCR amplified and gel purified.

treatment_protocol: Human PBMCs derived from an SLE patient were cultured with Medium (Control) or Medium supplemented with human hemoglobin (Test) for 24 hours.

value: Immune cells

value: Peripheral blood

tag: patient diagnosis

value: SLE


title: Test 1

source_name: Peripheral blood mononuclear cells_supplemented with human hemoglobin


title: Control 1


title: S2R+ N51A-replicate3

city: Montpellier FRANCE

first: Julie

last: Carnesecchi

address: 1919, route de Mende team JCL team JCL

department: IGMM

institute: CNRS

data_processing:
RNA-Seq analysis was performed by using genome 6 (dm6). The quality of the RNA-Seq reads was quantified via FastQC. Trimming was performed with java script "Trimmomatic 0.36"(Bolger et al., 2014), for the removal of the adapter (TruSeq-SE sequencing adapter), with the following command line: java -jar /path_to_java_script/Trimmomatic-0.36/trimmomatic-0.36.jar SE -phred33 <file.txt> <outputfile.txt> ILLUMINACLIP:TruSeq-SE.fa:2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36. The reads were aligned using STAR (genome generated: STAR --runThreadN 14 --runMode genomeGenerate --genomeFastaFiles Drosophila_melanogaster.BDGP6.dna.toplevel.fa --sjdbGTFfile Drosophila_melanogaster.BDGP6.86.gtf --genomeSAindexNbases 12; running STAR: STAR --runThreadN 14 --genomeDir /STAR/GenomeDir/ --readFilesIn /STAR/<file> --outSAMtype BAM Unsorted) (Dobin et al., 2013). Aligned reads were counted by HTSeq (htseq-count -f bam -r pos -m union -s no -t exon accepted_hits.bam Drosophila_melanogaster.BDGP6.86.gtf > count.txt) for further analysis in DESeq (Anders et al., 2015).
For the analysis in Junction-Seq the aligned reads were counted and the genome flattened with QoRTs by using the available java scripts (counting: java -jar /path/QoRTs.jar QC --singleEnded --minMAPQ 75 --nameSorted sorted.bam Drosophila_melanogaster.BDGP6.86.gtf rawCts/file; flattened: java -jar /path/QoRTs.jar makeFlatGff --stranded Drosophila_melanogaster.BDGP6.86.gtf annoFiles/JunctionSeq.flat.gff.gz) (Hartley and Mullikin, 2015). Differential expression analysis was performed with DESeq2 under standard conditions (Love et al., 2014). Different exon usage and the identification of splicing variants was identified by using the R tool Junction-Seq under standard conditions (Hartley and Mullikin, 2016). All data results were further processed in Excel and False Discovery Rate (FDR) was established at FDR 0.1.
Genome_build: Drosophila_melanogaster.BDGP6.86.gtf
Supplementary_files_format_and_content: Tables with raw gene counts for every gene and each sample

organism: Drosophila melanogaster

source_name: RNAs polyA enriched from Drosophila S2R+ cells

extract_protocol:
Total RNAs were extracted from four independent replicates from Drosophila S2R+ cells expressing GFPnls, myc-UbxWT, or myc-UbxN51A (Gal4-UAS, actin promoter) using Qiagen RNA extraction kit (RNeasy). RNA quality was assessed using BioAnalyzer 2100TM (Agilent Technologies).
Material handling and mRNA-Seq directional libraries were performed with the Deep-Sequencing facility in Heidelberg (Cell Networks), using the true-seq illumina standard protocol

value: embryonic cells

tag: overexpression

value: overexpressing GFP-Ubx-N51A


title: ChIP-Seq WT_4_H3


title: S2R+ N51A-replicate2


title: Control_eye_No.4

type: RNA

city: Hamburg

first: Julia

middle: Elisabeth

last: Neumann

state: Hamburg

address: Martinistr. 52

department: Institute of Neuropathology

institute: University Medical Center Hamburg-Eppendorf

description: regular eye tissue serves as healthy control reference

hyb_protocol: NanoString standard protocol

scan_protocol: NanoString standard protocol

data_processing: Raw expression data was processed using nSolverAnalysis Software 4.0.70: Background subtraction with geometric mean of negative controls; Normalization with geometric mean of positive controls and standard housekeeping genes; No ratios built.

source_name: orbita

label_protocol: NanoString standard protocol

growth_protocol: Murine Shh-MBs were obtained from 14 day old mice. RBS lesions and control eyes were obtained from E18.5 mouse embryos.

extract_protocol: Each sample represents a single tumor, lesion or eye from an individual animal. RNA was extracted from FF tissue using protocols of the RNeasy® Mini Kit (QIAGEN, 74136).

tag: strain

tag: disease state

value: control

tag: sample

value: FF


title: ChIP-Seq WT_3_H3


title: ChIP-Seq WT_3_H3K9ac


title: S2R+ N51A-replicate1


title: S2R+ WT-replicate3

value: overexpressing GFP-Ubx-WT


title: ChIP-Seq WT_2_H4K5ac

value: H4K5ac (Millipore 07327)


title: Control_eye_No.2


title: Control_eye_No.3


title: Test 2