title: Small intestine CD4 from SellTomato mice in OVA feeding, tolerance, and allergy - TCR 2

city: New York

first: Tiago

last: Castro

state: New York

address: 1230 York Avenue

institute: The Rockefeller University

description:
10x Genomics
230106_T

data_processing:
Raw fastq files from our 10x libraries were processed with Cellranger count (v6.2.0) using the 10x Genomics prebuilt mouse reference (v3.0.0 mm10) (for diets) or a customized mouse genome (mm10) that included the ai9Tomato sequence plasmid as an artificial chromosome with the Ai9Tomato and STOP sequences annotated as features (for SellTomato). Analyses were performed in R 4.2.2 (R Core Team, 2018). Quality control was performed by removing cells with high (> 10% for the antigen-free diet libraries, >5% for SellTomato) mitochondrial unique molecular identifier (UMI) content. Cells not expressing Trac or Cd4 were excluded from our analysis. We defined Tomato+ and Tomato– cells post-sequencing using normalized UMI counts for tdTomato and Stop (Tomato+: tdTomato > Stop. Tomato–: tdTomato < Stop), discarding ambiguous cells where tdTomato = Stop. The matrix of UMI counts was normalized by applying a regression model with a negative binomial distribution, available through the SCTransform function in the Seurat (v41.-4.3.) package (Hafemeister and Satija, 2019). The top 3000 variable genes were first used for dimensional reduction by PCA using the scaled data. The first 30 principal components were further used for visualization using the Manifold Approximation and Projection (UMAP) and cell clustering (Hafemeister and Satija, 2019; Stuart et al., 2019). TCR contigs and annotation were performed with the Cellranger vdj workflow from 10x Genomics and the prebuild mouse reference (v3.1.0 mm10). Contigs filtering, clonotype calling, and downstream TCR analysis was performed using scRepertoire (v1.5.2) (Borcherding et al., 2020). Further processing, statistical analysis, and visualizations were performed using ggplot2 (v3.4.1) (Wickham, 2016)  and rstatix (v0.7.2).
Assembly: mm10
Supplementary files format and content: Tab separated files and matrix of UMI counts.

source_name: Small intestine

extract_protocol:
Lymphocytes isolated from the small intestine epithelium or lamina propria were isolated and indexed with TotalSeqC Hashtag (BioLegend) cell surface antibodies, with 2 barcodes used per sample for deeper multiplexing. Total CD4+ T cells were sorted, pooled, and immediately loaded onto a Chromium Controller (10x Genomics). For SellTomato sorted Tomato+ and Tomato- (CD62L-, CD4+ T) cells were pooled at a ~1:3 ratio.
5’ Gene expression, VDJ, and Feature Barcode libraries were prepared using the Chromium Single Cell 5′ v2 Reagent Kit (10x Genomics) according to manufacturer’s protocol at the Genomics core of Rockefeller University. Libraries were sequenced on Illumina NextSeq500 or NovaSeq 6000. Hashtag indexing was used to demultiplex the sequencing data and generate gene-barcode matrices.

value: Small intestine

value: CD4 T cells

value: OVA feeding, tolerance, allergy


title: Small intestine CD4 from SellTomato mice in OVA feeding, tolerance, and allergy - TCR 1

status: Public on May 13 2023

type: SRA

country: USA

description:
10x Genomics
211203_T

library_source: transcriptomic single cell

molecule: total RNA

organism: Mus musculus

tag: tissue

tag: cell type

tag: treatment


title: Small intestine CD4 from SellTomato mice in OVA feeding, tolerance, and allergy - Gex 2

description:
10x Genomics
230106_G


title: Small intestine CD4 from GF mice fed OTIIp or Chow diet - TCR

description:
10x Genomics
211202_T

value: GF mice fed OTIIp or Chow diet


title: Small intestine CD4 from GF mice fed OTIIp or Chow diet - Gex

description:
10x Genomics
211202_G


title: Small intestine CD4 from SellTomato mice in OVA feeding, tolerance, and allergy - Gex 1

description:
10x Genomics
211203_G


title: Small intestine CD4 from SellTomato mice in OVA feeding, tolerance, and allergy - Hashtags 1

description:
10x Genomics
211203_H


title: Small intestine CD4 from SellTomato mice in OVA feeding, tolerance, and allergy - Hashtags 2

description:
10x Genomics
230106_H


title: Small intestine CD4 from GF mice fed OTIIp or Chow diet - Hashtags

description:
10x Genomics
211202_H


title: Small intestine CD4 from MM12 mice fed AA, OVA, or chow diet - TCR

description:
10x Genomics
211018_T

value: MM12 mice fed AA, OVA, or chow diet


title: Old-CMC-3

city: Beijing

first: zhenzhen

last: zhang

state: Haidian Distinct

address: No.30 Shuangqing Road

department: School of Medicine

country: China

institute: Tsinghua University

description: tRNA

library_source: transcriptomic

data_processing:
PCR duplication was removed with Fastx_toolkit v0.0.14.
The random barcode in the 5’ end was discarded with seqtk v1.3-r106.
Processed reads were further mapped to the genomic tRNA sequences from GtRNAdb atabase(http://gtrnadb.ucsc.edu/genomes/eukaryota/Mmusc10/) with Bowtie2 v2.3.5 (parameters: bowtie-a--best--strata--chunkmbs 2000).
To identify the pseudouridine sites of tRNA, the following criteria were considered: (1) the pseudouridine sites appearing in all independent replicates; (2) stop rate<1% in the BEU sample; (3) CMC coverage>50; (4) stop reads number>5 in the CMC sample; (5) stop rate (CMC−BEU) difference>4%; (6) Fisher test adjusted P value <0.05.
Assembly: mm10
Supplementary files format and content: tab-delimited text files include FPKM values and site calling for each group of samples.
Library strategy: DM-Ψ-seq

source_name: Hematopoietic stem and progenitor cell

extract_protocol:
Small RNA (<200nt) was extracted and purified using miRNeasy Mini Kit (Qiagen) and RNeasy MinElute Cleanup Kit (Qiagen).
Purified small RNA fragments were demethylated by wild-type and  mutant AlkBs, purified by phenol/chloroform extraction and ethanol precipitation, labeled by CMC. 50ng small RNA was denatured at 65 °C for 5 min and demethylated by wild-type and D135S mutant AlkBs. The purified small RNA was denatured at 80 °C for 5 min, added to BEU buffer with or without CMC, incubated at 37 °C for 20 min, then purified by ethanol precipitation.The  purified RNA was dissolved in sodium carbonate buffer and shaked at 37 °C for 6 h. The small RNA was dephosphorylated with CIP (NEB). The 3' adaptor ligation was added with T4 RNA ligase2, truncated KQ (NEB), followed by 5' Deadenylase (NEB) and RecJf (NEB) digestion. The RNA was reverse transcribed by SuperScript III reverse transcriptase (Invitrogen), then digested by RNase H. The 5' adaptor ligation was added with T4 RNA ligase 1, high concentration (NEB). The ligated cDNA was amplified by Q5 High-Fidelity 2× Master Mix (NEB).

value: Hematopoietic stem and progenitor cell

tag: genotype

value: wildtype


title: Old-CMC-1

description: tRNA


title: Old-BEU-1

description: tRNA


title: Old-BEU-2

description: tRNA


title: Young-CMC-3

description: tRNA


title: Young-CMC-2

description: tRNA


title: Young-BEU-3

description: tRNA


title: Young-BEU-2

description: tRNA


title: Small intestine CD4 from MM12 mice fed AA, OVA, or chow diet - Hashtags

description:
10x Genomics
211018_H


title: Small intestine CD4 from MM12 mice fed AA, OVA, or chow diet - Gex

description:
10x Genomics
211018_G


title: Old-CMC-2

description: tRNA


title: Old-BEU-3

description: tRNA


title: Young-CMC-1

description: tRNA


title: HepG2  cells,si-PTBP1-1

first: Chengzhi

last: Gao

address: Taiping Road

department: Genetics & Integrative Omics

institute: Beijing Institute of Radiation Medicine

data_processing:
The quality of raw sequencing reads was evaluated using FastQC.
Adaptor sequences and low-quality score bases were trimmed using Trim Galore (v.0.6.7, https://github.com/FelixKrueger/TrimGalore).
These reads were then mapped to the human genome reference GRCh38 from Ensembl release 105 using Bowtie2 (v.2.2.5).
The fragments per kilobase of exon per million mapped reads (FPKM) values and gene count values were computed using RSEM (v.1.2.8).
Assembly: GRCh38
Supplementary files format and content: tab-delimited text files include RPKM values for each Sample.

organism: Homo sapiens

extract_protocol:
RNA was harvested using Trizol Reagent (T9424, Sigma-Aldrich, USA).
RNA libraries for RNA-seq were prepared using VAHTS mRNA-seq v2 Library Prep Kit following manufacturer's protocols.

treatment_protocol: The total RNAs of HepG2 cells, which were transfected with si-scramble, si-IGFRIL or si-PTBP1, was extracted.

tag: cell line

value: Hepatocellular carcinoma cells

value: WT


title: HepG2  cells,si-PTBP1-2


title: HepG2  cells,si-PTBP1-3


title: HepG2  cells,si-IGFRIL-3

value: si-IGFRIL


title: Young-BEU-1

description: tRNA


title: Pus10-KO-3

data_processing:
Sequencing reads were mapped with Hisat2 v2.1.0.
Gene read counts were obtained with HTSeq v0.11.2.
The differential expression levels were estimated with R-package DESeq2.
Assembly: mm10
Supplementary files format and content: tab-delimited text files include raw read counts for each sample

source_name: Hematopoietic stem cell

extract_protocol:
HSCs were sorted into lysis buffer. RNA-Seq was performed according to Smart-seq2.
The RNA was reverse transcribed by SuperScript II reverse transcriptase (Invitrogen) and amplified to enrich the cDNA fragments. The cDNA was purified by VAHTS DNA Clean Beads (Vazyme). Tn5 transposase was used to tagment DNA, then stripped by 0.2% SDS. The DNA was amplified by Q5 High-Fidelity 2× Master Mix (NEB) followed by VAHTS DNA Clean Beads purification.

value: Hematopoietic stem cell


title: HepG2  cells,si-IGFRIL-2


title: HepG2  cells,si-IGFRIL-1


title: HepG2  cells,si-NC-1

value: si-NC


title: HepG2  cells,si-NC-2


title: Pus10-WT-2


title: Pus10-KO-2


title: HepG2  cells,si-NC-3


title: Pus10-WT-3


title: Pus10-WT-1


title: miR139–KD–6


title: miR139–KD–3


title: miR139–KD–5


title: miR139–KD–4


title: miR139–KD–2


title: Control 3

city: Yangzhou

first: Xiaolin

last: Wang

address: No98，Nantong West Road

institute: Department of Thoracic Surgery

data_processing:
Sequence reads were mapped to the human genome (hg19) using TopHat v2.0.11 with the default options with a TopHat transcript index built from Ensembl_GRCh37.
Count files of the aligned sequencing reads were generated by the htseq-count script from the Python package HTSeq with union mode, using the GTF annotation file
Genome_build: hg19
Supplementary_files_format_and_content: count file

growth_protocol: The human PC3 were cultured in RPMI-1640 medium (Gibco, USA) supplemented with 10 % fetal bovine serum at 37 °C in an atmosphere of 5 % CO2.

extract_protocol:
total RNA of PC3 lung  cancer cells with different treatment was harvested using Trizol reagent
RNA libraries were prepared for sequencing using standard Illumina protocols

treatment_protocol: The PC3 lung cancer  cells were transfected with control siRNA or siIGF2BP3 siRNA.

value: human PC3 cells

tag: passage

tag: transfection

value: control siRNA


title: miR139–KD–1


title: NTC-rep6

value: NTC


title: Control 2


title: siRNA treatment 2

value: siIGF2BP3 siRNA


title: siRNA treatment 3


title: Contra-4N-5_S8

city: Cambridge

first: Jessica

last: Whited

state: MA

address: 7 Divintiy Street, Sherman Fairchild - Whited Lab

institute: Harvard University

description: 2x100 bp

data_processing:
reads were psuedoaligned to the reference using Kallisto V0.48.0
the BUS files were converted into sparse matrix with BUSpaRse (V1.9.0)
counts were generated in Seurat (V4.2.0) and exported to sysAct_4N_barcodes.tsv sysAct_4N_genes.tsv sysAct_4N_matrix.mtx with DropletUtils (V1.19.3)
Assembly: ambMex60DD GCA_002915635.3
Supplementary files format and content: seurat object converted to 10X format

organism: Ambystoma mexicanum

source_name: limb tissue

extract_protocol:
Cells were dissociated in dissociation buffer (100 mg/ml liberase diluted in 0.7X PBS with 0.5 U/ml DNase I) for 30 min at room temperature with gentle mixing. Cells were then stained with 1 mg/ml Hoecsht, calcein AM, and propidium iodide for isolation of alive, dividing (4C) and non-dividing (2C) cells by fluorescence activated cell sorting. The 4C cells were used for library preparation.
10x Single Cell 3' v3.1 NextGEM library preparation protocol
NovaSeq_S4_single_lane 2 x 100 bp

value: limb tissue

value: wild-type


title: Contra-4N-3_S7

description: 2x100 bp


title: Contra-4N-2_S6

description: 2x100 bp


title: Intact-4N-5_S4

description: 2x100 bp


title: NTC-rep4


title: NTC-rep5


title: Contra-4N-1_S5

description: 2x100 bp


title: NTC-rep3


title: Pus10-KO-1


title: NTC-rep2


title: Intact-4N-1_S1

description: 2x100 bp


title: Intact-4N-3_S2

description: 2x100 bp


title: NTC-rep1


title: siRNA treatment 1


title: Control 1


title: KD652-3

data_processing:
Sequencing reads were mapped with Hisat2 v2.1.0.
Gene read counts were obtained with HTSeq v0.11.2.
The differential expression levels were estimated with R-package DESeq2.
Read count extraction and normalization were performed using CLC genomic benchwork
Assembly: mm10
Supplementary files format and content: tab-delimited text files include raw read counts for each sample


title: KD652-2


title: Contra-2N-4_S6

data_processing:
reads were psuedoaligned to the reference using Kallisto V0.48.0
the BUS files were converted into sparse matrix with BUSpaRse (V1.9.0)
counts were generated in Seurat (V4.2.0) and exported to sysAct_2N_barcodes.tsv sysAct_2N_genes.tsv sysAct_2N_matrix.mtx with DropletUtils (V1.19.3)
Assembly: ambMex60DD GCA_002915635.3
Supplementary files format and content: seurat object converted to 10X format

extract_protocol:
Cells were dissociated in dissociation buffer (100 mg/ml liberase diluted in 0.7X PBS with 0.5 U/ml DNase I) for 30 min at room temperature with gentle mixing. Cells were then stained with 1 mg/ml Hoecsht, calcein AM, and propidium iodide for isolation of alive, dividing (4C) and non-dividing (2C) cells by fluorescence activated cell sorting. The 2C cells were used for the downstream library preparation.
10x Single Cell 3' v3.1 NextGEM library preparation protocol
NovaSeq_S4_single_lane 2 x 100 bp


title: HepG2  cells,NC-PTB-1

data_processing:
For the analysis of CLIP-seq data, the reads were first trimmed of adaptor sequences by Trimmomatic (version 0.36).
Subsequently, the clean reads were mapped to the hg38 reference genome by Bowtie 2 (version 2.1.0) with parameters -p 10 -L 15 -N 1 -D 50 -R 50 --phred33 --qc-filter --very-sensitive --end-to-end
CLIP-seq peaks were identified by Piranha (version 1.2.1) with the following parameters: -s -b 20 -d Zero Truncated Negative Binomial -p 0.05 Homer (version 4.9.1) was used for peak annotation based on the hg38 genome assembly and for analysis of PTBP1-binding motifs.
Assembly: hg38
Supplementary files format and content: BigWig and BED format files that contain peaks and positions of cognate RNA

extract_protocol:
RNA and protein complexes were digested by proteinase K (9034, TaKaRa, Japan) and  RNAs were isolated with saturated phenol (AM9712, Ambion, USA).
cDNA libraries for CLIP-seq were prepared using SuperScript III First-Strand Kit (18080-051, Invitrogen, USA) following manufacturer's protocols.

treatment_protocol: The IGFRIL-depleted and control HepG2 cells were cross-linked by ultraviolet light (254 nm) to maintain covalent binding of RBPs to their cognate RNA. Subsequently, the PTBP1 and cross-linked RNAs were immunoprecipitated with anti-PTBP1 antibody (32-4800, Thermo Fisher, USA) and digested with Micrococcal nuclease (EN0181, Thermo Fisher Scientific, USA). CLIP assays with IgG antibody (ab172730, Abcam, USA) were used as negative control. An IR800-biotin adapter was ligated to the 3′ end of the RNA fragment. Then, the PTBP1/RNA complexes were separated by SDS-PAGE gel and transferred to the nitrocellulose membrane (HATF00010, Millipore, USA).


title: KD652-4


title: Contra-2N-2_S4


title: KD652-1


title: NTC-4


title: HepG2  cells,NC-PTB-2


title: HepG2  cells,NC-IgG-2

value: Anti-IgG


title: HepG2  cells,KD-PTB-1

value: IGFRIL-depleted


title: HepG2  cells,KD-PTB-2


title: HepG2  cells,KD-IgG-2


title: Intact-2N-4_S5


title: HepG2  cells,KD-IgG-1


title: NTC-2


title: Day30_LAPC4 KD4OE-3 (C3)

city: Houston

first: Danthasinghe

middle: Badrajee

last: Piyarathna

state: United States

address: One Baylor Plaza

institute: Baylor College of Medicine

data_processing:
Fastq files were run through fastqc for primary qc
Sequence reads were trimmed for adaptor sequence/low-quality sequence using cutadapt (parameter- Default settings)
Trimmed sequence reads were mapped to hg38 using STAR
Read count extraction and normalization were performed using htseq-counts
Assembly: hg38
Supplementary files format and content: tab-delimited text files include htseq counts

growth_protocol: LAPC4 cells were maintained in IMDM supplemented with 15% fetal bovine serum (FBS) and 1% penicillin streptomycin in humidified atmosphere with 5% CO2 at 37C

extract_protocol:
RNA was harvested using Rneasy mini plus kit (Qiagen). 1 ug of total RNA was used for the construction of sequencing libraries.
RNA libraries for RNA-seq were prepared using NovaSeq-PE-150 .

treatment_protocol: 3ug/mL Puromycin and 1 mg/mL G418 were used as respective selection antibiotics for knockdown and rescue respectively

value: Prostate epithelial cancer cell

value: UGT2B28 rescue

tag: time

value: Day 30


title: Intact-2N-5_S7


title: Intact-4N-4_S3

description: 2x100 bp


title: Day30_LAPC4 KD4OE-2 (C2)


title: Contra-2N-5_S8


title: NTC-1


title: Intact-2N-2_S3


title: Contra-2N-1_S2


title: Day30_LAPC4 KD4-3 (B3)

value: UGT2B28 knockdown


title: HepG2  cells,NC-IgG-1

value: Hepatocellular carcinoma cells


title: Intact-2N-1_S1


title: Day30_LAPC4 KD4OE-1 (C1)


title: Day30_LAPC4 KD4-2 (B2)


title: NTC-3


title: Aged LT-HSC Dlk1+, 3

city: Würzburg

first: Reyna Edith

last: Rosales Alvarez

address: Versbacher Str. 9

department: Quantitative Single-Cell Biology of the Immune System

country: Germany

institute: Würzburg Institute of Systems Immunology, Max Planck Research Group at the Julius-Maximilians-Universität Würzburg

data_processing:
Paired-end reads were aligned to the transcriptome using BWA (version 0.6.2-r126) with default parameters (Li and Durbin, 2010, Bioinformatics, DOI: 10.1093/bioinformatics/btp698). The transcriptome contained all gene models based on the mouse ENCODE VM9 release downloaded from the UCSC genome browser comprising 57,207 isoforms with 57,114 isoforms mapping to fully annotated chromosomes (1–19, X, Y, M). All isoforms of the same gene were merged to a single gene locus, and gene loci were merged to larger gene groups, if loci overlapped by >75%. This procedure resulted in 34,111 gene groups. The right mate of each read pair was mapped to the ensemble of all gene groups and to the set of 92 ERCC spike-ins in the sense direction. Reads mapping to multiple loci were discarded. The left mate contained the barcode information: the first six bases corresponding to the cell-specific barcode, followed by six bases representing the UMI. The remainder of the left read contained a poly(T) stretch and adjacent gene sequence. The left read was not used for quantification. For each cell barcode and gene locus, the number of UMIs was aggregated and, on the basis of binomial statistics, converted into transcript counts (Grün et al., 2014, Nat Methods, DOI: 10.1038/nmeth.2930).
Genome_build: mm10
Supplementary_files_format_and_content: CSV files, columns represent each cell barcode (total barcodes used = 192), rows represent the geneid and the values in the file are the quantified number of transcripts

molecule: polyA RNA

source_name: Bone marrow HSCs

growth_protocol: LT-HSCs were isolated from from C57BL/6J wildtype mice, aged 18 months old. Mice were purchesed from JAX

extract_protocol:
Murine bone marrow (BM) cells were isolated from pooled femura, tibiae, hips, ilia, and vertebrae by gentle crushing in PBS. Lineage negative (Lin-) cells were enriched by using Dynabeads Untouched Mouse CD4 Cells Kit (Invitrogen), according to the manufacturer’s instructions. Therefore, LT-HSCs were FACS sorted with the following surface markers: Lin-CD150+CD48-CD34-Kit+Sca1+ and either Dlk1+ or Dlk1-.
The CEL-Seq2 protocol with reduced volumes was used as previously described (Herman et al., 2018, Nat Methods, DOI: 10.1038/nmeth.4662) and modified using the following reagents.  Instead of 1.2 μl Vapour-Lock as hydrophobic encapsulation barrier mineral oil (Sigma, M8410-100ML) was used. For cDNA first-strand synthesis, Protoscript II and Protoscript II Reaction Buffer (NEB, M0368L) as well as murine RNase-Inhibitor (NEB, M0314S) was used instead of SuperScript II reverse transcriptase, first-strand synthesis buffer and RnaseOUT. E. coli DNA polymerase I, Escherichia coli DNA ligase, RNase H (Invitrogen; 18021071) and 5 x second-strand buffer were replaced with E.coli DNA polymerase (NEB, M0209L), E.coli DNA ligase (NEB, M0205L), RNaseH (NEB, M0297S) and 10x Second Strand Buffer (NEB, B6117S) respectively.  The water volume was adjusted to adequately dilute the 10x second strand buffer. After second strand synthesis 96 wells were pooled, which results in 96 single cells per library.   The library preparation was performed as previously described (Herman et al., 2018), but by using Protoscript II,  Protoscript II Reaction Buffer and murine RNase-Inhibitor as mentioned above instead of SuperScript II reverse transcriptase, first-strand synthesis buffer and RnaseOUT.

treatment_protocol: No treatment

tag: strain

tag: Sex

value: Male

tag: age

value: 18 months old


title: Day30_LAPC4 NT-3 (A3)

value: Control


title: Aged LT-HSC Dlk1+, 1


title: INT-2_S2

description:
intact_2
illumina

library_source: genomic

data_processing:
ATAC-seq reads were aligned to ambMex60DD GCA_002915635.3 using bowtie2 (V2.3.2)
Peaks were called using Genrich (V0.5) in ATAC mode
Assembly: ambMex60DD GCA_002915635.3
Supplementary files format and content: narrowPeak files containing the peak calls from Genrich (V0.5.0)

molecule: genomic DNA

extract_protocol:
Cells were dissociated in dissociation buffer (100 mg/ml liberase diluted in 0.7X PBS with 0.5 U/ml DNase I) for 30 min at room temperature with gentle mixing. Cells were then stained with 1 mg/ml Hoecsht, calcein AM, and propidium iodide for isolation of alive, dividing (4C) and non-dividing (2C) cells by fluorescence activated cell sorting. The 4C cells were used for library preparation.
Ten thousand sorted 4C cells from homeostatic, systemically-activated, and regenerating limbs were resuspended in 5 μL PBS. 42.5 μL of transposition buffer (38.8mM tris-acetate, 77.6mM potassium acetate, 11.8mM magnesium acetate, 0.12% NP-40, 18.8% DMF, 0.47% protease inhibitor cocktail) was added to the cells, mixed, and incubated 10 min at room temperature. 2.5 μL of pre-loaded Tn5 (Diagenode cat# C01070012) was added to the reaction mixture. The transposition reaction was carried out at 37°C for 30 min with shaking at 300 rpm. Following transposition, the samples were purified through Direct-zol RNA MiniPrep kit (Zymo Research cat# R2050). Purified library fragments were amplified with custom primers 1 and 2 using the following conditions: 72°C for 5min; 98°C for 30s; and thermocycling at 98°C for 10s, 72°C for 30s and 72°C for 1min. The libraries were amplified for five cycles. Soon after, 5μL aliquot of the PCR reaction was added with 10μL of the PCR cocktail with SYBR Green (0.6Xfinal concentration). 20 cycles of PCR was performed to determine the additional number of cycles needed for the remaining 45 μL reaction. The libraries were purified using Direct-zol RNA MiniPrep kit.
NovaSeq S4 2 x 100 bp lane

value: wild type


title: INT-1_S1

description:
intact_1
illumina


title: PUS10-OE_3

data_processing:
Data filtering was performed with SOAPnuke1.5.0.
The filtered sequencing data was mapped with Bowtie v1.1.2 with parameters: Bowtie -v 1 -k 20 --best –strata.
Basing on the read counts of each RNA, TPM (TPM = Reads count of snc-RNA * 1000000 / sample library size) was caculated to compare the expression of individual RNA.
Assembly: mm10
Supplementary files format and content: tab-delimited text files include raw read counts for each sample

extract_protocol:
Small RNA (<200nt) was extracted and purified using miRNeasy Mini Kit (Qiagen) and RNeasy MinElute Cleanup Kit (Qiagen). The small RNA sequence was performed by BGI (Shenzhen, China).
18-30nt RNA was selected by PAGE gel. The 3' adaptor ligation and 5' adaptor ligation was added. The RNA was reverse transcribed and amplified to enrich the cDNA fragments. The DNA fragments were purified by PAGE gel. The double stranded DNA was denatured and circularised to generate single strand circle DNA.

value: Hematopoietic stem cell


title: Aged LT-HSC Dlk1-, 4


title: Small intestine CD4 from GF mice fed AA or OVA diet - TCR

description:
10x Genomics
190821_T

value: Small intestine

value: CD4 T cells

value: GF mice fed AA or OVA diet


title: Aged LT-HSC Dlk1+, 4


title: CTL-5_S10

description:
systemically activated_5
illumina

value: limb tissue


title: CTL-4_S9

description:
systemically activated_4
illumina


title: Vector_3

value: Vector


title: Day30_LAPC4 KD4-1 (B1)


title: Small intestine CD4 from GF mice fed AA or OVA diet - Gex

description:
10x Genomics
190821_G


title: Vector_2


title: Day30_LAPC4 NT-2 (A2)


title: Aged LT-HSC Dlk1+, 2


title: Aged LT-HSC Dlk1-, 2


title: Vector_1


title: Aged LT-HSC Dlk1-, 1


title: Day30_LAPC4 NT-1 (A1)


title: Small intestine CD4 from GF mice fed AA or OVA diet - Hashtags

description:
10x Genomics
190821_H


title: BLA-1_S11

description:
blastema_1
illumina

source_name: blastema and wound epidermis

value: blastema and wound epidermis


title: PUS10-OE_2


title: BLA-2_S12

description:
blastema_2
illumina


title: RNAseq__KURAMOCHI__HGSOC__WT1_Cell_Line_biorep2

status: Public on May 12 2023

city: San Antonio

first: Kate

last: Lawrenson

address: 7703 Floyd Curl Dr.

institute: Cedars Sinai Medical Center

data_processing:
hisat2
htseq
Assembly: grch37
Supplementary files format and content: MTF_KD_KURAMOCHI_FT246.sorted.htseq-count.TPM.txt

source_name: KURAMOCHI

extract_protocol: BGI

treatment_protocol: siRNA reverse transfection protocol

value: KURAMOCHI

value: High-grade serous "ovary" cancer


title: RNAseq__KURAMOCHI__HGSOC__WT1-AS_Cell_Line_biorep2


title: RNAseq__KURAMOCHI__HGSOC__ZNF217_Cell_Line_biorep2


title: PUS10-OE_1


title: RNAseq__KURAMOCHI__HGSOC__MECOM_Cell_Line_biorep2

value: siMECOM


title: RNAseq__KURAMOCHI__HGSOC__SOX17_Cell_Line_biorep2


title: Aged LT-HSC Dlk1-, 3


title: RNAseq__KURAMOCHI__HGSOC__WT1_Cell_Line_biorep1


title: RNAseq__KURAMOCHI__HGSOC__NT2_Cell_Line_biorep2


title: RNAseq__KURAMOCHI__HGSOC__PAX8_Cell_Line_biorep1


title: RNAseq__KURAMOCHI__HGSOC__ZNF217_Cell_Line_biorep1


title: RNAseq__KURAMOCHI__HGSOC__NT2_Cell_Line_biorep1


title: RNAseq__KURAMOCHI__HGSOC__NT1_Cell_Line_biorep1


title: RNAseq_022221_FT246_11_FTSEC_1_SOX17_Cell_Line_biorep2

value: Fallopian Tube Secretory Epithelium


title: RNAseq_022221_FT246_14_FTSEC_1_ZNF217_Cell_Line_biorep2


title: RNAseq_022221_FT246_9_FTSEC_1_NT2_Cell_Line_biorep2


title: RNAseq_022221_FT246_8_FTSEC_1_NT1_Cell_Line_biorep2


title: RNAseq_022221_FT246_10_FTSEC_1_PAX8_Cell_Line_biorep2


title: RNAseq_012521_FT246_7_FTSEC_1_ZNF217_Cell_Line_biorep1


title: RNAseq_022221_FT246_12_FTSEC_1_MECOM_Cell_Line_biorep2

value: MECOM


title: RNAseq_012521_FT246_6_FTSEC_1_WT1_Cell_Line_biorep1


title: RNAseq__KURAMOCHI__HGSOC__PAX8_Cell_Line_biorep2


title: RNAseq_012521_FT246_5_FTSEC_1_MECOM_Cell_Line_biorep1


title: RNAseq_012521_FT246_4_FTSEC_1_SOX17_Cell_Line_biorep1


title: RNAseq__KURAMOCHI__HGSOC__WT1-AS_Cell_Line_biorep1


title: RNAseq__KURAMOCHI__HGSOC__MECOM_Cell_Line_biorep1


title: RNAseq__KURAMOCHI__HGSOC__NT1_Cell_Line_biorep2


title: RNAseq_012521_FT246_1_FTSEC_1_NT1_Cell_Line_biorep1


title: RNAseq_012521_FT246_2_FTSEC_1_NT2_Cell_Line_biorep1


title: RNAseq__KURAMOCHI__HGSOC__SOX17_Cell_Line_biorep1


title: clipseq_H1_YTHDC2_UV_IP_1

city: Durham

first: Tongyu

last: Sun

state: NC

address: 307 research drive

institute: Duke university

data_processing:
Reads were trimmed using fastp (0.20.0) using default settings.
Reads were aligned to the hg38 genome using HISAT2 (2.1.0) with default settings.
Bam files were converted to bigwig files using bamCoverage and renormalized using RPKM (bamCoverage -b file.bam -o file.bw --normalizeUsing RPKM)
Assembly: hg38
Supplementary files format and content: bigwig

source_name: H1 hESCs

growth_protocol: Human pluripotent stem cells are maintained in the chemically defined feeder- free, serum-free mTseR plus medium(Stemcell Tech, 100-0276) according to manufacturer's instructions and detached using 5mM EDTA/PBS or accutase. 10 μM Rho-associated protein kinase (ROCK) inhibitor Y-27632 was added into the culture medium if the cells were single cells.

extract_protocol:
Cells were sonicated with Bioruptor(30S ON, 30S OFF, 10cycles)
Adptors were ligated to the 3' ends. 5' end of cDNA was liagted to a adaptor. Libraries were PCR amplified.

treatment_protocol: Cells were UV crosslinked on ice.

value: H1 hESCs

value: UV crosslink

tag: rip antibody


title: RNA_H1_YTHDC2KO_YTHDC2E317Q_rescue1

data_processing:
RNA-seq reads were trimmed using fastp (0.20.0) using default settings.
RNA-seq reads were aligned to the hg38 genome using HISAT2 (2.1.0) with default settings.
Bam files were converted to bigwig files using bamCoverage and renormalized using RPKM (bamCoverage -b file.bam -o file.bw --normalizeUsing RPKM)
Reads on each gene were counted using featureCounts (2.0.1).
Differentially expressed genes were determined using DESeq2.
Assembly: hg38
Supplementary files format and content: bigwig

extract_protocol:
RNA was harvested using Direct-zol RNA MiniPrep Kit (Zymo Research, 11-331). 10 ng of total RNA was used for the construction of sequencing libraries.
RNA libraries for RNA-seq were prepared following the Smart-seq2 protocol.

treatment_protocol: Neurons were induced using STEMdiff™ SMADi Neural Induction Kit(Stemcell Technologies, 08581) following monolayer cell culture protocol. In brief, human stem cells were dissociated into single cells using accutase.  Cells were transferred to a single well of 24 well plate in complete induction medium with 10 μM Y-27632. Medium was changed daily. Cells were collected at day0 (Ctrl) and day3.

value: overexpression of YTHDC2 (E317Q) in YTHDC2 KO cells


title: RNA_H1_YTHDC2KO_YTHDC2WT_rescue1

value: overexpression of YTHDC2 (WT) in YTHDC2 KO cells


title: clipseq_H1_YTHDC2_UV_Input_1

value: wild type


title: RIP_H1_YTHDC1_1

data_processing:
RIP-seq reads were trimmed using fastp (0.20.0) using default settings.
RIP-seq reads were aligned to the hg38 genome using HISAT2 (2.1.0) with default settings.
To create RIP-seq coverage plots, bam files were converted to bigwig files using bamCoverage and renormalized using RPKM (bamCoverage -b file.bam -o file.bw --normalizeUsing RPKM).
Peaks were called uing MACS3 (3.0.0a7) with the significance cut-off q-value <=0.05
Assembly: hg38
Supplementary files format and content: bigwig

extract_protocol:
Cells were lysed in lysis buffer on ice to extract native RNA-protein complexes. The soluble complexes were enriched by YTHDC2 antibodies. RNA was extracted using the Direct-zol RNA MiniPrep Kit (Zymo Research, 11-331). 10 ng of total RNA was used for the construction of sequencing libraries.
RNA libraries for RNA-seq were prepared following the Smart-seq2 protocol.


title: RIP_H1_STM2457_1


title: RIP_H1_YTHDC2E317Q_rescue_1

value: Flag


title: RIP_H1_DMSO_1

value: DMSO


title: RIP_H1_YTHDC2WT_rescue_1


title: RNAseq_022221_FT246_13_FTSEC_1_WT1_Cell_Line_biorep2


title: RNAseq_012521_FT246_3_FTSEC_1_PAX8_Cell_Line_biorep1


title: ChIP_H1_YTHDC2KO_YTHDC2WTrescue_YTHDC2_1

data_processing:
ChIP-seq reads were trimmed using fastp (0.20.0) using default settings.
ChIP-seq reads were aligned to the hg38 genome using Bowtie version 2.4.4. Only uniquely mapping reads without mismatches were retained.
To create ChIP-seq coverage plots, bam files were converted to bigwig files using bamCoverage and renormalized using RPKM (bamCoverage -b file.bam -o file.bw --normalizeUsing RPKM).
Peaks were called uing MACS2 v2.2.7.1 with the significance cut-off q-value <=0.05
Assembly: hg38
Supplementary files format and content: bigwig

extract_protocol: Cells were crosslinked with the crosslinking buffer (10mM NaCl, 0.1 mM EDTA, 0.05 mM EGTA, 5 mM Hepes pH8.0, 1% formaldehyde) at room temperature for 10 min. The crosslinking reaction was stopped by adding 2.5M glycine to a final concentration of 0.125M and incubation at room temperature for another 5 min. Cells were washed with ice cold PBS twice and transferred to 15 mL tubes in PBS. After centrifugation at 500g for 5 min, the PBS was discarded. Cell pellets were resuspended in 50 μl lysis buffer (1% SDS, 50 mM Tris-HCl, pH 8.0, 20 mM EDTA supplemented with 10x protease inhibitor) and incubated for10 min on ice. The cell lysates were diluted to 500 μl with the cold TE buffer. The lysates were sonicated using Covaris M220. The sonicated lysates were centrifuged at 12000 g for 15 min at 4 °C. The supernatants were transferred into new tubes and DNA concentrations were measured on a NanoDrop. For each replicate, 20 μg of chromatin was used. To pre-clear the chromatin solutions, 20 μl lgG Dynabeads (Life Technologies, 11204D) were washed and added. The chromatin beads solutions were rotated gently in the cold room for 2 hours. After the incubation, place tubes on a magnetic stand to collect beads. 100 μl supernatants were transferred into new tubes. 100 μl Binding Buffer (1% Triton-X, 01% Sodium Deoxycholate supplemented with protease inhibitor in TE buffer) was added to the 100 μl chromatin. The mixture was incubated in the cold room overnight on a rotator. Next day, 20 μl lgG pre-washed Dynabeads were added into each reaction and incubated for another 2 hours. After incubation, the beads were washed five times with wash buffer (50 mM Hepes, pH 8.0, 1% NP-40, 0.70% Sodium Deoxycholate, 0.5 M LiCl, 1 mM EDTA supplemented with protease inhibitor) and twice with cold 1xTE. After the final wash, 150 μl ChIP elution buffer (10 mM, Tris-HCl pH 8.0, 1 mM EDTA, 1% SDS) were added into tubes. The beads mixture was transferred to new tubes and incubated at 65°C for 20 minutes at 1300 rpm on a Thermomixer. The supernatants were transferred into tubes and incubated at 65°C overnight to reverse crosslinks. Next day, 250μL1xTE and 8 μl of 10 mg/mL RNase A was added to each sample and incubated at 37°C for 1 hour. 8 μl of 20 mg/mL Proteinase K was added and incubated at 55°C for 1 hour. After that, the DNA was purified and measured on a NanoDrop. To generate ChIP libraries, DNA ends were repaired. ployA tails were added to the ends and then adaptors were ligated at room temperature. Last, the DNA was amplified with TruSeq primers and purified with magnetic beads. Libraries were sequenced on the MGISEQ-2000 platform in pair-end mode with 100 base pair (bp) per read.

tag: chip antibody


title: ChIP_H1_YTHDC2KO_YTHDC2MUTrescue_YTHDC2_1


title: ChIP_H1_YTHDC2KO_YTHDC2WTrescue_K9me3_1


title: ChIP_H1_YTHDC2KO_YTHDC2MUTrescue_K9me3_1


title: ChIP_H1_YTDC2KO_H3K4me3_1

value: YTHDC2 KO


title: ChIP_H1_YTHDC1_1

value: WT


title: ChIP_H1_YTHDC2WT_H3K4me3_1

value: YTHDC2 WT


title: ChIP_H1_YTDC2KO_H3K4me1_1


title: ChIP_H1_YTHDC2WT_H3K4me1_1


title: RNA, neuron, day3, R2

data_processing:
RNA-seq reads were trimmed using fastp (0.20.0) using default settings.
RNA-seq reads were aligned to the hg38 genome using HISAT2 (2.1.0) with default settings.
Reads on each gene were counted using featureCounts (2.0.1).
Differentially expressed genes were determined using DESeq2.
Assembly: hg38
Supplementary files format and content: bigwig

source_name: neuron

value: neuron

value: complete induction


title: ChIP_H1_YTHDC2WT_H3K27me3_1


title: ChIP_H1_STM2457_K9me3_1


title: ChIP_H1_YTDC2KO_H3K27me3_1


title: RNA, H1, dcas9KRAB-CARGO, R1

value: hESCs


title: ChIP_H1_DMSO_K9me3_1


title: RNA, neuron, day3, R1


title: RNA, neuron, Ctrl, R1

value: Ctrl


title: RNA, H1, dcas9KRAB-Ctrl, R2


title: RNA, neuron, Ctrl, R2


title: RNA, H1, dcas9KRAB-CARGO, R2


title: RNA, H1, dcas9KRAB-Ctrl, R1


title: RNA, H1, YTHDC2-KO, R1


title: RNA, H1, YTHDC2-Ctrl, R1


title: RNA, H1, YTHDC2-Ctrl, R2


title: RIP, H1, YTHDC2-IP, R1

data_processing:
RIP-seq reads were trimmed using fastp (0.20.0) using default settings.
RIP-seq reads were aligned to the hg38 genome using HISAT2 (2.1.0) with default settings.
To create RIP-seq coverage plots, bam files were converted to bigwig files using bamCoverage and renormalized using RPKM.
Peaks were called uing MACS3 (3.0.0a7) with the significance cut-off q-value <=0.05
Assembly: hg38
Supplementary files format and content: bigwig


title: RIP, H1, YTHDC2-IP, R2


title: RIP, H1, YTHDC2-Input, R2


title: RIP, H1, YTHDC2-Input, R1


title: 5mC, H1, YTHDC2-KO, R2

data_processing:
MeDIP-seq reads were trimmed using fastp (0.20.0) using default settings.
MeDIP-seq reads were aligned to the hg38 genome using Bowtie version 2.4.4. Only uniquely mapping reads without mismatches were retained.
To create MeDIP-seq coverage plots, bam files were converted to bigwig files using bamCoverage and renormalized using RPKM.
Peaks were called uing MACS2 v2.2.7.1 with the significance cut-off q-value <=0.05
Assembly: hg38
Supplementary files format and content: bigwig

extract_protocol:
Genomic DNA was extracted using Quick-DNA kits following manufacturer's protocols and sonicated using Covaris M220.
Fragmented genomic DNA was enriched using 5hmC or 5mC antibodies and PCR amplified.

tag: antibody


title: 5mC, H1, YTHDC2-KO, R1


title: 5mC, H1, YTHDC2-Ctrl, R2


title: 5mC, H1, YTHDC2-Ctrl, R1


title: 5hmC, H1, YTHDC2-Ctrl, R2


title: 5hmC, H1, YTHDC2-KO, R2


title: 5hmC, H1, YTHDC2-KO, R1


title: 5hmC, H1, YTHDC2-Ctrl, R1


title: BRD4, H1, YTHDC2-WT, R1

data_processing:
CUT&RUN reads were trimmed using fastp (0.20.0) using default settings.
CUT&RUN reads were aligned to the hg38 genome using Bowtie version 2.4.4. Only uniquely mapping reads without mismatches were retained.
To create CUT&RUN coverage plots, bam files were converted to bigwig files using bamCoverage and renormalized using RPKM.
Peaks were called uing MACS2 v2.2.7.1 with the significance cut-off q-value <=0.05
Assembly: hg38
Supplementary files format and content: bigwig
Library strategy: CUT&RUN

extract_protocol:
Cells were collected at RT and slightly crosslinked in 0.1% formaldehyde.
Cells were treated with primary antibodies and pAG-Mnase. Genomic DNA was extracted and PCR amplified.


title: BRD4, H1, YTHDC2-WT, R2


title: RNA, H1, YTHDC2-KO, R2


title: Roots, control,  biol rep 2

first: Tao

last: Chen

state: Jiangsu

address: 48 Wenhui East Road

department: School of Horticulture and Plant Protection

institute: Yangzhou University

description: CK-b

data_processing:
llumina Casava1.8 sotware used for bascealing
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence, then mapped to mm8 whole genome using hisat2
Fragment Per Kilobase per Million bases of library size (FPKM) were calculated using cufflinks v2.2.1
The differentially expressed genes were detected as previously described based on the parameters of DeSeq2: Fold change > = 2.00 and Probability > = 0.8 with a significant false discovery rate-adjusted P value (FDR) <0.05
Assembly: mm9
Supplementary files format and content: tab-delimited text files include RPKM values for each sample

organism: Pyrus betulifolia

source_name: root tip

extract_protocol:
RNA of each sample was extracted using the Trizol kit (Promega, MA, USA).  1 ug of total RNA was used for the construction of sequencing libraries.
RNA libraries for RNA-seq were prepared using NEBNext® UltraTM RNA Library Prep Kit for Illumina® following manufacturer's protocols.

value: root tip

value: root tip cell

value: Control


title: Roots, boron deficiency, biol rep 2

value: Boron deficiency


title: Roots, control,  biol rep 3

description: CK-c


title: H3K9me3, H1, YTHDC2-WT, R2

data_processing:
ChIP-seq reads were trimmed using fastp (0.20.0) using default settings.
ChIP-seq reads were aligned to the hg38 genome using Bowtie version 2.4.4. Only uniquely mapping reads without mismatches were retained.
To create ChIP-seq coverage plots, bam files were converted to bigwig files using bamCoverage and renormalized using RPKM.
Peaks were called uing MACS2 v2.2.7.1 with the significance cut-off q-value <=0.05
Assembly: hg38
Supplementary files format and content: bigwig


title: H3K9me3, H1, dCas9-CARGO, R1


title: H3K9me3, H1, YTHDC2-KO, R1


title: TET1, H1, YTHDC2-KO, R1


title: TET1, H1, YTHDC2-WT, R2


title: TET1, H1, YTHDC2-KO, R2


title: Roots, control,  biol rep 1

description: CK-a


title: H3K9me3, H1, dCas9-CARGO, R2


title: Liver_5_CMO

city: Madrid

first: Rui

last: Benedito

state: Madrid

address: C. de Melchor Fernández Almagro, 3

country: Spain

institute: CNIC

description: 10x Genomics

data_processing:
Control (CMO302) and Dll4iDEC (CMO303) groups non-endothelial cell (EC) data are displayed in Extended Data Figure 6.
The demultiplexing, barcoded processing, gene counting and aggregation were made using the Cell Ranger software v4.0 or v6.1 (https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/what-is-cell-ranger)
For alignment and quantification of gene expression the reference transcriptome was built using mouse genome GRCm38 and ensembl gene build version 98 (sep2019.archive.ensembl.org). The phiYFP-sv40pA, MbTomato-2A-Cre-WPRE-sv40pa or CreERT2 transgenes sequences expressed in the samples were added to the reference
Liver ECs: The following quality control steps were performed to minimize low-quality cells and improve posterior normalization and analysis: i) A minimum of normalized counts per cell of 1500 and a maximum of 40000 was applied. ii) A minimum gene detection filter of 600 genes. iii) A maximum 25% of MT content. iv) A minimum cell fraction in the sample of 0.1%. The cell fraction refers to the proportion of UMIs the cell contributes with to the total UMIs in the sample. v) Cells with high numbers of reads in just a few genes were filtered out. vi) Cells with more than 0.1% reads in HBB genes were removed. vii) Cells with < 100 UMIs in the hashtags were removed to improve sample calling. Viii) Only single cells were selected; doublets were filtered out.
Cells were de-multiplexed using the pipeline provided with the Seurat package. Counts were normalized and scaled for posterior analysis. Clusters were identified using the nearest neighbors algorithm at different resolutions. Clusters and cells were classified based on the SingleR method using Blueprint Encode and the Human Primary Cell Atlas cell type profiles collection
Following cluster identification with the starting dataset, the remaining Liver EC datasets were mapped using the FindTransferAnchors() function from the Seurat package using 30 PCA dimensions with the default settings.
Liver non ECs: Cells were de-multiplexed by aplying the cellranger multi pipeline. The following quality control steps were performed to minimize low-quality cells and improve posterior normalization and analysis: i) A minimum of normalized counts per cell of 2000 and a maximum of 30000 was applied. ii) A minimum gene detection filter of 500 genes and a maximum of 6000. iii) A maximum 5% of MT content. iv) A maximum 35% ribosomal content. v) a maximum 1% of Hbb content. Viii) Only single cells were selected; doublets were filtered out in the cellranger multi demultiplexing step.
Counts were log-normalized and scaled followed by PCA and clustering using the shared nearest neighbours algorithm and Louvain clustering (settings as defaults except for the 2000 most variable genes, 30 principal components and a resolution of 0.8). Clusters and cells were classified based on the Seurat method using Blueprint Encode, Human Primary Cell Atlas and mouseRNAseq datasets available in the celldex package, as well as a recent dataset from Ben-Moshe et al 2019 in order to classify each cluster to a different cell type. Final clustering was later manually refined based on markers expression.
Assembly: mm10
Supplementary files format and content: fasta file
Supplementary files format and content: gtf file
Supplementary files format and content: csv expression matrix
Supplementary files format and content: csv metadata

source_name: Liver

extract_protocol:
To isolate ECs for scRNAseq experiments, livers were dissected, minced, and digested for 30 min with pre-warmed (37C) dissociation buffer (2.5mg/ml collagenase I (Thermo Fisher 17100017 ), 2.5mg/ml dispase II (Thermo Fisher 17105041), 1 μl /ml DNAse in PBS containing Ca2+ and Mg2+ (Gibco)). The digestion tube was agitated every 3-5 minutes in a water bath. At the end of the 30 minutes incubation, sample tubes were filled up to 15 ml with sorting buffer (PBS containing no Ca2+ or Mg2+ and supplemented with 10% FBS (Sigma, F7524)) and centrifuged (450g, 5 min, 4°C). The supernatant was aspirated, and cell pellets were resuspended in 2ml 1x RBC lysis buffer (BioLegend, 420301) and incubated for 5 min on ice. To each sample were added 6 ml of sorting buffer, and samples were then passed through a 70um filter. Live cells were counted in a Neubauer Chamber using trypan blue exclusion. Cells from each condition (4x10^6/condition) were collected in separate tubes, and cells were incubated for 30 min with horizontal rotation in 300 μl antibody incubation buffer (PBS + 1% BSA) containing 1 μl CD31-APC, 1 μl CD45-APC-Cy7, and 1μl of hash tag oligo (HTO) conjugated antibodies (Biolegend). HTOs were used to label and distinguish the different samples when loaded on the same 10x genomics port, and in this way also guarantee the absence of batch effects. After the antibodies incubation, samples were transferred to 15 ml Falcon tubes, 10 ml sorting buffer was added, and samples were centrifuged (450g, 5min, 4°C). The supernatant was aspirated, pellets were resuspended in 1.5 ml sorting buffer and transferred to Eppendorf tubes, and the resulting suspensions were centrifuged (450g, 5min, 4°C). The resulting pellets were resuspended in 300 μl sorting buffer containing DAPI. Cells were sorted by FACS with an Aria Cell Sorter (BD Biosciences); ECs were identified as CD31+, CD45- MbTomato+ cells. After sorting, cells were centrifuged at 500g, 5min, 4ºC and the cell pellet was resuspended in cell capture buffer (PBS + 0.04% ultra-pure BSA (Thermo Fisher, AM2616) in order to get a concentration of 700-1200 cells/μl.
Single cell suspensions of freshly isolated ECs were resuspended in Cell Capture buffer to a final concentration of 800 to 1600 cells/μl. scRNA-seq libraries were prepared using the Chromium Single Cell 3´ Reagent Kits v3.1 (10x Genomics; Pleasanton, CA, USA) according to the manufacturer’s instructions.

treatment_protocol:
1.5 mg tamoxifen was injected on 4 consecutive days in each mouse. Mice were processed following day 14 after the first tamoxifen injection.
Genotypes for the mice were: Control (Tg(Cdh5-CreERT2), Tg(iSuRe-Cre) and Dll4iDEC (Dll4flox/flox, Tg(Cdh5-CreERT2), Tg(iSuRe-Cre)

value: Misc

value: Liver

tag: library type

value: CMO

value: Cdh5-CreERT2 + iSuRe-Cre

value: 10-20 weeks old

tag: hto demultiplexing_conditions

value: CMO303=Dll4iDEC (Dll4LOF)


title: H3K9me3, H1, YTHDC2-WT, R1


title: Input, H1, WT, R1


title: H3K9me3, H1, YTHDC2-KO, R2


title: Roots, boron deficiency, biol rep 3


title: H3K9me3, H1, dCas9-Ctrl, R2


title: Roots, boron deficiency, biol rep 1


title: Liver_5_GEX_CD31Neg_non_ECs_Control_Dll4iDEC

description: 10x Genomics

extract_protocol:
To isolate non ECs for scRNAseq experiments, livers were dissected, minced, and digested for 30 min with pre-warmed (37C) dissociation buffer (2.5mg/ml collagenase I (Thermo Fisher 17100017), 2.5mg/ml dispase II (Thermo Fisher 17105041), 1ul/ml DNAse in PBS containing Ca2+ and Mg2+ (Gibco)). The digestion tube was agitated every 3-5 minutes in a water bath. At the end of the 30 minutes incubation, sample tubes were filled up to 15 ml with sorting buffer (PBS containing no Ca2+ or Mg2+ and supplemented with 10% FBS (Sigma, F7524)) and centrifuged (450g, 5 min, 4°C). The supernatant was aspirated, and cell pellets were resuspended in 2ml 1x RBC lysis buffer (BioLegend, 420301) and incubated for 5 min on ice. To each sample were added 6 ml of sorting buffer, and samples were then passed through a 70um filter. Live cells were counted in a Neubauer Chamber using trypan blue exclusion. Cells from each condition (5x10^5) were collected, were collected in separate tubes, and cells were incubated for 30 min with horizontal rotation in 300 μl diluted antibody incubation buffer (PBS + 1% BSA) containing 0.1 μl CD31-APC, 0.1 μl CD45-APC-Cy7. After the incubation, 1ml of cell capture buffer (PBS + 0.04% ultra-pure BSA (Thermo Fisher, AM2616) was added and cells were centrifuged at 450g, 5min 4ºC. The cell pellet was then resuspended in 25 μl Cell Multiplexing Oligo (CMO) conjugated lipids (CellPlex Kit 3’, 10x Genomics, PN-1000261) and incubated for 5 mins at room temperature before adding 1ml cell capture buffer and centrifuging at 450g, 5 min, 4ºC. The resulting pellets were resuspended in 300 μl sorting buffer containing DAPI. Cells were sorted by FACS with an Aria Cell Sorter (BD Biosciences); non-ECs were identified as CD31- cells with different levels of CD45+ signal. After sorting, cells were centrifuged at 500g, 5min, 4ºC and the cell pellet was resuspended in cell capture buffer in order to get a concentration of 700-1200 cells/ μl.
Single cell suspensions of freshly isolated ECs were resuspended in Cell Capture buffer to a final concentration of 800 to 1600 cells/μl. scRNA-seq libraries were prepared using the Chromium Single Cell 3´ Reagent Kits v3.1 (10x Genomics; Pleasanton, CA, USA) according to the manufacturer’s instructions.

value: mRNA


title: H3K9me3, H1, dCas9-Ctrl, R1


title: Liver_4_HTO

description: 10x Genomics

data_processing:
Control + Vehicle (BC305), Dll4iDEC+Vehicle (BC302) and Dll4iDEC+SL327 (BC303) data is displayed in UMAP of Figure 7n.
Dll4iDEC+Vehicle data was also shown in UMAP of Extended Data Figure 5i.
Dll4/MYCiDEC (BC304) data is shown in UMAP of Figure 6j and related violin/dot plots and heatmaps.
The demultiplexing, barcoded processing, gene counting and aggregation were made using the Cell Ranger software v4.0 or v6.1 (https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/what-is-cell-ranger)
For alignment and quantification of gene expression the reference transcriptome was built using mouse genome GRCm38 and ensembl gene build version 98 (sep2019.archive.ensembl.org). The phiYFP-sv40pA, MbTomato-2A-Cre-WPRE-sv40pa or CreERT2 transgenes sequences expressed in the samples were added to the reference
Liver ECs: The following quality control steps were performed to minimize low-quality cells and improve posterior normalization and analysis: i) A minimum of normalized counts per cell of 1500 and a maximum of 40000 was applied. ii) A minimum gene detection filter of 600 genes. iii) A maximum 25% of MT content. iv) A minimum cell fraction in the sample of 0.1%. The cell fraction refers to the proportion of UMIs the cell contributes with to the total UMIs in the sample. v) Cells with high numbers of reads in just a few genes were filtered out. vi) Cells with more than 0.1% reads in HBB genes were removed. vii) Cells with < 100 UMIs in the hashtags were removed to improve sample calling. Viii) Only single cells were selected; doublets were filtered out.
Cells were de-multiplexed using the pipeline provided with the Seurat package. Counts were normalized and scaled for posterior analysis. Clusters were identified using the nearest neighbors algorithm at different resolutions. Clusters and cells were classified based on the SingleR method using Blueprint Encode and the Human Primary Cell Atlas cell type profiles collection
Following cluster identification with the starting dataset, the remaining Liver EC datasets were mapped using the FindTransferAnchors() function from the Seurat package using 30 PCA dimensions with the default settings.
Liver non ECs: Cells were de-multiplexed by aplying the cellranger multi pipeline. The following quality control steps were performed to minimize low-quality cells and improve posterior normalization and analysis: i) A minimum of normalized counts per cell of 2000 and a maximum of 30000 was applied. ii) A minimum gene detection filter of 500 genes and a maximum of 6000. iii) A maximum 5% of MT content. iv) A maximum 35% ribosomal content. v) a maximum 1% of Hbb content. Viii) Only single cells were selected; doublets were filtered out in the cellranger multi demultiplexing step.
Counts were log-normalized and scaled followed by PCA and clustering using the shared nearest neighbours algorithm and Louvain clustering (settings as defaults except for the 2000 most variable genes, 30 principal components and a resolution of 0.8). Clusters and cells were classified based on the Seurat method using Blueprint Encode, Human Primary Cell Atlas and mouseRNAseq datasets available in the celldex package, as well as a recent dataset from Ben-Moshe et al 2019 in order to classify each cluster to a different cell type. Final clustering was later manually refined based on markers expression.
Assembly: mm10
Supplementary files format and content: fasta file
Supplementary files format and content: gtf file
Supplementary files format and content: csv expression matrix
Supplementary files format and content: csv metadata

treatment_protocol:
2mg tamoxifen was injected on 3 consecutive days in mice belonging to the groups Control, Dll4iDEC+Vehicle and Dll4iDEC+SL327. Starting on the same day as the first tamoxifen injection, Dll4iDEC mice were injected daily (for 4 days) with vehicle or SL327 120mg/kg (S1066, Selleckchem). The last injection of SL327 was done 2 hours before tissue harvest.  1.5 mg tamoxifen was injected on 4 consecutive days in Dll4/MYCiDEC group mice and these were processed on day 14 after the first tamoxifen injection.
Genotypes for the mice were: Control (Tg(Cdh5-CreERT2), Tg(iSuRe-Cre)); Dll4iDEC (Dll4flox/flox, Tg(Cdh5-CreERT2), Tg(iSuRe-Cre) and Dll4/MYCiDEC (Dll4flox/flox, MYCflox/flox, Tg(Cdh5-CreERT2, Tg(iSuRe-Cre);

value: Endothelial cells

value: HTO

value: BC305=Control + Vehicle

value: BC304=Dll4/MYCiDEC (Dll4/MycKO)


title: Liver_4_GEX_CD31+ECs_Ctrl+Veh_Dll4iDEC+Veh_Dll4iDEC+SL327_Dll4/MyciDEC

description: 10x Genomics

treatment_protocol:
2mg tamoxifen was injected on 3 consecutive days in mice belonging to the groups Control, Dll4iDEC+Vehicle and Dll4iDEC+SL327. Starting on the same day as the first tamoxifen injection, Dll4iDEC mice were injected daily (for 4 days) with vehicle or SL327 120mg/kg (S1066, Selleckchem). The last injection of SL327 was done 2 hours before tissue harvest.  1.5 mg tamoxifen was injected on 4 consecutive days in Dll4/MYCiDEC group mice and these were processed on day 14 after the first tamoxifen injection.
Genotypes for the mice were: Control (Tg(Cdh5-CreERT2), Tg(iSuRe-Cre)); Dll4iDEC (Dll4flox/flox, Tg(Cdh5-CreERT2), Tg(iSuRe-Cre) and Dll4/MYCiDEC (Dll4flox/flox, MYCflox/flox, Tg(Cdh5-CreERT2, Tg(iSuRe-Cre); Cre); Dll4Het (Dll4flox/wt, Tg(Cdh5-CreERT2); Tg(iSuRe-Cre)); Notch1/2/4iDEC (Notch1/2/4flox/flox, Tg(Cdh5-CreERT2); Tg(iSuRe-Cre)); Jag1/Jag2/Dll1iDEC (Jag1/2/Dll1flox/flox, Tg(Cdh5-CreERT2); Tg(iSuRe- Cre); Dll4/MyciDEC (Dll4/Mycflox/flox, Tg(Cdh5-CreERT2), Tg(iSuRe-Cre)).


title: Liver_3_HTO

description: 10x Genomics

data_processing:
Jag1/Jag2/Dll1iDEC sample (BC304) is displayed in UMAP of Figure 5k and related violin/dot plots. The other samples in the raw data file were NOT analysed in this study.
The demultiplexing, barcoded processing, gene counting and aggregation were made using the Cell Ranger software v4.0 or v6.1 (https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/what-is-cell-ranger)
For alignment and quantification of gene expression the reference transcriptome was built using mouse genome GRCm38 and ensembl gene build version 98 (sep2019.archive.ensembl.org). The phiYFP-sv40pA, MbTomato-2A-Cre-WPRE-sv40pa or CreERT2 transgenes sequences expressed in the samples were added to the reference
Liver ECs: The following quality control steps were performed to minimize low-quality cells and improve posterior normalization and analysis: i) A minimum of normalized counts per cell of 1500 and a maximum of 40000 was applied. ii) A minimum gene detection filter of 600 genes. iii) A maximum 25% of MT content. iv) A minimum cell fraction in the sample of 0.1%. The cell fraction refers to the proportion of UMIs the cell contributes with to the total UMIs in the sample. v) Cells with high numbers of reads in just a few genes were filtered out. vi) Cells with more than 0.1% reads in HBB genes were removed. vii) Cells with < 100 UMIs in the hashtags were removed to improve sample calling. Viii) Only single cells were selected; doublets were filtered out.
Cells were de-multiplexed using the pipeline provided with the Seurat package. Counts were normalized and scaled for posterior analysis. Clusters were identified using the nearest neighbors algorithm at different resolutions. Clusters and cells were classified based on the SingleR method using Blueprint Encode and the Human Primary Cell Atlas cell type profiles collection
Following cluster identification with the starting dataset, the remaining Liver EC datasets were mapped using the FindTransferAnchors() function from the Seurat package using 30 PCA dimensions with the default settings.
Liver non ECs: Cells were de-multiplexed by aplying the cellranger multi pipeline. The following quality control steps were performed to minimize low-quality cells and improve posterior normalization and analysis: i) A minimum of normalized counts per cell of 2000 and a maximum of 30000 was applied. ii) A minimum gene detection filter of 500 genes and a maximum of 6000. iii) A maximum 5% of MT content. iv) A maximum 35% ribosomal content. v) a maximum 1% of Hbb content. Viii) Only single cells were selected; doublets were filtered out in the cellranger multi demultiplexing step.
Counts were log-normalized and scaled followed by PCA and clustering using the shared nearest neighbours algorithm and Louvain clustering (settings as defaults except for the 2000 most variable genes, 30 principal components and a resolution of 0.8). Clusters and cells were classified based on the Seurat method using Blueprint Encode, Human Primary Cell Atlas and mouseRNAseq datasets available in the celldex package, as well as a recent dataset from Ben-Moshe et al 2019 in order to classify each cluster to a different cell type. Final clustering was later manually refined based on markers expression.
Assembly: mm10
Supplementary files format and content: fasta file
Supplementary files format and content: gtf file
Supplementary files format and content: csv expression matrix
Supplementary files format and content: csv metadata

treatment_protocol:
1.5 mg tamoxifen was injected on 4 consecutive days in Jag1floxed/Jag2floxed/Dll1floxed Cdh5-CreERT2 + iSuRe-Cre mice and on day 14 after the first injection, livers were processed for cell isolation.
Genotypes of the mice were: Control (Tg(Cdh5-CreERT2), Tg(iSuRe-Cre));  Jag1/2/Dll1iDEC (Jag1flox/flox, Jag2flox/flox, Dll1flox/flox Tg(Cdh5-CreERT2, Tg(iSuRe-Cre)


title: H3K27ac, H1, dCas9-CARGO, R2

value: hESCs


title: H3K27ac, H1, dCas9-CARGO, R1


title: Liver_3_CD31+ECs _Jag1/2/Dll1iDEC

description: 10x Genomics


title: TET1, H1, YTHDC2-WT, R1


title: H3K27ac, H1, dCas9-Ctrl, R1


title: H3K27ac, H1, dCas9-Ctrl, R2


title: Liver_2_HTO

description: 10x Genomics

data_processing:
Control group data of Liver 2 (BC301) was merged with control data of Liver1 experiment.
Notch1/2/4iDEC group data (BC306) is shown in UMAP of Figure 4o and related violin/dot plots.
Dll4 HETiDEC group data (BC307) and DBZ (BC302) is shown in UMAP of Figure 4s and related violin/dot plots.
Dll4iDEC+anti-VEGF group data (BC305) is shown in UMAP of Figure 7e and related violin/dot plots.
The demultiplexing, barcoded processing, gene counting and aggregation were made using the Cell Ranger software v4.0 or v6.1 (https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/what-is-cell-ranger)
For alignment and quantification of gene expression the reference transcriptome was built using mouse genome GRCm38 and ensembl gene build version 98 (sep2019.archive.ensembl.org). The phiYFP-sv40pA, MbTomato-2A-Cre-WPRE-sv40pa or CreERT2 transgenes sequences expressed in the samples were added to the reference
Liver ECs: The following quality control steps were performed to minimize low-quality cells and improve posterior normalization and analysis: i) A minimum of normalized counts per cell of 1500 and a maximum of 40000 was applied. ii) A minimum gene detection filter of 600 genes. iii) A maximum 25% of MT content. iv) A minimum cell fraction in the sample of 0.1%. The cell fraction refers to the proportion of UMIs the cell contributes with to the total UMIs in the sample. v) Cells with high numbers of reads in just a few genes were filtered out. vi) Cells with more than 0.1% reads in HBB genes were removed. vii) Cells with < 100 UMIs in the hashtags were removed to improve sample calling. Viii) Only single cells were selected; doublets were filtered out.
Cells were de-multiplexed using the pipeline provided with the Seurat package. Counts were normalized and scaled for posterior analysis. Clusters were identified using the nearest neighbors algorithm at different resolutions. Clusters and cells were classified based on the SingleR method using Blueprint Encode and the Human Primary Cell Atlas cell type profiles collection
Following cluster identification with the starting dataset, the remaining Liver EC datasets were mapped using the FindTransferAnchors() function from the Seurat package using 30 PCA dimensions with the default settings.
Liver non ECs: Cells were de-multiplexed by aplying the cellranger multi pipeline. The following quality control steps were performed to minimize low-quality cells and improve posterior normalization and analysis: i) A minimum of normalized counts per cell of 2000 and a maximum of 30000 was applied. ii) A minimum gene detection filter of 500 genes and a maximum of 6000. iii) A maximum 5% of MT content. iv) A maximum 35% ribosomal content. v) a maximum 1% of Hbb content. Viii) Only single cells were selected; doublets were filtered out in the cellranger multi demultiplexing step.
Counts were log-normalized and scaled followed by PCA and clustering using the shared nearest neighbours algorithm and Louvain clustering (settings as defaults except for the 2000 most variable genes, 30 principal components and a resolution of 0.8). Clusters and cells were classified based on the Seurat method using Blueprint Encode, Human Primary Cell Atlas and mouseRNAseq datasets available in the celldex package, as well as a recent dataset from Ben-Moshe et al 2019 in order to classify each cluster to a different cell type. Final clustering was later manually refined based on markers expression.
Assembly: mm10
Supplementary files format and content: fasta file
Supplementary files format and content: gtf file
Supplementary files format and content: csv expression matrix
Supplementary files format and content: csv metadata

treatment_protocol:
2mg tamoxifen was injected on 3 consecutive days in each mouse. Mice were processed on day 14 after the first tamoxifen injection. For anti-VEGFA experiments, mouse anti-VEGFA G6-31, developed by Genentech, was administered four times over 14 days at a concentration of 5 mg/kg. The γ-secretase inhibitor DBZ (YO-01027, S2711, Selleckchem) was injected at 30µmol/kg in adult animals every day in the morning for the last 4 days, and 16h (in the evening) before collection of the tissues. Control animals were injected with tamoxifen and vehicle only.
Genotypes of the mice were: Control (Tg(Cdh5-CreERT2), Tg(iSuRe-Cre) mice treated with Vehicle); DBZ (Tg(Cdh5-CreERT2), Tg(iSuRe-Cre) mice treated with DBZ); Dll4iDEC+antiVEGF (Dll4flox/flox, Tg(Cdh5-CreERT2), Tg(iSuRe-Cre) treated with anti-VEGF; Notch1/2/4iDEC (Notch1flox/flox, Notch2 flox/flox Notch4KO/KO Tg(Cdh5-CreERT2, Tg(iSuRe-Cre); Dll4HETiDEC (Dll4flox/Wt, Tg(Cdh5-CreERT2), Tg(iSuRe-Cre);

value: BC305=Dll4iDEC+antiVEGF (D4KO_aVEGF)


title: dCas9, H1, CARGO, R2

value: CARGO


title: dCas9, H1, CARGO, R1


title: ATACseq, H1, WT, R2

data_processing:
ATAC-seq reads were trimmed using fastp (0.20.0) using default settings.
ATAC-seq reads were aligned to the hg38 genome using Bowtie version 2.4.4. Only uniquely mapping reads without mismatches were retained.
To create ATAC-seq coverage plots, bam files were converted to bigwig files using bamCoverage and renormalized using RPKM.
Peaks were called uing MACS2 v2.2.7.1 using default settings.
Assembly: hg38
Supplementary files format and content: bigwig

extract_protocol:
Nucleus were isolated in lysis buffer on ice.
Nucleus were treated by Tn5 tranposase. Genomic DNA was extracted and PCR amplified.

value: WT


title: H3K27ac, H1, WT, R1


title: H3K27ac, H1, WT, R2


title: H3K9me3, H1, WT, R1


title: YTHDC2, H1, WT, R1


title: H3K9me3, H1, WT, R2


title: S11_HSCs_GFI1-36N_2

city: Luebeck

first: Pradeep

middle: Kumar

last: Patnana

state: Schleswig-Holstein

address: Ratzeburger Allee 160

institute: Universitätsklinikum Schleswig-Holstein

data_processing:
RNA-seq data quantification: RNA-seq data (single-end fastq files) quantification was performed using the pseudo-aligner SALMON (v1.9.0) against the full Mus musculus cDNA database (GRCm38, Ensembl release 102; kmer size set to 31). The algorithm was run using 30 bootstrap intervals, GC bias, and sequence-specific bias correction. Additionally, positional bias correction was performed, and the library type was inferred automatically by SALMON.
Differential expression analysis and pathway enrichment: Pseudo-alignments were converted into sleuth format (wasabi package v1.0.1) and Ensembl transcript IDs were mapped to gene symbols using annotables (v0.1.91). Effect sizes (log2 fold changes) of gene expression differences were estimated using Wald-test in sleuth (v0.30.0) and p values were retrieved from the likelihood-ratio test.   Pathway enrichment analysis against combined REACTOME and HALLMARK gene sets (msigdf R package v7.4) on effect sizes was performed using a rank-MANOVA-based approach as implemented in mitch (v1.8.0; priority on significance); gene sets with an adjusted p-value below 0.05 and an absolute enrichment above 0.1 were considered as significant.
Statistical analysis: Analysis was performed using R (v4.2.1) and for data handling the tidyverse (v1.3.2) package. Plots were generated with the packages ggplot2 (v3.3.6).
Assembly: GRCm38

source_name: Hematopoietic Stem Cells

extract_protocol:
RNA was extracted from the sorted HSCs of mice using RNeasy Micro Kit (74004, Qiagen, Germany) as per manufacturer instructions.
The samples were ssequenced at core facility genomics (CFG MFM) in University Hospital Münster. The NEBNext  Poly(A) mRNA Magnetic Isolation module (NEB #E7490) protocol was used for library preparation.  Module (NEB #E7490)

value: Hematopoietic Stem Cells

value: No treatment


title: S10_HSCs_GFI1-36N_1


title: S15_HSCs_Gfi1-WT_3

value: Gfi1 WT/WT


title: V5-TET1, H1, WT, R1


title: S9_HSCs_GFI1-36S_3

value: GFI1 KI /KI


title: S13_HSCs_Gfi1-WT_1


title: S12_HSCs_GFI1-36N_3


title: S7_HSCs_GFI1-36S_1


title: ATACseq, H1, WT, R1


title: vehicle_4

city: Vienna

first: Martin

last: Direder

state: Vienna

address: Lazarettgasse 14

department: Orthopedic and Trauma Surgery

country: Austria

institute: Medical University Vienna

description: Single-cell RNAseq data

data_processing:
Raw sequencing data were demultiplexed, aligned to a reference genome and counted using Cell Ranger (version 3.0.2; except 5.0.1 [skin_7]; 10x Genomics).
Assembly: mm10
Supplementary files format and content: tsv files contain lists of genes and barcodes for each sample, mtx files count gene UMI counts for each sample

extract_protocol:
4mm tissue-biopsies were dissociated using MACS Miltenyi Whole Skin Dissociation Kit for mouse (Miltenyi)
libraries were prepared using Chromium Next GEM Single Cell 3´GEM, Library & Gel Bead Kit v3.1, Chromium Nexxt GEM Chip G Single Cell Kit and Single Index Kit T Set A (all 19x Genomics, Pleasanton, CA, USA)

value: dorsal skin

value: female

tag: age (weeks)

value: BALB/c


title: vehicle_3

description: Single-cell RNAseq data

tag: tissue


title: S5_HSCs_GFI1-KD_2

tag: cell type

value: GFI1 KD /KD


title: S4_HSCs_GFI1-KD_1

tag: treatment


title: vehicle_2

description: Single-cell RNAseq data


title: S6_HSCs_GFI1-KD_3

tag: genotype


title: S3_HSCs_Gfi1-KO_3

value: GFI1 KO /KO


title: vehicle_1

description: Single-cell RNAseq data


title: Liver_2_GEX_CD31+ECs _Control_DBZ_Dll4iDEC+Anti-VEGF_ Notch1/2/4iDEC_ Dll4 HETiDEC

description: 10x Genomics

value: Endothelial cells

value: Liver

value: mRNA

value: Cdh5-CreERT2 + iSuRe-Cre

value: 10-20 weeks old


title: PBMCsec_4

description: Single-cell RNAseq data


title: S14_HSCs_Gfi1-WT_2


title: S2_HSCs_Gfi1-KO_2


title: S8_HSCs_GFI1-36S_2


title: S1_HSCs_Gfi1-KO_1


title: PBMCsec_2

description: Single-cell RNAseq data


title: PBMCsec_1

description: Single-cell RNAseq data


title: L2-3 Replicate 2

first: Jiexi

last: Li

state: TX

address: 1881 East Road

institute: The University of Texas MD Anderson Cancer Center

data_processing:
The NextSeq RTA (version 2.4.11.0) was used for base calling
bcl2fastq (version 2.20.0) was used to convert the .bcl files into .fastq files
Fastq files were aligned to the mouse reference genome (mm9) using HISAT2 (version 2.1.0) and assembled using StringTie (version 2.1.1)
R (version 3.5.2) and R package DESeq2 (version 1.32.0) was used for data normalization and differential expression analysis
Assembly: mm9
Supplementary files format and content: raw counts

growth_protocol: Cells were cultured in DMEM with 10% tetracycline-free Fetal Bovine Serum (FBS), 1X penicillin/streptomycin (Invitrogen) and 1 µg/ml doxycycline.

extract_protocol:
RNA was isolated using the RNeasy Kit (Qiagen)
Libraries were generated using Illumina’s TruSeq Kit

tag: cell line

value: mouse CRC cell line


title: KO Replicate 3

source_name: KO

value: KO


title: HCT116, P53 Null, APR-246 plus irradiation, Rep2

city: Linkoping

first: Chuanwen

last: Fan

address: US campus

country: Sweden

institute: Linkoping university

data_processing:
RAW reads from 3 cell lines and 2 different conditions were analysed for their quality using fastqc. Then STAR aligner was used to map the filtered reads on the human genome (GRCh38.p13).
BAM files were sorted by coordinates within STAR (parameter: --outSAMtype BAM SortedByCoordinate). Gene counts were generated with the featureCounts tool from the subread package.
Unexpressed genes (those not represented by enough reads) were removed to reduce the problem associated with multiple testing in the DEG calculation phase retaining only those genes represented by at least 10 cpm (cpm=counts per million) reads in at least two samples.
The data were normalized to scale the raw library sizes. In order to assess the overall similarity between samples, a multidimensional scaling (MDS) analysis was performed before and after normalization.
The resulting lists were annotated through the Ensembl biomaRt library (Release-102) in R.
Assembly: GRCh38.p13
Supplementary files format and content: tab-delimited text files include CPM values for each Sample

source_name: Colon cancer cell line

growth_protocol: All 3 cell lines were grown in McCoy 5A medium, supplemented with 10% fetal bovine serum (FBS), and penicillin/streptomycin at 37°C in 5% CO2 and 95% humidity.

extract_protocol:
Total RNA were extracted from samples using TRIzol (Invitrogen, USA) according to manufactures's instructions. 1 µg total RNA was sent to BGI for quality testing and library construction based on the standard workflow.
Oligo(dT)-attached magnetic beads were used to purified mRNA. Purified mRNA was fragmented into small pieces with fragment buffer at appropriate temperature. Then First-strand cDNA was generated using random hexamer-primed reverse transcription, followed by a second-strand cDNA synthesis. afterwards, A-Tailing Mix and RNA Index Adapters were added by incubating to end repair. The cDNA fragments obtained from previous step were amplified by PCR, and products were purified by Ampure XP Beads, then dissolved in EB solution. The product was validated on the Agilent Technologies 2100 bioanalyzer for quality control. The double stranded PCR products from previous step were heated denatured and circularized by the splint oligo sequence to get the final library. The single strand circle DNA (ssCir DNA) was formatted as the final library. The final library was amplified with phi29 to make DNA nanoball (DNB) which had more than 300 copies of one molecular, DNBs were loaded into the patterned nanoarray and pair end 100 bases reads were generated on BGIseq500 platform (BGI-Shenzhen, China).

treatment_protocol: Cells were irradiated at 6Gy with or without pretreatment with 20 uM APR-246, and collected at 72 hours after irradiation.

value: Colon cancer cell line

value: APR-246 plus irradiation


title: KO Replicate 2


title: HCT116, P53 Null, irradiation, Rep1

value: Irradiation


title: HCT116, P53 Null, Solvent control, Rep2

value: Solvent


title: HCT116, P53 Null, APR-246, Rep1


title: HCT116, P53 Null, irradiation, Rep2


title: HCT116, P53 Null, APR-246, Rep2


title: L2-3 Replicate 3


title: L2-3 Replicate 1


title: 3346 Replicate 3


title: HCT116, P53 Mutant, APR-246 plus irradiation, Rep2

value: Mut


title: HCT116, P53 Null, Solvent control, Rep1


title: HCT116, P53 Null, APR-246 plus irradiation, Rep1


title: 3346 Replicate 2


title: 3346 Replicate 1


title: HCT116, P53 Mutant, irradiation, Rep2


title: 2A3 HEK293 RNaseP ic Rep2 SS2 Modified

first: Anthony

middle: McDowell

last: Mustoe

address: 1 Baylor Plaza

department: Biochemistry

data_processing:
Fastq files were aligned and parsed using ShapeMapper2 (v.2.1.5; https://github.com/Weeks-UNC/shapemapper2) for files designated Orig or Shapemapper2.2 for files designated 22dev-DMS
Parsed mutation files were analyzed using RingMapper (v1)
Assembly: sequence files used for alignment are included as supplementary files
Supplementary files format and content: Processed data archives (.tar) include Shapemapper reactivity profiles (profile.txt), normalized DMS reactivites (.dms), and pairmap analysis files (pairmap.txt). A complete description of these files is provided in the ShapeMapper and RingMapper documentation

source_name: adherent cell culture

growth_protocol: E. coli cells were grown in liquid LB media, while HEK293 cells were grown on 10 cm cell culture dishes in DMEM media.

extract_protocol:
Reverse-transcription was performed using specific primers, and DNA libraries were generated using a two-step PCR protocol.  Sample-specific modification and extraction details are provided in the SAMPLES section. All samples were DNase treated and purified using Qiagen RNeasy prior to reverse transcription.  Mutational profiling (MaP) reverse transcription (RT) was performed using an updated protocol that substantially improves yield and product length. In an 11 μL volume, RNA was mixed with 200 nM specific primer or 20 ng/μL random 9mer and 2 mM dNTPs and incubated at 65 °C for 10 min followed by 4 °C for 2 min. 8 uL 3x MaP buffer [1x MaP buffer consists of 6 mM MnCl2, 1 M betaine, 50 mM Tris (pH 8.0), 75 mM KCl, 10 mM DTT] was added and the combined solution incubated at 23 °C for 2 min. Finally, 1 μL SuperScript II Reverse Transcriptase (Invitrogen) was added and the RT reaction was performed according to the following temperature program: 25 °C for 10 min, 42 °C for 90 min, 10x[50 °C for 2 min, 42 °C for 2 min], 72 °C for 10 min. For the adenine riboswitch, ~1 pmol RNA was input into RT. For E. coli and human short RNAs, 0.5-2 μg total RNA was input into RT. For randomly primed E. coli RNA samples (16S and 23S rRNA), 1.5-3 μg total RNA was input into RT. RT products were then purified (Agencourt RNAclean XP beads, Beckman Coulter). Sequencing adaptors were added according to protocols listed in the SAMPLES section.
RNA was converted to cDNA using mutational profiling (MaP) reverse transcription, and libraries were prepared by 2-step PCR.

treatment_protocol:
HEK293 cell cultures were supplemented with bicine buffer [200 mM bicine (pH 8.0), final concentration], and modified with 170 mM DMS for 6 minutes at 37 °C. Total RNA was then extracted using Trizol.
RNA was folded at 30 C in 300 mM bicine (pH 8.0), 100 mM NaCl, 5 mM MgCl2 with variable adenine and modified with DMS (or ethanol) for 10 minutes, quenched with 20% 2-mercaptoethanol, and ethanol precipitated.

tag: probing condition

value: in-cell


title: HCT116, P53 Mutant, irradiation, Rep1


title: PBMCsec_3

description: Single-cell RNAseq data

value: dorsal skin

value: female


title: HCT116, P53 Mutant, APR-246, Rep2


title: 2A3 HEK293 RMRP ic Rep2 SS2 Untreated

value: DMSO


title: 2A3 HEK293 RNaseP ic Rep1 SS2 Modified


title: HCT116, P53 Mutant, APR-246, Rep1


title: HCT116, P53 Mutant, APR-246 plus irradiation, Rep1


title: 2A3 HEK293 RMRP ic Rep2 SS2 Modified


title: KO Replicate 1


title: HCT116, P53 Mutant, Solvent control, Rep2


title: HCT116, P53 wild type, APR-246 plus irradiation, Rep2

value: WT


title: HCT116, P53 Mutant, Solvent control, Rep1


title: DMS pH-nobuff HEK293 RMRP ic Rep2 Mara Modified 2A3 HEK293 RNaseP ic Rep2 SS2 Untreated

extract_protocol:
Reverse-transcription was performed using specific primers, and DNA libraries were generated using a two-step PCR protocol.  Sample-specific modification and extraction details are provided in the SAMPLES section. All samples were DNase treated and purified using Qiagen RNeasy prior to reverse transcription.  Mutational profiling (MaP) reverse transcription (RT) was performed using an updated protocol that substantially improves yield and product length. In a 5.8 μL volume, RNA was mixed with 200 nM specific primer or 20 ng/μL random 9mer and 2 mM dNTPs and incubated at 65 °C for 10 min followed by 4 °C for 2 min. 12.2 uL 3x MaP buffer [1x MaP buffer consists of 1 mM MnCl2, 50 mM Tris (pH 8.3), 200 mM KCl, 5 mM DTT, 20% glycerol] was added and the combined solution incubated at 23 °C for 2 min. Finally, 1 μL MarathonRT Reverse Transcriptase (Kerafast) was added and the RT reaction was performed according to the following temperature program: 42 °C for 3 h. For E. coli and human short RNAs, 0.5-2 μg total RNA was input into RT. For randomly primed E. coli RNA samples (16S and 23S rRNA), 1.5-3 μg total RNA was input into RT. RT products were then purified (Agencourt RNAclean XP beads, Beckman Coulter). Sequencing adaptors were added according to protocols listed in the SAMPLES section.
RNA was converted to cDNA using mutational profiling (MaP) reverse transcription, and libraries were prepared by 2-step PCR.

value: DMS

value: DMSO treated


title: 2A3 HEK293 RMRP ic Rep1 SS2 Modified


title: HCT116, P53 wild type, APR-246 plus irradiation, Rep1


title: HCT116, P53 wild type, irradiation, Rep2


title: HCT116, P53 wild type, APR-246, Rep2


title: DMS pH-nobuff HEK293 RMRP ic Rep1 Mara Modified 2A3 HEK293 RNaseP ic Rep1 SS2 Untreated


title: DMS pH8 HEK293 RMRP ic Rep2 Mara Modified


title: 2A3 HEK293 RMRP ic Rep1 SS2 Untreated


title: DMS pH7 HEK293 RMRP ic Rep2 Mara Modified


title: DMS pH8 HEK293 RMRP ic Rep1 Mara Untreated

value: EtOH


title: DMS pH8 HEK293 RMRP ic Rep1 Mara Modified


title: DMS pH7 HEK293 RMRP ic Rep1 Mara Modified


title: HCT116, P53 wild type, Solvent control, Rep1


title: HCT116, P53 wild type, Solvent control, Rep2


title: HCT116, P53 wild type, APR-246, Rep1


title: HEK293 RNaseP ic Rep2 TGIRT Untreated

extract_protocol:
Reverse-transcription was performed using specific primers, and DNA libraries were generated using a two-step PCR protocol.  Sample-specific modification and extraction details are provided in the SAMPLES section. All samples were DNase treated and purified using Qiagen RNeasy prior to reverse transcription.  Mutational profiling (MaP) reverse transcription (RT) was performed using an updated protocol that substantially improves yield and product length. In a 10 μL volume, RNA was mixed with 200 nM specific primer or 20 ng/μL random 9mer and 2 mM dNTPs and incubated at 65 °C for 10 min followed by 4 °C for 2 min. 8 uL of 5x MaP buffer [final concentration 50 mM Tris–HCl (pH 8.3), 75 mM KCl, and 3 mM MgCl2] was added and the combined solution incubated at 23 °C for 2 min. Finally, 1 μL TGIRT-III Reverse Transcriptase (InGex) was added and the RT reaction was performed according to the following temperature program: 60 °C for 2 h. For E. coli and human short RNAs, 0.5-2 μg total RNA was input into RT. For randomly primed E. coli RNA samples (16S and 23S rRNA), 1.5-3 μg total RNA was input into RT. RT products were then purified (Agencourt RNAclean XP beads, Beckman Coulter). Sequencing adaptors were added according to protocols listed in the SAMPLES section.
RNA was converted to cDNA using mutational profiling (MaP) reverse transcription, and libraries were prepared by 2-step PCR.


title: CR_TN_2248

city: Gdansk

first: Jakub

last: Mieczkowski

address: Debinki 12

country: Poland

institute: Medical University of Gdansk

data_processing:
RAW data (BCL) was converted to fastq files using bcl2fastq v2.20.0.422
fastq files were aligned to human reference genome hg38 and number of counts and spatial transcriptomics information were obtained using spaceranger count
For downstream analysis (data pre-processing, normalization, clustering, label transfering etc.), the processed data was read as Seurat (version 4.0.3) object
Genome_build: hg38
Supplementary_files_format_and_content: hdf5-spatial transcriptomics data

source_name: Temporal Neocortex

growth_protocol: Not applicable

extract_protocol:
Tissue permeabilization according to Vissum Spatial Gene Expression USER GUIDE CG30239RevC
Library construction according to Vissum Spatial Gene Expression USER GUIDE CG30239RevC
Vissum Spatial Gene Expression library

treatment_protocol: Hematoxylin eosin / Hematoxylin Congo Red

value: Brain

tag: brain

value: Temporal Neocortex


title: HEK293 RNaseP ic Rep2 TGIRT Modified


title: HEK293 RNaseP ic Rep2 SS2 Untreated


title: CR_TN_2339


title: CR_ON_2248

source_name: Orbitofrontal Neocortex

value: Orbitofrontal Neocortex


title: CR_ON_2339


title: HEK293 RNaseP ic Rep2 Mara Untreated


title: HE_TN_2248


title: HEK293 RNaseP ic Rep2 SS2 Modified


title: vsxMo_3

city: Napoli

first: Lorena

last: Buono

address: via Emanuele Gianturco 32

country: Italy

institute: IRCSS-SDN

data_processing:
Each sample was mapped against the index genome built from GRCz10 assembly using Hisat2 (Kim et al., 2015)
The aligned read SAM files were assembled into transcripts, their abundance was estimated and tested for differential expression by Cufflinks  v2.2.1(Trapnell et al., 2012)
Assembly: GRCz10
Supplementary files format and content: fpkm values (cufflinks output)

organism: Danio rerio

source_name: head

growth_protocol: zebrafish embryos were kept in E3 medium at 28°C until 18 hpf stage

extract_protocol:
Trizol extraction as manufacturer's instruction; DNAse treatment
RiboZero stranded RNA

treatment_protocol: vsx2 morpholino was injected in zebrafish fertilized embryos at 1-cell stage. Injected embryos were kept in E3 medium at 28 °C until 18 hpf. Head of zebrafish embryos at 18 hpf were dissected and used for RNA extraction.

value: head

tag: developmental stage

value: 18 hpf

value: injection with vsx2 morpholino


title: HE_TN_2339


title: HCT116, P53 wild type, irradiation, Rep1

value: Colon cancer cell line

value: Irradiation


title: HEK293 RNaseP ic Rep2 Mara Modified

tag: strain


title: HE_ON_2248


title: vsxMo_2


title: HEK293 RNaseP ic Rep1 TGIRT Modified


title: HEK293 RNaseP ic Rep1 TGIRT Untreated


title: vsxKO_2 [atac-seq]

data_processing:
data aligned with Bowtie2 (Langmead et al., 2012)
peakcalling with MACS2 (Zhang et al., 2008)
Genome_build: danRer10
Supplementary_files_format_and_content: narrowPeak files (MACS2 output)

extract_protocol:
DNA tagmented as in Buenrostro et al., 2015
Library prepared as in Buenrostro et al., 2015


title: vsxMo_1


title: HEK293 RNaseP ic Rep1 SS2 Untreated


title: HEK293 RNaseP ic Rep1 SS2 Modified


title: HE_ON_2339


title: WT_2 [atac-seq]


title: vsxKO_1 [atac-seq]


title: THP1_repI

city: Ribeirão Preto

first: Ana

middle: Lucia

last: Fachin

state: SP

address: Av. Costabile Romano, 2201

department: Biotechonology  Unit

country: Brazil

institute: Unaerp

data_processing:
FastQC v0.11.7 was used to visualize the libraries quality before and after trimming.
Trimmomatic v0.36 was used to remove adapters and quality filtered setting the cutoff threshold for average base quality score at 15 over a window of 4 bases. Reads shorter than 36 bases post-trimming were excluded.
Filtered reads were mapped onto the human genome (hg38) using the STAR v2.5.2b.
Gene level read-counts were quantified using STAR’s –quantMode GeneCounts parameter.
The data were analyzed using the R package DESeq2. The samples were normalized, and the dispersions were estimated using the default settings. The package was then used to identify differentially expressed genes for each pairwise comparison of interest. Genes with an adjusted P value of < 0.05 and a log2(foldchange) >= 1.0 (upregulation) or <= -1.0 (downregulation) were called as differentially expressed.
Genome_build: Human genome (hg38).
Supplementary_files_format_and_content: tab-delimited text files include reads count values for each sample and DESeq output in CSV format containing mean expression, fold changes and p value.

source_name: THP1 - control, replicate I

growth_protocol: The Trichophyton rubrum strain CBS 118892 was cultured on Sabouraud dextrose agar for 15 days at 28ºC. Macrophages were cultured in RPMI medium supplemented with 10% fetal bovine serum (FBS) at 37ºC in a humidified atmosphere containing 5% CO2.

extract_protocol:
RNA extraction of T. rubrum and THP-1 were performed using an Illustra RNAspin Mini RNA Isolation according to GE Healthcare guidelines.
RNA libraries were prepared for sequencing using standard Illumina protocols.

treatment_protocol: Pre-cultured conidia and heat inactive of T. rubrum were added to THP-1 cell line and the co-culture was incubated for 24 h at 37ºC in a humidified atmosphere containing 5% CO2.

value: human THP-1 macrophages


title: THP1_repIII

source_name: THP1 - control, replicate III


title: HEK293 RNaseP ic Rep1 Mara Untreated

value: in-cell


title: THP1_repII

source_name: THP1 - control, replicate II


title: HEK293 RNaseP ic Rep1 Mara Modified


title: vsxKO_3

data_processing:
Each sample was mapped against the index genome built from GRCz10 assembly using Hisat2 (Kim et al., 2015)
The aligned read SAM files were assembled into transcripts, their abundance was estimated and tested for differential expression by Cufflinks  v2.2.1(Trapnell et al., 2012)
Genome_build: GRCz10
Supplementary_files_format_and_content: fpkm values (cufflinks output)


title: Ecoli tmRNA cf Rep1 TGIRT Untreated

organism: Escherichia coli

source_name: liquid cell culture

treatment_protocol:
Cell-free RNA from E. coli was obtained by gently extracting total RNA from exponential E. coli cultures that had been treated for 20 minutes with rifampicin to chase RNAs and RNA-protein complexes to fully assembled states. Cell-free RNA was equilibrated in 200 mM bicine (pH 8.0), 200 mM KOAc, 5 mM MgCl2 and modified with 170 mM DMS for 6 minutes at 37 °C.
RNA was folded at 30 C in 300 mM bicine (pH 8.0), 100 mM NaCl, 5 mM MgCl2 with variable adenine and modified with DMS (or ethanol) for 10 minutes, quenched with 20% 2-mercaptoethanol, and ethanol precipitated.

tag: substrain

value: cell-free


title: Ecoli tmRNA cf Rep1 SS2 Modified


title: Ecoli tmRNA cf Rep1 TGIRT Modified


title: WT_1 [atac-seq]


title: Ecoli tmRNA ic Rep2 TGIRT Untreated

treatment_protocol:
Exponential E. coli cultures were treated for 20 minutes with rifampicin, pelleted, resuspended in 200 mM bicine (pH 8.0), 200 mM KOAc, 5 mM MgCl2, and modified with 700 mM DMS for 6 minutes at 37 °C. Total RNA was then extracted using Trizol.
RNA was folded at 30 C in 300 mM bicine (pH 8.0), 100 mM NaCl, 5 mM MgCl2 with variable adenine and modified with DMS (or ethanol) for 10 minutes, quenched with 20% 2-mercaptoethanol, and ethanol precipitated.


title: Ecoli tmRNA ic Rep2 SS2 Untreated


title: HEK293 RMRP ic Rep2 HIV Untreated

extract_protocol:
Reverse-transcription was performed using specific primers, and DNA libraries were generated using a two-step PCR protocol.  Sample-specific modification and extraction details are provided in the SAMPLES section. All samples were DNase treated and purified using Qiagen RNeasy prior to reverse transcription.  Mutational profiling (MaP) reverse transcription (RT) was performed using an updated protocol that substantially improves yield and product length. In a 9 μL volume, RNA was mixed with 500 nM specific primer and 2 mM dNTPs and incubated at 70 °C for 10 min followed by 4 °C for 2 min. 9 uL 5x MaP buffer [1x MaP buffer consists of 200 mM Tris-HCl (pH 8.3), 400 mM KCl, 20 mM MgCl2] was added and the combined solution incubated at 23 °C for 2 min. Finally, 2 μL eHIV Reverse Transcriptase was added and the RT reaction was performed according to the following temperature program: 42 °C for 3 h, 95 °C for 1 min. For human short RNAs, 0.5-2 μg total RNA was input into RT. RT products were then purified (Agencourt RNAclean XP beads, Beckman Coulter). Sequencing adaptors were added according to protocols listed in the SAMPLES section.
RNA was converted to cDNA using mutational profiling (MaP) reverse transcription, and libraries were prepared by 2-step PCR.


title: Ecoli tmRNA cf Rep1 Mara Untreated


title: Ecoli tmRNA ic Rep2 TGIRT Modified


title: Ecoli tmRNA cf Rep1 SS2 Untreated


title: Ecoli tmRNA ic Rep2 SS2 Modified


title: vsxKO_2


title: Ecoli tmRNA ic Rep2 Mara Untreated


title: Ecoli tmRNA cf Rep1 Mara Modified


title: Ecoli tmRNA ic Rep2 Mara Modified


title: HEK293 RMRP ic Rep2 HIV Modified


title: vsxKO_1


title: WT_3


title: Ecoli tmRNA ic Rep1 SS2 Untreated


title: Ecoli tmRNA ic Rep1 TGIRT Untreated


title: HEK293 RMRP ic Rep2 TGIRT Modified


title: Ecoli tmRNA ic Rep1 Mara Untreated


title: WT_2


title: Ecoli tmRNA ic Rep1 Mara Modified


title: HEK293 RMRP ic Rep2 SS2 Untreated


title: Ecoli tmRNA ic Rep1 TGIRT Modified


title: WT_1


title: HEK293 RMRP ic Rep2 SS2 Modified


title: SMYD2 KO rep3

city: Guangzhou

first: Yu

last: Zhou

state: Guangdong

address: No 58, Zhongshan 2nd street

department: Division of Vascular Surgery

institute: The First Affiliated Hospital, Sun Yat-sen University

data_processing:
We used BGISEQ-500 platform for basecalling.
We use internal software SOAPnuke (version v1.5.2)
Reads filtered with parameters: -l 15 -q 0.2 -n 0.05
We mapped clean reads to reference using Bowtie2 (Version: v2.2.5; Parameters: -q --phred64 --sensitive --dpad 0 --gbar 99999999 --mp 1,1 --np 1 --score-min L,0,-0.1 -I 1 -X1000 --no mixed --no-discordant -p 1 -k 200)
Calculate gene expression level with RSEM(Version: v1.2.12; Parameters: default).
Supplementary_files_format_and_content: FPKM

source_name: Aortic smooth muscle cell

growth_protocol: Primary Aortic smooth muscle cells isolated from FLox and SMYD2 KO mice were cultured in 10% FBS DMEM and passage 6 of the cells were collected for RNA extraction

extract_protocol:
Aortic smooth muscle cells were collected and RNA was harvested using Promega RNA extraction kit. Illumina TruSeq RNA Sample Prep Kit (Cat#FC-122-1001) was used with 1 ug of total RNA for the construction of sequencing libraries.
RNA libraries were prepared for sequencing using standard Illumina protocols

tag: tissue source

value: Aorta

value: smooth muscle

value: SMYD2 KO


title: SMYD2 KO rep2


title: HEK293 RMRP ic Rep2 Mara Untreated


title: SMYD2 KO rep1


title: Ecoli rRNA cf Rep2 Mara Untreated


title: Ecoli rRNA cf Rep2 Mara Modified

value: DMS


title: Ecoli rRNA cf Rep1 Mara Untreated


title: Ecoli rRNA cf Rep1 SS2 Untreated


title: Ecoli rRNA cf Rep1 SS2 Modified


title: HEK293 RMRP ic Rep2 Mara Modified


title: Ecoli rRNA cf Rep2 SS2 Modified


title: HEK293 RMRP ic Rep1 HIV Untreated


title: Ecoli rRNA ic Rep1 TGIRT Untreated


title: Flox rep3

value: SMYD2 Flox


title: Ecoli rRNA cf Rep1 Mara Modified


title: HEK293 RMRP ic Rep1 HIV Modified


title: Ecoli rRNA ic Rep1 TGIRT Modified


title: Ecoli tmRNA ic Rep1 SS2 Modified


title: Flox rep2


title: Ecoli rRNA ic Rep1 SS2 Untreated

value: EtOH


title: HEK293 RMRP ic Rep1 TGIRT Untreated


title: Flox rep1


title: Ecoli rRNA cf Rep2 SS2 Untreated


title: HEK293 RMRP ic Rep1 TGIRT Modified


title: HEK293 RMRP ic Rep1 SS2 Untreated


title: amhc:EGFP+;myl7:dsRED-NLS+ WT control

city: Cincinnati

first: Joshua

last: Waxman

state: OH

address: 240 Albert Sabin Way

department: Molecular Cardiovascular Biology Division

institute: Cincinnati Children's Hospital Medical Center

data_processing:
RNA-seq reads in FASTQ format were subjected to quality control to assess the need for trimming of adapter sequences or bad quality segments using FastQC v0.11.5 , Trim Galore! v0.4.2  and cutadapt v1.9.1.
The trimmed reads were aligned to the reference zebrafish genome version danRer10 with the program STAR v2.5.2.
Aligned reads were stripped of duplicate reads with the program picard v1.89.
Gene-level expression was assessed by counting features for each gene, as defined in the NCBI's RefSeq database.
Read counting was done with the program featureCounts v1.5.3 from the Rsubread package.
Raw counts were normalized as transcripts per million (TPM).
ATAC-seq reads in FASTQ format were first subjected to quality control to assess the need for trimming of adapter sequences or bad quality segments using FastQC v0.11.5, Trim Galore! v0.4.2 and cutadapt v1.9.1
The trimmed reads were aligned to the reference zebrafish genome version danRer10 with the program STAR v2.5.2 with splice awareness turned off.
Aligned reads were stripped of duplicate reads with the program picard v1.89.
Peaks were called using the program MACS v2.1.2 using the broad peaks mode.
Peaks with q-value < 0.01 were selected for further analysis.
Common peaks among all samples were obtained by merging called peaks at 50% overlap using BEDtools v2.27.0.
The common peaks, originally in BED format were converted to a Gene Transfer Format (GTF) to enable fast counting of reads under the peaks with the program featureCounts v1.5.3 (Rsubread package).
BAM files are converted into wig format using bedtools v2.27.0.
The normalization is performed using a scaling factor proportional to the total number of per million mapped reads in the BAM file.
Wig files are converted to bigWig format using the tool wigToBigWig.
Genome_build: GRCz10/danRer10
Supplementary_files_format_and_content: text file of matrix table with normalized transcripts per million read counts
Supplementary_files_format_and_content: text file of matrix table with raw read counts of ATAC peak coordinates
Supplementary_files_format_and_content: bigwig file of the ATAC-Seq data from wt and nr2f1a mutant samples

source_name: amhc:EGFP+;myl7:dsRED-NLS cells

extract_protocol:
For amhc:EGFP+ and amhc:EGFP+/myl7:DsRED-NLS cells, they were flow sorted following digestion of whole embryos at 48 hours post-fertlization. For RNA, the RNA was harvested using TRIZOL and a PureLink RNA Micro Kit (Invitrogen). The libary was then amplified using a Single Cell Purification Kit (Norgen Biotek). For ATAC-seq, the harvested cells were processed for ATAC-seq with the Tn protein and libraries generated.
RNA and ATAC libraries were prepared for sequencing using standard protocols

value: AB/TU

value: sorted amhc:EGFP+;myl7:dsRED-NLS cells

tag: age

value: 48 hours post-fertilization


title: HEK293 RMRP ic Rep1 SS2 Modified


title: amhc:EGFP+ nr2f1a mutant

source_name: amhc:EGFP+ cells

value: sorted amhc:EGFP+ cells


title: HEK293 RMRP ic Rep1 Mara Untreated


title: amhc:EGFP+ WT control