title: Grace, drought, F.avenaceum, 96hpi, rep3

value: Fusarium avenaceum


title: IGROV-1-EPZ-5676 treated replicate 2

status: Public on Sep 15 2023

type: SRA

city: Birmimgham

first: Romi

last: Gupta

state: Alabama

address: 720, 20th street south

country: USA

institute: University of Alabama at Birmingham

library_source: transcriptomic

data_processing:
Illumina bcl2fastq version 2.18.0.12 was used for basecalling
The raw fastq sequences were aligned to the human hg38 reference genome (GenBank assembly accession: GCA_000001405.28). The alignments were carried out using STAR (version 2.7.1a) (Dobin et al 2013) with default parameters. Gene expression was quantified as gene level counts using the htseq-count function (version 0.12.3) (Anders et al 2015); the UCSC gene annotations for the human genome were used. The htseq-count default parameters were used, except for the strand parameter which was set to reverse to consider the strandedness of library. Genes for which there are less than 3 samples with normalized counts greater than or equal to 4 were filtered out. Differentially expressed genes were identified using DESeq2 (version 1.28) (Love et al 2014).
Genome_build: human hg38 reference genome (GenBank assembly accession: GCA_000001405.28)
Supplementary_files_format_and_content: tab-delimited text file containing raw counts for each gene in each sample

molecule: total RNA

organism: Homo sapiens

source_name: IGROV-1 cell line

growth_protocol: IGROV-1 and SK-OV-3 cells were grown in RPMI medium and DMEM medium respectively with 10% Fetal bovine serum and Penicilline and Streptomycin at  37 C under 5% CO2 condition.

extract_protocol:
Total RNA was extracted using Trizol reagent (Invitrogen) as per the instruction from the manufacture and then purified using RNAeasy mini columns (Qiagen) as per the manufactures instructions.
mRNA is purified from approximately 500ng of total RNA with oligo-dT beads and sheared by incubation at 94C.  Following first-strand synthesis with random primers, second strand synthesis is performed with dUTP for generating strand-specific sequencing libraries.  The cDNA library is then end-repaired, and A-tailed, adapters are ligated and second-strand digestion is performed by Uricil-DNA-Glycosylase. Indexed libraries that meet appropriate cut-offs for both are quantified by qRT-PCR using a commercially available kit (KAPA Biosystems) and insert size distribution determined with the LabChip GX or Agilent Bioanalyzer. Samples with a yield of ≥0.5 ng/ul are used for sequencing.

treatment_protocol: IGROV-1 and SK-OV-3 cells were treated with  DOT1L inhibitor (10 micomolar) EPZ-5676 for 48 hours,  were harvested and were used for preparing the total RNA and to perfrom RNA Seq.

tag: cell line

tag: agent

value: EPZ-5676 treated, 10 micromolar, 48 hours


title: EpiSC_WT2_rep2 [rEpiSC_WT#2_2]

city: Kashihara

first: Hisato

last: Kobayashi

state: Nara

address: 840 Shijo-Cho

department: Department of Embryology

country: Japan

institute: Nara Medical University

data_processing:
>Basecalls were performed using NextSeq 500/550 RTA software,v2.11.3.
>FASTQ files were generated using bcl2fastq,v2.17.1.14
>TPM valuses were calculated using RaNA-seq program (https://ranaseq.eu/index.php).
Assembly: rn6
Supplementary files format and content: A single text file that lists coding gene log2(TPM+1) values for each sample

organism: Rattus norvegicus

source_name: Epiblast stem cell

growth_protocol:
rESCs were maintained on mitomycin-C-treated mouse embryonic fibroblasts (MEFs) in rESC medium; N2B27 medium supplemented with 1 µM PD0325901 (Axon, Groeningen, The Netherlands), 3 µM CHIR99021 (Axon), and 1000 U/ml of rat LIF (Millipore, Bedford, MA). For passaging, confluent rESC colonies were dissociated by 0.25% Trypsin-EDTA and re-plated to a well of 12-well plates (IWAKI, Shizuoka, Japan) containing 1 ml of rESC medium per well. The rESCs are routinely passaged every 3 days.
For derivation of rEpiSCs, E7.5-8.5 rat epiblasts were isolated as described previously (Kobayashi et al., 2020). Briefly, the ectoplacental cone and extra-embryonic ectoderm were mechanically dissected out and used for genotyping. The embryonic visceral endoderm surrounding the epiblast was removed by enzyme solution containing 1.0 mg/ml collagenase type IV (Worthington Biochemical Corporation), 1.0 mg/ml dispase (FUJIFILM Wako, Osaka, Japan), and 0.3 mg/ml hyaluronidase (Sigma-Aldrich) for 5 min at 37°C followed by repeated pipetting with a glass capillary. The isolated epiblasts were individually explanted on MEF in rEpiSC medium; N2B27 medium supplemented with 5% KSR, 20 ng/ml Activin-A (Peprotech, Cranbury, NJ), 12 ng/ml bFGF (Peprotech), two Wnt inhibitors (2 µM IWP2 (Tocris Bioscience, Bristol, UK) and 5 µM XAV-939 (Abcam), and 5 µM ROCKi (Tocris Bioscience). After 5 days, the expanded epiblast outgrowth was mechanically dissociated using a glass capillary. From the second passage, rEpiSC colonies were dissociated by Accutase (Innovative Cell Technologies) and re-plated to a well of MEF coated 12-well plates containing 1 ml of rEpiSC medium per well. The rEpiSCs were routinely passaged every 3-4 days.
rEpiLCs and rPGCLCs inductions were performed according to our recent report (Oikawa et al., 2022). Briefly, trypsinized rESCs were seeded in 96-well Nunclon Sphera-Treated U-shaped microplate containing 100 µl of rEpiLC medium (N2B27 medium supplemented with 1% KSR, 20 ng/ml Activin-A, and 12 ng/ml bFGF) at 4x10e3 cells per well. After culture for 48-60 h, rEpiLC-aggregates were transferred into a well of 96-well Nunclon Sphera-Treated U-shaped microplate containing 100 µl of rPGCLC medium [N2B27 medium supplemented with 500 ng/ml BMP4 (Peprotech), 1000 U/ml rLIF, 100 ng/ml SCF (R&D Systems), and 50 ng/ml EGF (R&D Systems)]. After 3 days, rEpiLC-derived rPGCLCs (LC-rPGCLCs) were analyzed by FACS, IF and RT-qPCR. To directly induce rPGCLCs from rEpiSCs, trypsinized rEpiSCs were seeded in 96-well Nunclon Sphera-Treated U-shaped microplate containing 100 µl of rPGCLC medium at 0.5-1.5x10e4 cells per well. After 3 days, rEpiSC-derived rPGCLCs (SC-rPGCLCs) were analyzed.
For the reprogramming of rEpiSCs, the 6 candidate genes (rKlf2, rKlf4, rEsrrb, rTbx3, rTfcp2l1, rNanog) were cloned individually into a PiggyBac transposon vector containing Doxycycline (Dox)-inducible Tet-On system. The plasmids were transfected into rEpiSCs by reverse transfection protocol using lipofectamine 2000 (Kobayashi et al., 2017). Transfected rEpiSCs were harvested on neomycin-resistant feeder (Kitayama labes, Nagano, Japan). Two days after transfection, 400 µg/ml G418 (Sigma-Aldrich) was added in the medium and rEpiSCs were cultured for 3-4 days. After G418 selection, 6 transcription factors-transfected rEpiSCs (6TFs-rEpiSCs) were re-plated at 2~10 x 10e4 cells per well of MEF coated 12-well plates containing 1 ml of rEpiSC medium supplemented with 1 µg/ml Dox (Sigma-Aldrich). On the next day, the medium was replaced by rESC medium supplemented with Dox. The PV(+) dome-shaped colonies were picked manually at day 5-7, and then dissociated by 0.25% Trypsin-EDTA into single cells, and re-plated into a well of 12-well plates containing 1 ml of rESC medium without Dox. PV(+) rESCLCs were routinely passaged every 3 days.

extract_protocol:
Total RNA was extracted using PicoPure RNA Isolation Kit (Thermo Fisher Scientific) according to manufacturer’s protocols.
RNA-seq libraries were prepared using the SMART-Seq HT PLUS Kit (Takara Bio).

tag: strain

tag: genotype

value: Wild type

tag: cell type

value: Epiblast stem cell


title: Grace, drought, F.avenaceum, 96hpi, rep2

city: Freising

first: Ralph

last: Hückelhoven

address: Emil-Ramann-Str. 2

department: Chair of Phytopathology

country: Germany

institute: Technische Universität München

data_processing:
Data processing steps for the CPM_Horvu_Morex_V2
The quality of the raw RNAseq data was analyzed with FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc).
The trimming step was done with Trimmomatic using the parameters ILLUMINACLIP:Illumina-SE.fasta:2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:40.
We used Hisat2 with  parameters -U --rna-strandness FR to map the trimmed data to the Morex v2 assembly. In order to add an artificial 3’UTR because of the 3’-RNAseq reads, we used a custom script to elongate the last exon either by 3 kb or until the next gene in the gff file of Morex v2.
The counts of the generated bam files were assigned to genes using featureCounts with the parameters -t gene -s 1 -M -O.
CPM values were calculated using edgeR with "calcNormFactors" and "cpm( log = FALSE, normalized.lib.sizes = TRUE)"

Data processing steps for the CPM_Horvu_Morex_V3 and CPM_Fusarium_culmorum_UK99
The RNAseq data was analyzed using the nf-core/rnaseq pipeline version 3.9 with default settings. An artificially merged genome was used for mapping consisting of MorexV3 and Fc-UK99
The gene counts were split again into the two species and CPM values were calculated separately for each species using edgeR with "calcNormFactors" and "cpm( log = FALSE, normalized.lib.sizes = TRUE)"

Assembly: MorexV2 (Mascher et al., 2019), MorexV3 (Mascher et al., 2020), Fc UK99 (Urban et al., 2016)

Supplementary files format and content: CPM_Horvu_Morex_V2.csv is a comma-separated file containing the counts per million values for all samples, mapped against MorexV2
Supplementary files format and content: CPM_Horvu_Morex_V3.csv is a comma-separated file containing the counts per million values for all samples, mapped against MorexV3
Supplementary files format and content: CPM_Fusarium_culmorum_UK99.csv is a comma-separated file containing the counts per million values for all samples, mapped against Fusarium culmorum UK99

organism: Hordeum vulgare

source_name: spike

growth_protocol: The pots were placed on flooding tables for daily automatic watering and with additional lightning for 16 h per day. Plants were held upright with support devices and were randomized twice a week to assure uniform growth. Temperature was set to 18 °C (day) and 16 °C (night) with a relative air humidity of 60 %. Drought conditions were set on by a stop of automatic watering on separate flooding tables. To prevent plants from early and premature plant death due to fast desiccation of the substrate, each pot received little amounts of water (50 - 100 ml) in the first three days after stopping daily irrigation.

extract_protocol:
Each individual spike sample of each biological triplicate per treatment variation was divided into two pieces using one part for individual RNA extraction. After separation, the spike samples were immediately ground in liquid nitrogen and stored at minus 70 °C. RNA extraction was performed with Direct-zol RNA Miniprep Plus Kit (ZymoResearch, USA) according to manufacturer’s protocol.
Preparation of libraries for 3’mRNA sequencing was carried out with Lexogen QuantSeq 3'mRNA-Seq Library Prep Kit (FWD) (Lexogen, Austria) for Illumina sequencing according to manufacturer’s protocol. Quantification of input RNA was conducted with Qubit Fluorometer 2.0 (Invitrogen, USA) and Qubit RNA BR (broad range) Assay Kit (Invitrogen, USA) according to manufacturer’s protocol in a range of 10 to 1200 ng total RNA. Quantification of library size was performed with Qubit Fluorometer 2.0 und Qubit DNA High Sensitivity Assay Kit (Invitrogen, USA). A check for distribution of mRNA fragment size of each library was performed with am Agilent 2100 Electrophoresis Bioanalyzer (Agilent Technologies, USA). Final quantification of each library was determined according to Illumina qPCR guide with a KAPA SYBR Fast Mastermix Low ROX (Peqlab, Germany) in a QuantStudio 5 real-time PCR system (Applied Biosystems, USA). Final libraries were normalized with elution buffer (Qiagen, Germany) to a final concentration to 2 nM and pooled with an equal amount of each sample library. Denaturation and dilution of libraries for HiSeq Clustering was performed according to protocol A of user guide (Illumina) with a concentration for clustering of 10 pM.

treatment_protocol: Fugal inoculum was cultured and propagated according to Linkmeyer et al. (2013). Therefore, three isolates of F. culmorum (Fc002, Fc03, Fc06 – culture collection, Chair of Phytopathology, Technical University of Munich) and three isolates of F. avenaceum (Fa002, Fa02, Fa04 – culture collection, Chair of Phytopathology, Technical University of Munich) known to strongly infect barley spikes and to produce DON (Linkmeyer et al., 2013; Hofer et. al., 2016, Hoheneder et al., 2022) were used in equal amounts. Spore solution was adjusted to 50.000 conidia per ml tap water and contained 1 ml/L Tween 80 to improve wetting of the spike tissue. The respective mock solution contained the same amount of Tween 80. The spikes of the plants were sprayed with handheld spray flasks till run-off either with F. culmorum spore solution or mock solution. To maintain optimal moist conditions for infection (99 % relative air humidity), spikes were covered and sealed with transparent polythene bags for two days. Respective mock sprayed plants were similarly treated. 48h and 96h after inoculation, individual spikes were cut from each pot and flash frozen in liquid nitrogen.

tag: cultivar

value: Grace

tag: tissue

value: spike

tag: abiotic stress

value: drought

tag: inoculation

tag: harvest time point [hpi]


title: IGROV-1-EPZ-5676 treated replicate 3


title: SC_PGCLC_d3_rep1 [SC_rPGCLC_d3_1]

source_name: Primordial germ cell-like cell

value: Primordial germ cell-like cell


title: IGROV-1-EPZ-5676 treated replicate 1


title: IGROV-1-DMSO treated replicate 3

value: DMSO treated


title: Grace, drought, F.avenaceum, 96hpi, rep1


title: Grace, drought, F.culmorum, 96hpi, rep3

value: Fusarium culmorum


title: EpiSC_WT2_rep1 [rEpiSC_WT#2_1]


title: SK-OV-3-EPZ-5676 treated replicate 3

source_name: SK-OV-3 cell line


title: EpiSC_WT1_rep2 [rEpiSC_WT#1_2]


title: EpiSC_WT1_rep1 [rEpiSC_WT#1_1]


title: IGROV-1-DMSO treated replicate 1


title: IGROV-1-DMSO treated replicate 2


title: Grace, drought, F.culmorum, 96hpi, rep1


title: Grace, drought, F.culmorum, 96hpi, rep2


title: D3-20

city: Basel

first: Antoine

last: Peters

address: Maulbeerstrasse 66

country: Switzerland

institute: Friedrich Miescher Institute

library_source: transcriptomic single cell

data_processing:
RNA-Seq datasets were aligned to a custom genome containing the Mus musculus genome assembly (GRCm38/mm10 Dec. 2011) and ERCC92 sequences using STAR (Dobin et al., 2013) with parameters "-outFilterMultimapNmax 300 -outMultimapperOrder Random -outSAMmultNmax 1 -alignIntronMin 20 -alignIntronMax 1000000", allowing multimappers with up to 300 matches in the genome and choosing positions for multimappers randomly
A random transcript isoform for each gene was chosen from Bioconductor annotation package TxDb.Mmusculus.UCSC.mm10.knownGene (version 3.2.2) and exonic genomic regions for the random transcript isoforms for each gene were used in further analyses.
Expression quantification for selected exonic genomic regions was done using QuasR R package (Gaidatzis et al., 2015) selecting only uniquely mapped reads (mapqMin=255).
Assembly: mm10
Supplementary files format and content: CSV file contatining read count table for exonic regions of each gene (column ENTREZID), total length of genomic regions for each gene (column width) and each single oocyte.

molecule: polyA RNA

organism: Mus musculus

source_name: in vivo-derived FGO

extract_protocol:
Each GRO and FGO derived from BVSC-iPSCs, E12.5 gonads, 6 dpp follicles and in vivo samples was dissociated from follicular somatic cells using 30G needles, followed by treatment with Accumax (Innovative Cell Technologies) for 5 min and by pipetting to remove somatic cells completely. The zona pellucida of each GRO and FGO was removed by acid tyrode’s solution (Merck) and samples were individually washed in PBS supplemented with 0.01% PVA (PBS-PVA) and lysed in the lysis buffer composed of 0.09% Triton-X 100, 2 U SUPERase IN RNase inhibitor (Invitrogen, AM2694), 2.5 μM Oligo-dT primer (Microsynth AG), dNTP mix (2.5 mM each, Promega), ERCC RNA Spike-In Mix (1 : 3.2 x 107, Thermo Fischer Scientific 4456740) in individual tubes of a 8-well strip, then immediately frozen on the dry ice and kept at -80C for longer storage.
RNA sequencing libraries for single oocytes were prepared according to the Smartseq2 protocol

value: in vivo-derived FGO


title: D3-21


title: D3-19


title: EpiLC_rep2 [rEpiLC_2]

source_name: Epiblast-like cell

value: Epiblast-like cell


title: EpiSC_PV_rep1 [rEpiSC_PV_1]


title: D3-18


title: EpiLC_rep1 [rEpiLC_1]


title: D3-17


title: Grace, irrigated, mock, 48hpi, rep3

value: irrigated

value: Mock


title: ESC_N3T_rep2 [rESC_N3T_2]

source_name: Embryonic stem cell

value: Embryonic stem cell


title: D3-16


title: D3-15


title: D3-22


title: Grace, irrigated, mock, 48hpi, rep2


title: ESC_N3T_rep1 [rESC_N3T_1]


title: D3-14


title: D3-13


title: D3-12


title: Grace, irrigated, mock, 48hpi, rep1


title: D3-11


title: ESC_PV_rep1 [rESC_PV_1]


title: D3-9


title: D3-10


title: Grace, irrigated, F.avenaceum, 48hpi, rep2


title: ESC_WT_rep2 [rESC_WT_2]


title: ESC_WT_rep1 [rESC_WT_1]


title: Grace, irrigated, F.avenaceum, 48hpi, rep1


title: Grace, irrigated, F.culmorum, 48hpi, rep3


title: D3-3


title: D3-4


title: bovine, control, scRNAseq

city: Gliwice

first: Anna

last: Papiez

address: Akademicka 16

department: Department of Data Science and Engineering

country: Poland

institute: Silesian University of Technology

description: 10x Genomics

data_processing:
The demultiplexing, barcoded processing, gene counting were made using the Cell Ranger software v3.1.0 (https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/what-is-cell-ranger)
Assembly: hg38; bt12
Supplementary files format and content: Tab-separated values files and matrix files

source_name: blood

extract_protocol:
PBMCs sample from cattle and human were isolated using pancoll (Pan-Biotech) gradient density centrifugation following the manufacturer’s protocol. Purified PBMCs were counted using a Neubauer chamber and resuspended in RPMI-1640 + 10% FCS at a concentration of 1 - 5x10^6 PBMCs / ml medium for downstream applications.  Cells were rested overnight at 37°C and 5% CO2 before stimulation with 10 µg/ml Mtb H37Rv whole cell lysate (BEI Resources Repository NIAID) for 3h at 37°C and 5 %CO2. Control samples were left untreated. For qPCR CD14+ monocytes were isolated using an anti-human CD14 antibody (Biolegend, Clone: M5E2) or an anti-bovine CD14 antibody (Kingfisher Biotech, Clone: CAM66A) using magnetic-based enrichment.
Library quality and concentration was assessed by Bioanalyzer (Agilent Technology) and the KAPPA library quantification kit (Roche) following the manufacturer’s instructions. Libraries were pooled and sequenced using a on an Illumina HiSeq1500 instrument using paired-end sequencing (26 bp Read 1 and 98 bp Read 2) with a single sample index (8bp i7 index) and using a HiSeq Rapid SBS Kit V2 (Illumina).

value: PBMC

value: blood

tag: treatment

value: control


title: D3-1


title: Grace, irrigated, F.culmorum, 48hpi, rep2


title: Grace, irrigated, F.culmorum, 48hpi, rep1


title: C3-23

source_name: 6dpp follicle-derived FGO

value: 6dpp follicle-derived FGO


title: human, control, scRNAseq

description: 10x Genomics


title: ESC_PV_rep2 [rESC_PV_2]

value: Wild type


title: C3-22


title: C3-19


title: Grace, irrigated, F.avenaceum, 48hpi, rep3

value: Fusarium avenaceum


title: C3-20


title: C3-21


title: D3-7


title: C3-18


title: D3-6


title: Grace, drought, mock, 48hpi, rep3

value: Grace


title: Grace, drought, mock, 48hpi, rep2

value: spike


title: C3-16


title: C3-17


title: Grace, drought, mock, 48hpi, rep1

value: drought


title: D3-5


title: C3-15


title: C3-14


title: Grace, drought, F.avenaceum, 48hpi, rep3


title: C3-12


title: Grace, drought, F.avenaceum, 48hpi, rep2


title: C3-24


title: C3-13


title: C3-11


title: C3-10


title: Grace, drought, F.avenaceum, 48hpi, rep1


title: C3-9


title: C3-7


title: C3-6


title: Grace, drought, F.culmorum, 48hpi, rep3


title: C3-5


title: C3-3


title: C3-4


title: C3-2


title: Barke, irrigated, mock, 96hpi, rep3

value: Barke


title: Grace, drought, F.culmorum, 48hpi, rep2


title: Grace, drought, F.culmorum, 48hpi, rep1


title: C3-1


title: B3-23

source_name: FGO derived from E12.5 embryonic gonad

value: FGO derived from E12.5 embryonic gonad


title: B3-24


title: C3-8


title: Barke, irrigated, mock, 96hpi, rep1


title: B3-22


title: B3-21


title: Barke, irrigated, F.avenaceum, 96hpi, rep3


title: B3-20


title: Barke, irrigated, F.avenaceum, 96hpi, rep2


title: Barke, irrigated, mock, 96hpi, rep2


title: B3-19


title: B3-18


title: Barke, irrigated, F.avenaceum, 96hpi, rep1


title: B3-15


title: Barke, irrigated, F.culmorum, 96hpi, rep3

value: Fusarium culmorum


title: B3-14


title: B3-17


title: B3-13


title: Barke, irrigated, F.culmorum, 96hpi, rep2


title: B3-16


title: B3-12


title: B3-10


title: Barke, irrigated, F.culmorum, 96hpi, rep1


title: B3-11


title: B3-9


title: B3-8


title: B3-7


title: Barke, drought, mock, 96hpi, rep3


title: Barke, drought, mock, 96hpi, rep2


title: B3-5


title: Barke, drought, mock, 96hpi, rep1


title: Cingulate cortex, Mvp KO, rep3

city: Dijon

first: Binnaz

last: Yalcin

address: 15 boulevard Maréchal De Lattre de Tassign

country: France

institute: INSERM

data_processing:
Image analysis and base calling were performed using RTA 2.7.3 and bcl2fastq 2.17.1.14.
Reads were preprocessed using cutadapt 1.10 in order to remove adapter, polyA and low-quality sequences (Phred quality score below 20), reads shorter than 40 bases were discarded for further analysis.
Reads mapping to rRNA and spike-in sequences were also discarded (this mapping was performed using bowtie 2.2.8).
Reads were then mapped onto the mm10 assembly of Mus musculus genome using STAR version 2.5.3a.
Gene expression was quantified using htseq-count 0.6.1p1 and gene annotations from Ensembl release 91.
Assembly: mm10
Supplementary files format and content: Tab-delimitated text file including raw read counts in each sample.

source_name: Cingulate cortex

extract_protocol:
Total RNA was extracted from samples by using a phenol-chloroform technique. Samples were transferred and homogenized with TRI Reagent (T9424, Sigma Aldrich) in tubes containing ceramic beads (Precellys Lysing Kit). The phase separation was done by addition of chloroform followed by centrifugation at 12000g for 5 minutes. Supernatant was collected and RNA extraction was conducted with RNeasy Plus Mini Kit (74134, Qiagen) as told by the manufacturer. RNA concentration and quality were assessed by spectrophotometry (Nanodrop 2000, Thermo Scientific). Only RNA samples with a ratio A260/A280 close to 1.8 and a ratio A260/A230 above 2.0, to exclude guanidine thiocyanate-containing buffer contamination, were further processed. RNA integrity number (RIN) was assessed using the Bioanalyser (Agilent 2100). Samples with RIN < 8 were not used in downstream experimental settings.
Libraries were generated from 200 ng of total RNA using TruSeq Stranded mRNA LT Sample Preparation Kit (Illumina, San Diego, CA), according to manufacturer's instructions.

value: Cingulate cortex

value: Mvp KO


title: Cingulate cortex, Mvp KO, rep4


title: B3-4


title: Barke, drought, F.avenaceum, 96hpi, rep3


title: Cingulate cortex, Mvp KO, rep2


title: B3-3


title: whole_blood_donor35

type: genomic

city: Bayswater

first: Brady

last: Owen

state: Victoria

address: 885 Mountain Hwy

country: Australia

institute: Systematic Medicine

hyb_protocol: standard Illumina protocol

scan_protocol: Arrays were imaged using iScan System using standard recommended Illumina scanner setting

data_processing: Background subtracted beta values were generated using GenomeStudio v2011.1

label: Cy3 and Cy5

molecule: genomic DNA

source_name: Whole blood

label_protocol: standard Illumina protocol

extract_protocol: RNA was extracted using the Tempus Spin RNA isolation kit according to the manufacturer's instructions. Genomic DNA was isolated from the supernatant after peleting the RNA using the QIAamp DNA micro kit  (Qiagen 56304) with a modified protocol.  40 µL of proteinase K and 400 µL of buffer AL to 400 µL of Tempus RNA blood tube supernatant. Samples were pulse vortexed for 15 seconds then incubated at 56 °C for 10 minutes with shaking at 1500 RPM. After adding 200 μL of ethanol and pulse vortexing for 15 seconds, samples were incubated at room temperature for 3 minutes before loading 700 μL into a QIAamp MinElute column. Samples were centrifuge at 6000 g for 1 minute and the flow through discarded. The remaining sample was loaded on the same column and the column was centrifuged again at 6000 g for 1 minute. The wash and elution steps were performed according to the QIAamp DNA micro kit manufacturer’s instructions. Samples were eluted in a volume of 50 µL and typically had concentrations of 75-125 ng/µL (3750 – 6250 ng total recovery) as measured using the Qubit 1x broad range dsDNA kit (Thermo Fisher Scientific Q33266).

treatment_protocol: Whole blood was collected in Tempus RNA blood tubes and stored at -20 °C until DNA extraction.

tag: age

tag: Sex

value: Male


title: Barke, drought, F.avenaceum, 96hpi, rep1


title: Cingulate cortex, Mvp KO, rep1


title: B3-2


title: whole_blood_donor33

value: Female


title: whole_blood_donor28


title: Cingulate cortex, WT, rep4

value: WT


title: whole_blood_donor34


title: Barke, drought, F.avenaceum, 96hpi, rep2


title: Barke, drought, F.culmorum, 96hpi, rep3


title: B3-1


title: whole_blood_donor31


title: Barke, drought, F.culmorum, 96hpi, rep2


title: whole_blood_donor27


title: Cingulate cortex, WT, rep2


title: whole_blood_donor25


title: Barke, drought, F.culmorum, 96hpi, rep1


title: A3-23

source_name: FGO derived from BVSC-iPSC

value: FGO derived from BVSC-iPSC


title: A3-24


title: B3-6


title: whole_blood_donor32


title: whole_blood_donor29


title: whole_blood_donor30


title: Barke, irrigated, mock, 48hpi, rep3

value: irrigated

value: Mock


title: whole_blood_donor22


title: A3-22


title: Barke, irrigated, mock, 48hpi, rep2


title: Cingulate cortex, WT, rep3


title: A3-21


title: small-intestine LP ILC2s, S3

city: Hamburg

first: Nikhat

last: Shaikh

address: Martinistraße 52

institute: University Medical Center Hamburg-Eppendorf

data_processing:
The reads were trimmed to remove sequencing adapters using Trimmomatic 0.38
data was aligned to the mouse reference assembly (GRCM38.92) using STAR (v2.5.4b)
A transcript database was generated by using the same reference genome and counting reads per gene with the summarizeOverlaps function from GenomicAlignments package (Version 1.26.0) in R (version 4.03).
Genes with less than 5 reads in total were filtered out and differential expression analysis was performed by using the DESeq function of R package DEseq2 (version 1.39)
Assembly: Mus_musculus.GRCm38.92
Supplementary files format and content: xlsx (countMatrix), excel file

source_name: small-intestine LP

extract_protocol:
Leukocytes were isolated from kidneys by digestion, percoll gradient, erythrocyte lysis and CD45 enrichment using CD45-microbeads (Miltenyi); from lungs by digestion, percoll gradient, and erythrocyte lysis; and from SILP by digestion and percoll gradient. After sorting of kidneys and lungs ILC2s as CD45+Lin−CD127+CD25+Sca 1+ and of ILC2s from SILP as CD45+Lin−CD127+CD25+KLRG1+, total RNA was extracted using using the RNeasy Plus Micro Kit (QIAGEN) according to the manufacturer’s instructions
250-300 bp cDNA insert library, 6G raw data/sample on average

value: small-intestine LP

value: naive


title: Barke, irrigated, mock, 48hpi, rep1


title: A3-20


title: Cingulate cortex, WT, rep1


title: Barke, irrigated, F.avenaceum, 48hpi, rep3

value: Fusarium avenaceum


title: small-intestine LP ILC2s, S4


title: whole_blood_donor21


title: A3-19


title: whole_blood_donor18


title: whole_blood_donor26


title: small-intestine LP ILC2s, S2


title: whole_blood_donor24


title: small-intestine LP ILC2s, S1


title: whole_blood_donor16


title: whole_blood_donor19


title: whole_blood_donor23


title: A3-18


title: Barke, irrigated, F.avenaceum, 48hpi, rep2


title: whole_blood_donor17


title: Lung ILC2s, S4

source_name: Lung

value: Lung


title: Barke, irrigated, F.avenaceum, 48hpi, rep1

value: spike


title: A3-17


title: Barke, irrigated, F.culmorum, 48hpi, rep3


title: Lung ILC2s, S2


title: A3-16


title: Barke, irrigated, F.culmorum, 48hpi, rep2


title: whole_blood_donor15


title: Barke, irrigated, F.culmorum, 48hpi, rep1


title: Lung ILC2s, S1


title: rat#16-EV _hippocampus7d

type: RNA

city: Moscow

first: Diana

last: Salikhova

address: Miklukho-Maklaya, 8

department: Institute of molecular and cellular medecine

country: Russia

institute: RUDN University

description:
Test
SAMPLE 16

data_processing:
For the normalization it uses the average of housekeeping genes: SNORD61, SNORD72 (small nucleolar RNA, C/D box 61/72).
Target gene signals normalized to housekeeping genes;  ΔСt = Ct(miRNA) – Ct(SNORD)
Fold Change worksheet reports control/test ( all samples PBS treatment/ all samples EV treatment) ratios at 7 day for brain regions (cortex, striatum, hippocampus).

label: SYBR Green

source_name: brain tissue

label_protocol: cDNA was synthesized with miScript II RT Kit (Qiagen, Hilden, Germany) using 250 ng of total RNA isolated from brain tissue samples as described in the previous section. Synthesized cDNA was quantified using miScript SYBR Green PCR Kit (Qiagen, Hillen, Germany) and a сustom miScript miRNA PCR Array (Cat. No./ID: 331231, Qiagen, Hilden, Germany) in the 96-well format for analysis of 45 miRNAs, SNORD61, SNORD72 (small nucleolar RNA, C/D box 61/72) in 2 samples per plate, and PPC (PCR positive control) offered by the manufacturer. The real-time PCR was carried out on a CFX96 system (BioRad, USA) according to the program: 95°С – 15 min, 45 cycles: 94°С – 15 s, 55°С – 30 s, 70°С – 30 s, followed by analysis of the melting curves of the obtained PCR-products plotted in the range of 55 °С – 95 °С.

extract_protocol: Total RNA was extracted with RNeasy Mini Kit followed by DNase I treatment.

tag: animal

value: male

value: Wistar

value: brain

tag: brain region

value: hippocampus

value: TBI

tag: activation agent

value: EV treatment

tag: time

value: 7 days


title: rat#12-EV _hippocampus_7d

description:
Test
SAMPLE 15


title: whole_blood_donor14

value: Female


title: Barke, drought, mock, 48hpi, rep3

value: Barke

value: drought


title: whole_blood_donor13


title: whole_blood_donor10


title: whole_blood_donor12

value: Male


title: A3-13

value: FGO derived from BVSC-iPSC


title: Kidney ILC2s, S2

source_name: Kidney

value: Kidney


title: A3-12


title: Kidney ILC2s, S1

value: naive


title: A3-15


title: Barke, drought, mock, 48hpi, rep1

value: Mock


title: whole_blood_donor9


title: Barke, drought, mock, 48hpi, rep2


title: rat#13-control_hippocampus_7d

description:
control
SAMPLE 13

value: PBS treatment


title: A3-11


title: rat#1-EV _hippocampus_7d

description:
Test
SAMPLE 14


title: A3-14


title: Npm1c_Mut_2

city: Cambridge

first: Bertie

last: Gottgens

address: Jeffrey Cheah Biomedical Centre

country: United Kingdom

institute: University of Cambridge

description:
10x Genomics
SLX12472_SIGAB5

data_processing:
Barcode processing, mapping and gene counting were performed using the Cell Ranger pipeline v6.0.1
Assembly: mm10
Supplementary files format and content: h5 file

source_name: bone marrow

extract_protocol:
Mouse bone marrow cells were collected from the femurs, tibiae and iliac crest and depleted of red blood cells by an ammonium chloride lysis step (STEMCELL Technologies). The cells were stained with the fluorophore-conjugated monoclonal antibodies and lineage− c-Kit+ (LK) cells were then isolated using an LSRII Fortessa (BD).
Single-cell sequencing libraries were generated using 10x Chromium (10x Genomics, Pleasanton, CA) reagent kit v2 according to the manufacturer’s protocol.

value: bone marrow

value: LK

value: F

value: 15 weeks

value: Npm1c Het

value: pIpC


title: rat#7-control_hippocampus_7d

description:
control
SAMPLE 12

value: male

value: Wistar

value: brain

value: hippocampus

value: TBI


title: A3-10


title: Npm1c_Mut_1

description:
10x Genomics
SLX12472_SIGAA5


title: Barke, drought, F.avenaceum, 48hpi, rep3

source_name: spike

value: Barke

value: spike

value: drought

value: Fusarium avenaceum


title: Barke, drought, F.avenaceum, 48hpi, rep2


title: whole_blood_donor20

value: Female


title: whole_blood_donor7

value: Male


title: Npm1_WT_2

description:
10x Genomics
SLX12472_SIGAD5

value: bone marrow

value: WT


title: rat#12-EV_striatum_7d

description:
Test
SAMPLE 10

value: striatum

value: EV treatment

value: 7 days


title: rat#5-control_hippocampus_7d

description:
control
SAMPLE 11

value: PBS treatment


title: A3-8

value: FGO derived from BVSC-iPSC


title: Lung ILC2s, S3

value: Lung

value: naive


title: Npm1_WT_1

description:
10x Genomics
SLX12472_SIGAC5

value: LK

value: F

value: 15 weeks

value: pIpC


title: Barke, drought, F.culmorum, 48hpi, rep3

value: Barke

value: spike

value: drought

value: Fusarium culmorum


title: A3-9


title: A3-7


title: Barke, drought, F.avenaceum, 48hpi, rep1

value: Fusarium avenaceum


title: whole_blood_donor11

value: Female


title: Jak2_WT_3

description:
10x Genomics
SLX12969_SIGAA8

value: bone marrow

value: WT


title: whole_blood_donor8

value: Male


title: Barke, drought, F.culmorum, 48hpi, rep2


title: rat#7-control_striatum_7d

description:
control
SAMPLE 8

value: male

value: Wistar

value: brain

value: striatum

value: TBI

value: PBS treatment

value: 7 days


title: A3-6

value: FGO derived from BVSC-iPSC


title: Jak2_WT_2

description:
10x Genomics
SLX11525_SIGAH2

value: LK

value: F

value: 12 weeks


title: rat#5-control_striatum_7d

description:
control
SAMPLE 7


title: rat#1-EV_striatum_7d

description:
Test
SAMPLE 9

value: EV treatment


title: whole_blood_donor3


title: A3-5


title: A3-4


title: Jak2_WT_1

description:
10x Genomics
SLX11516_SIGAC2


title: whole_blood_donor6


title: eWAT, cachexia, rep3

city: Shanghai

first: Chenyang

last: Jin

address: 180 Fenglin Road

department: Department of General Surgery

country: China

institute: Zhongshan Hospital of Fudan University

data_processing:
Raw data (raw reads) of fastq format were firstly processed through in-house perl scripts. In this step, clean data (clean reads) were obtained by removing reads containing adapter, reads containing ploy-N and low quality reads from raw data. At the same time, Q20, Q30 and GC content the clean data were calculated.
featureCounts v1.5.0-p3 was used to count the reads numbers mapped to each gene. And then FPKM of each gene was calculated based on the length of the gene and reads count mapped to this gene.
Index of the reference genome was built using Hisat2 v2.0.5 and paired-end clean reads were aligned to the reference genome using Hisat2 v2.0.5
Assembly: ensembl release-94 mus_musculus

source_name: epididymal white adipose tissue

extract_protocol:
Total RNA was extracted using the TRIzol method, according to the official guidelines provided by Invitrogen. RNA degradation and contamination were detected by 1% agarose gel electrophoresis. RNA concentration was measured using a NanoPhotometer spectrophotometer (Implen, Germany) for OD260/280 and OD260/230. RNA integrity was measured by Agilent 2100 Bioanalyzer (Agilent, USA).
we used NEBNext® Poly(A) mRNA Magnetic Isolation Module (#E7490, NEB, USA) to isolate mRNA and NEBNext® Ultra™ II RNA Library Prep Kit for Illumina® (#E7775, NEB, USA) to construct cDNA libraries. The specific experimental methods for library construction are described in the official guidelines and technical documents.

value: epididymal white adipose tissue

value: cachexia


title: eWAT, cachexia, rep2


title: A3-3


title: Jak2_Mut_3

description:
10x Genomics
SLX12969_SIGAB8

value: bone marrow

value: 14 weeks

value: Jak2 V617F Hom


title: eWAT, cachexia, rep1


title: A3-2


title: rat#16-EV _cortex_7d

description:
Test
SAMPLE 6

value: male

value: cortex


title: rat#12-EV_cortex_7d

description:
Test
SAMPLE 5

value: Wistar

value: brain


title: whole_blood_donor5

value: Female


title: rat#13-control_cortex_7d

description:
control
SAMPLE 3

value: TBI

value: PBS treatment


title: eWAT, control, rep3

value: control


title: rat#1-EV _cortex_7d

description:
Test
SAMPLE 4

value: 7 days


title: A3-1

value: FGO derived from BVSC-iPSC


title: Dnmt3a_WT_2

description:
10x Genomics
SLX12969_SIGAF5

value: LK

value: F

value: 11 weeks

value: WT

value: pIpC


title: Jak2_Mut_2

description:
10x Genomics
SLX11525_SIGAB3

value: bone marrow

value: 12 weeks


title: eWAT, control, rep2

value: epididymal white adipose tissue


title: whole_blood_donor4


title: whole_blood_donor2


title: Kidney ILC2s, S3

value: Kidney

value: naive


title: D2-24

source_name: in vivo-derived GRO

value: in vivo-derived GRO


title: rat#7-control_cortex_7d

description:
control
SAMPLE 2

value: male

value: Wistar

value: brain

value: cortex


title: Jak2_Mut_1

description:
10x Genomics
SLX11516_SIGAB2

value: Jak2 V617F Hom


title: Dnmt3a_WT_1

description:
10x Genomics
SLX12969_SIGAE5


title: rat#5-control_cortex_7d

description:
control
SAMPLE 1

value: TBI

value: PBS treatment

value: 7 days


title: eWAT, control, rep1

value: control


title: D2-23


title: whole_blood_donor1

value: Female


title: SPT2 KO-6, replicate 2

first: Francesco Nicola

last: Carelli

address: Henry Wellcome Building of Cancer and Developmental Biology,  Tennis Court Road

library_source: genomic

data_processing:
Reads were trimmed using trim_galore (version 0.6.7), and aligned using bwa mem (version 0.7.17)
Low-quality (q<10) reads and reads mapping to non-assembled contigs mtDNA were discarded.
Normalised coverage was calculated using MACS2:  --nomodel --keep-dup all --extsize 150 --shift -75 --bdg --SPMR --gsize hs --nolambda
Assembly: hg38
Supplementary files format and content: BigWig tracks, SPMR normalised, 1bp resolution

source_name: U-2 OS

growth_protocol: Cells were grown at 37C with 5% CO2 in DMEM media supplemented with  100μg/ml G418

extract_protocol:
ATAC-seq  was performed with the Active Motif ATAC-seq kit (53150) according to the manfacturers' protocol. Briefly, 80,000 cells for each sample were resuspended in ATAC Lysis buffer, and cells were resuspended in Tagmentation Master Mix containing Tn5 transposomes.
The tagmented DNA was then amplified with Q5 polymerase (10 cycles), according to the Active Motif ATAC-seq kit, using i5 and i7 Indexed primers. The PCR reaction products were then purified using SPRI beads to obtain fragments between 150 and 600bp.

tag: cell line

value: U-2 OS

value: female osteosarcoma cells

value: SPT2 KO-6


title: Calr_WT_1

description:
10x Genomics
SLX14832_SIGAG10

value: bone marrow

value: LK

value: F

value: 12 weeks

value: WT


title: SPT2 KO-6, replicate 3


title: Small-intestine LP ILC2s, donors, scRNAseq

description: 10X Genomics

data_processing:
Demultiplexing and mapping reads to the reference genome was performed using CellRanger (version 4.0.0)
Data was analysed using single Seurat object (v.4.0.2).
After excluding low quality cells, low expressed genes and cells with high mitochondrial gene number, unsupervised clustering was performed using standard workflow by Seurat
Assembly: Mus_musculus.(mm10-Ensemble 98)
Supplementary files format and content: Tab-separated values files
Supplementary files format and content: Matrix files

source_name: Small intestine LP

extract_protocol:
Leukocytes were isolated from kidneys by digestion and CD45 enrichment using CD45-microbeads (Miltenyi) and from SILP by digestion and percoll gradient. The ILC2s were sorted as CD45+Lin-CD127+IL-13fm+
Single-cell libraries of sorted cells were generated with the 10X Genomics Chromium Single Cell 5’v1.1 reagents kit according to the manufacturer’s protocol.

value: Small intestine LP

value: BALB/c IL-13fm

value: rm IL-33


title: Calr_Mut_1

description:
10x Genomics
SLX14832_SIGAE10

value: Calr 52-bp del Hom


title: Dnmt3a_Mut_2

description:
10x Genomics
SLX12969_SIGAH5

value: 11 weeks

value: Dnmt3a R882H Het

value: pIpC


title: Small-intestine LP ILC2s, 8 week recipients, scRNAseq

description: 10X Genomics

value: BALB/c Rag2-/-Il2rg-/-

value: Kid ILC2 recipients


title: D2-21

value: in vivo-derived GRO


title: SPT2 KO-4, replicate 3

value: U-2 OS

value: female osteosarcoma cells

value: SPT2 KO-4


title: Kidney ILC2s, donors, scRNAseq

description: 10X Genomics

value: Kidney


title: SPT2 KO-4, replicate 1


title: D2-20


title: Barke, drought, F.culmorum, 48hpi, rep1

value: Barke

value: spike

value: drought

value: Fusarium culmorum


title: D2-17


title: Mus musculus retina, Dram2 KO, rep1

city: South San Francisco

first: Christine

last: Clarke

state: California

address: 1 DNA Way

department: OMNI Bioinformatics

institute: Genentech

data_processing:
The demultiplexing, barcoded processing, gene counting and aggregation were made using the Cell Ranger software v3.1.0 (https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/what-is-cell-ranger)
Assembly: GRCm38
Supplementary files format and content: Comma-separated values files

source_name: retina

extract_protocol:
Single-cell RNAseq libraries prepared using 10x protocol from mouse retinas derived from DRAM2 wt/wt (1 sample) and DRAM2 ko/ko mice (1 sample). Total of n=2 samples.
Library was performed according to the manufacter’s instructions (single cell 3’ v2 protocol, 10x Genomics). Briefly, GCs were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the apropiated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.

value: retina

tag: replicate

tag: batch

value: Dram2 KO

value: 12 months

value: male


title: Mus musculus retina, Dram2 WT, rep2

tag: genotype

value: Dram2 WT

tag: strain


title: SPT2 KO-3, replicate 2

tag: cell type

value: SPT2 KO-3


title: mESCs_inAGTuC_Exp261_c16

first: Dig

middle: B

last: Mahat

email: mahat@mit.edu, dbm222@cornell.edu

state: MA

address: Koch Institute, MIT, 500 Main St, Building 76, #417

department: Koch Institute for Integrative Cancer Research

institute: Massachusetts Institute of Technology

description: Nascent RNA

data_processing:
Adaptor sequences were removed from paired-end fastq files using Cutadapt. Specifically, the Read 1 sequence CCCCTGTCTCTTATACACAT and the Read 2 sequence AGATCGGAAGAGCGTCGTGT were trimmed with a maximum error rate of 0.15, requiring a minimum overlap of 12 nucleotides between the read and adapter.
The resulting adapter-trimmed reads were subjected to demultiplexing using Flexbar. Cell barcodes and Unique Molecular Identifiers (UMIs) were extracted from the 5’ end of Read 1, applying a barcode error rate of 0.15 and retaining reads of at least 14 nucleotides in length.
The adapter-clipped and demultiplexed reads were first mapped to the mouse ribosomal genome using bowtie2 in --very-sensitive mode. The reads unmapped to the ribosomal genome were mapped to the mouse genome (mm10 build) also in --very-sensitive mode.
After mapping, duplicate reads were identified and removed utilizing UMI and mapping coordinates with UMI-tools.
Assembly: mm10
Supplementary files format and content: Quality filtered mapped reads in GRange format as R data object (for files with *_consolidated.rds)
Supplementary files format and content: Features X cells matrix as R data object (for files with *_mapq3qc_filtered_counts.rds)
Library strategy: scGRO-seq, inAGTuC, AGTuC

source_name: embryonic stem cells

growth_protocol: mESCs were cultured in Dulbecco’s Modified Eagle Medium plus 10% fetal bovine serum supplemented with 1x penicillin/streptomycin, 1x non-essential amino acids, 1x L-Glutamine, 1x β-mercaptoethanol, and 1000 U/ml leukemia inhibitory factor on tissue culture-treated 10 cm plates pre-coated with 0.2% gelatin prepared in phosphate-buffered saline. Cells were grown at 37°C and 5% CO2 and passed with Hepes buffered saline solution and 0.25% Trypsin-EDTA when 70% confluency was reached (every two days).

extract_protocol:
For nuclei isolation, the pellet was resuspended in ice-cold douncing buffer (10 mM Tris-Cl pH 7.4, 300 mM Sucrose, 3 mM CaCl2, 2 mM MgCl2, 0.1 % Triton X-100, 0.5 mM DTT, 0.1x Halt Protease inhibitor, and 0.02 U/ul RNase inhibitor) and transferred to a 7 ml dounce homogenizer. After incubation on ice for 5 minutes, the cells were dounced 25 times with a tight pestle, transferred back to the 15 ml conical tube, and centrifuged to pellet the nuclei. The pellet was washed twice in douncing buffer. For cell permeabilization, the pellet was resuspended in ice-cold permeabilization buffer (10 mM Tris-Cl pH 7.4, 300 mM Sucrose, 10 mM KCl, 5 mM MgCl2, 1 mM EGTA, 0.05 % Tween-20, 0.1% NP-40, 0.5 mM DTT, 0.1x Halt Protease inhibitor, and 0.02 U/ul RNase inhibitor). After incubation on ice for 5 minutes, the cells were centrifuged to pellet the nuclei. The pellet was washed twice in the permeabilization buffer.
A 96-well plate containing 5’-AzScBc DNA with a unique cell barcode in each well previously synthesized and aliquoted is thawed from -80°C. Sodium Ascorbate, PEG 8000, CuSO4, and accelerating ligand BTTAA was prepared and dispensed into each well of the 96-well plate containing 5’-AzScBc DNA. The CuAAC reaction mix was dispensed into individual wells containing single cells in Urea using a multi-channel pipette or a 96-well pipettor. The 96-well plates are sealed, vortexed for 10 seconds in an orbital vortexer, and centrifuged for 1 min at 500 g prior to incubation for 2 hours at 50°C. After completion of incubation, the CuAAC reaction was quenched with 5 mM EDTA and pooled from 96-wells into a single 1.5 ml eppendorf tube. PEG 8000 was removed with Trizol. Remaining CuAAC reagents (Sodium Ascorbate, CuSO4, and mM BTTAA) were removed with a centrifugal filter with 3 kDa cellulose membrane (Amicon, 2020-04). The purified RNA was fragmented with 10 mM ZnCl2 for 5 min at 65°C. Reverse transcription of the clicked RNA-DNA conjugate was performed Reverse Transcriptase lacking RNase H activity but capable of RNA-dependent and DNA-dependent polymerase activity, non-templated addition and template switching. RT reaction was incubated with RNA-DNA conjugate for 2 hours at 50°C. The cDNA was size-selected in 10% denaturing polyacrylamide gel electrophoresis (PAGE) away from the unclicked 5’-AzScBc DNA and empty cDNA formed between the 5’-AzScBc DNA and TSO. library was PCR amplified with Q5 High-Fidelity 2X Master Mix (NEB, M0492S) and 0.5 uM PCR primers with unique dual index using the two-step PCR cycles.

value: embryonic stem cells


title: mESCs_inAGTuC_Exp234_c11

description: Nascent RNA


title: D2-22


title: D2-16


title: Dnmt3a_Mut_1

description:
10x Genomics
SLX12969_SIGAG5

tag: tissue

value: bone marrow

value: LK

value: F

value: 11 weeks

value: Dnmt3a R882H Het

value: pIpC


title: mESCs_scGROseq_Exp264a_c16

description: Nascent RNA


title: SPT2 KO-3, replicate 1

value: U-2 OS

value: female osteosarcoma cells


title: Small-intestine LP ILC2s, 2 week recipients, scRNAseq

description: 10X Genomics

value: Small intestine LP

value: BALB/c Rag2-/-Il2rg-/-

value: Kid ILC2 recipients


title: SPT2 KO-4, replicate 2

value: SPT2 KO-4


title: D2-19

value: in vivo-derived GRO


title: D2-15


title: Mus musculus retina, Dram2 WT, rep1

value: retina

value: 12 months

value: male


title: Mus musculus retina, Dram2 WT, rep3

value: Dram2 WT


title: Mus musculus retina, Dram2 KO, rep2

value: Dram2 KO


title: mESCs_scGROseq_Exp264a_c07

description: Nascent RNA

value: embryonic stem cells


title: mESCs_scGROseq_Exp264a_c14

description: Nascent RNA


title: D2-13


title: mESCs_scGROseq_Exp264a_c10

description: Nascent RNA


title: WT, replicate 1

value: WT


title: D2-14


title: Mus musculus retina, Dram2 KO, rep3


title: D2-18


title: Mus musculus choroid, Dram2 KO, rep3

description:
SAM24373238
NGS2918_counts.csv
NGS2918_meta.csv

source_name: choroid

extract_protocol:
Single-cell RNAseq libraries prepared using 10x protocol from mouse choroid derived from DRAM2 wt/wt (3 sample) and DRAM2 ko/ko mice (3 sample). Total of n=6 samples.
Library was performed according to the manufacter’s instructions (single cell 3’ v2 protocol, 10x Genomics). Briefly, GCs were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the apropiated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.

value: choroid

value: 12-16 months


title: WT, replicate 2


title: D2-12


title: mESCs_scGROseq_Exp264a_c06

description: Nascent RNA


title: Mus musculus choroid, Dram2 KO, rep2

description:
SAM24373237
NGS2918_counts.csv
NGS2918_meta.csv


title: D2-11


title: mESCs_scGROseq_Exp264a_c05

description: Nascent RNA


title: Mus musculus choroid, Dram2 KO, rep1

description:
SAM24373236
NGS2918_counts.csv
NGS2918_meta.csv


title: mESCs_scGROseq_Exp264a_c04

description: Nascent RNA


title: D2-10


title: FOXA1 siRNA rep 3

city: Chiyoda-ku

first: Shogo

last: Okazaki

state: Tokyo

address: 1-8-13 Kanda-Surugadai

department: School of Dentistry

institute: Nihon University

data_processing:
Sequenced reads were mapped to GRCh38 genome using HISAT2
Normalized counts were calculated using DESeq2.
Assembly: GRCm38
Supplementary files format and content: tab-delimited text files include normalized count values for each sample

source_name: Oral squamous cell carcinoma cell line

growth_protocol: Cells were maintained under 5% CO2 at 37°C in DMEM (Sigma-aldrich) supplemented with 10% fetal bovine serum.

extract_protocol:
Total RNA was extracted and purified with the use of Reliaprep RNA Miniprep System (Promega).
RNA libraries were prepared with NEBNext Ultra RNA Library Prep Kit for Illumina (NEB).

treatment_protocol: OSC19SSZR cells were transfected with control or FOXA1 siRNA using Lipofectamine RNAi max and cultured for 48h.

value: FOXA1 siRNA


title: FOXA1 siRNA rep 2


title: FOXA1 siRNA rep 1


title: WT, replicate 3

value: U-2 OS


title: Mus musculus choroid, Dram2 WT, rep3

description:
SAM24373235
NGS2918_counts.csv
NGS2918_meta.csv


title: SPT2 KO-3, replicate 3

value: female osteosarcoma cells

value: SPT2 KO-3


title: control siRNA rep 3

value: control siRNA


title: mESCs_scGROseq_Exp263b_c10

description: Nascent RNA


title: D2-9


title: mESCs_scGROseq_Exp263b_c07

description: Nascent RNA


title: control siRNA rep 2


title: Mus musculus choroid, Dram2 WT, rep2

description:
SAM24373234
NGS2918_counts.csv
NGS2918_meta.csv


title: D2-8


title: Mus musculus choroid, Dram2 WT, rep1

description:
SAM24373233
NGS2918_counts.csv
NGS2918_meta.csv


title: D2-7


title: mESCs_scGROseq_Exp263_c08

description: Nascent RNA


title: mESCs_scGROseq_Exp263b_c04

description: Nascent RNA


title: D2-5


title: HSC_Rep4

city: St Louis

first: Bhuvic

last: Patel

state: MO

address: 660 S. Euclid Ave

department: Neurosurgery

institute: Washington University in St. Louis School of Medicine

data_processing:
Fragmented libraries were sequenced on an Illumina NovaSeq-6000 using paired end reads extending 150 bases.
Basecalls and demultiplexing were performed with Illumina’s bcl2fastq software and a custom python demultiplexing program with a maximum of one mismatch in the indexing read.
RNA-seq reads were then aligned to the Ensembl release 76 primary assembly with STAR version 2.5.1a1.
Gene counts were derived from the number of uniquely aligned unambiguous reads by Subread:featureCount version 1.4.6-p52.
Assembly: GRCh38
Supplementary files format and content: A single tab-delimited text file including non-normalized transcript counts for all samples. Column names correspond to sample names and rows correspond to genes.

source_name: Immortalized human Schwann cells (HSC)

extract_protocol:
HSC cells were plated at a density of 10,000 cells per ml of growth media in a 6-well plate and expanded for 2 days prior to RNA extraction. RNA extraction was performed with RNeasy Mini (Qiagen) per manufacturer protocol. Samples were submitted to the GTAC core laboratory at Washington University. Total RNA integrity was determined using Agilent Bioanalyzer or 4200 Tapestation.
Library preparation was performed with 500ng to 1ug of total RNA. Ribosomal RNA was removed by an RNase-H method using RiboErase kits (Kapa Biosystems). mRNA was then fragmented in reverse transcriptase buffer and heated to 94 degrees for 8 minutes. mRNA was reverse transcribed to yield cDNA using SuperScript III RT enzyme (Life Technologies, per manufacturer's instructions) and random hexamers. A second strand reaction was performed to yield ds-cDNA. cDNA was blunt ended, had an A base added to the 3' ends, and then had Illumina sequencing adapters ligated to the ends. Ligated fragments were then amplified for 12-15 cycles using primers incorporating unique dual index tags.

value: Immortalized human Schwann cells (HSC)

value: Schwann cell


title: D2-6

value: in vivo-derived GRO


title: D2-4


title: PGC1a-1, scRNAseq

city: Philadelphia

first: Jianfu

last: Zhou

state: Pennsylvania

address: 3400 Civic Center Boulevard, 12th floor

institute: University of Pennsylvania

description: 10x Genomics

data_processing:
The demultiplexing, barcoded processing, gene counting and aggregation were made using the Cell Ranger software v3.1.0 (https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/3.1/what-is-cell-ranger)
Assembly: mm10
Supplementary files format and content: Tab-separated values files and matrix files

source_name: adult kidney

extract_protocol:
Euthanized mice were perfused with chilled 1× PBS via the left ventricle. Kidneys were harvested, minced into approximately 1 mm3 cubes and digested using Multi Tissue dissociation kit (Miltenyi, 130-110-201). The tissue was homogenized using 21G and 26.5G syringes. Up to 0.25g of the tissue was digested with 50μl of Enzyme D, 25μl of Enzyme R and 6.75 μl of Enzyme A in a ml of RPMI and incubated of 30 minutes at 37°C. Reaction was deactivated by 10% FBS. The solution was then passed through a 40 μm cell strainer. After centrifugation at 400g for 5 minutes, cell pellet was incubated with 1ml of RBC lysis buffer on ice for 3 minutes. Cell number and viability were analyzed using Countess AutoCounter (Invitrogen, C10227). This method generated single cell suspension with greater than 80% viability.
10,000 cells were loaded into the Chromium Controller (10X Genomics, PN-120223) on a Chromium Single Cell B Chip (10X Genomics, PN-120262) and processed to generate single-cell gel beads in the emulsion (GEM) according to the manufacturer’s protocol (10x Genomics, CG000183). Libraries were generated using Chromium Single Cell 3’ Reagent Kits v3 (10x Genomics, PN-1000092) and Chromium i7 Multiplex Kit (10x Genomics, PN-120262) according to the manufacturer’s manual. Quality control for constructed library was performed by Agilent Bioanalyzer High Sensitivity DNA kit (Agilent Technologies, 5067-4626) for qualitative analysis. Quantification analysis was performed by Illumina Library Quantification Kit (KAPA Biosystems, KK4824). The library was sequenced on Illumina HiSeq 4000 system with 2 × 150 paired-end kits using the following read length: 28 bp Read1 for cell barcode and UMI, 8 bp I7 index for sample index, and 91 bp Read2 for transcript.

value: adult kidney

value: transgenic overexpression of PGC1a

value: 6 weeks old


title: HSC_Rep2


title: Notch3, scRNAseq

description: 10x Genomics

value: male

value: transgenic overexpression of Notch1


title: mESCs_scGROseq_Exp263_c03

description: Nascent RNA

value: embryonic stem cells


title: D2-3


title: mESCs_scGROseq_Exp263_c02

description: Nascent RNA


title: mESCs_scGROseq_Exp261_c16

description: Nascent RNA


title: MMctrl8, scRNAseq

description: 10x Genomics

value: wild type control

value: 6-8 weeks old


title: D2-2


title: NC_4

city: Beijing

first: mengxia

last: fu

address: xicheng road

institute: fuwai hospital

description: 10x Genomics

data_processing:
For 10x data, Cell Ranger 6.1.2 was used to quantify gene expression leve with human reference version GRCh38.
The output of Cell Ranger filtered gene expression matrix were analyzed by R software (Version 3.6.1) with the Seurat package (Version 3.2.0).
Before the downstream analysis, low-quality cells were excluded and retained the high-quality single-cell data that met the following criterial: Briefly, cells with high likelihood of being doublets were filtered out by employing DoubletFinder (2.0.3). Then, we merged all samples and removing all cells with >3 median absolute deviations difference in read numbers and mitochondrial reads3 as the reviewer suggested. Finally, we excluded HBB or MYL2 positive cells to remove the red blood cells and cardiomyocytes.
After removal of low-quality cells, the gene expression matrices were normalized by the NormalizeData function in Seurat.
Assembly: GRCh38
Supplementary files format and content: Matrix table with raw gene counts for every gene and every cell

source_name: perivascular adipose tissue

extract_protocol:
Samples were washed by PBS to remove the remaining blood cells and then cut into small pieces. These small pieces were washed by PBS again and digested in DPBS (Gibco, 14040133) containing 0.28 U/mL Liberase TM (Roche, 05401119001) at 37 °C for 45 minutes, followed by gently shaking. The single-cell suspension was filtered with a 40 μm cell strainer and collected by centrifugation at 400g, 4 °C for 5 minutes. The supernatant was discarded, and the cell pellets were resuspended in 1 mL ACK lysing buffer (Gibco, A1049201) and incubated for 5 minutes at room temperature. The resulting suspension was further diluted to 10x the initial volume with cold PBS, and centrifuged at 400g, 4 °C for 5 minutes. The cell precipitate was resuspended in 500 μl cold DMEM containing 10% fetal bovine serum and use immediately.
Single-cell suspensions were processed through the 10x Genomics Chromium System (10x Genomics, Pleasanton, CA, USA), followed by the construction of 3’ gene expression v3.1 libraries and sequencing on an Illumina Noveseq6000 sequencer. Briefly, cells (>90% viability), reagents, gel beads, and partitioning oil are loaded onto 10x Chromium Chip B. Ideally, each individual cell is wrapped with an oil drop that contains gel beads and reagents. This creates an independent reaction space for each cell called gel bead in emulsion (GEM). The primers provided by the gel bead contain a sequencing primer, barcode, UMI, and poly (dT) sequence. The cell in the GEM is lysed, and incubation of the GEM produces barcoded, full-length cDNA from poly-adenylated mRNA. Next, all cDNAs are pooled together, and the steps of general library construction are performed including amplification, fragmentation, end repair, adapter ligation, and sample index PCR (98 °C for 45 seconds; [98 °C for 20 seconds, 67 °C for 30 seconds, and 72 °C for 1 minute] x 14 cycles; 72 °C for 1 minute). The libraries were then sequenced using Illumina Noveseq6000 by CapitalBio Technology (Beijing, China).

value: perivascular adipose tissue

value: stromal vascular fraction

tag: disease state

value: non-atherosclerosis

value: heart transplant


title: MMctrl7, scRNAseq

description: 10x Genomics


title: control siRNA rep 1


title: D2-1

extract_protocol:
Each GRO and FGO derived from BVSC-iPSCs, E12.5 gonads, 6 dpp follicles and in vivo samples was dissociated from follicular somatic cells using 30G needles, followed by treatment with Accumax (Innovative Cell Technologies) for 5 min and by pipetting to remove somatic cells completely. The zona pellucida of each GRO and FGO was removed by acid tyrode’s solution (Merck) and samples were individually washed in PBS supplemented with 0.01% PVA (PBS-PVA) and lysed in the lysis buffer composed of 0.09% Triton-X 100, 2 U SUPERase IN RNase inhibitor (Invitrogen, AM2694), 2.5 μM Oligo-dT primer (Microsynth AG), dNTP mix (2.5 mM each, Promega), ERCC RNA Spike-In Mix (1 : 3.2 x 107, Thermo Fischer Scientific 4456740) in individual tubes of a 8-well strip, then immediately frozen on the dry ice and kept at -80C for longer storage.
RNA sequencing libraries for single oocytes were prepared according to the Smartseq2 protocol


title: ESRRA, scRNAseq

description: 10x Genomics

value: Esrra KO

value: 8 weeks old


title: PTC-2_D20 (Patient corrected, rep2, day20) [RD24]

city: Houston

first: Annie

middle: W.

last: Shieh

state: Texas

address: 1825 Pressler St

department: Brown Foundation Institute Of Molecular Medicine

institute: University of Texas Health Science Center

description: muscle_diff_rnaseq_2ln.v37.featurecounts.pub.txt

data_processing:
Sequence reads were mapped to the GRCh38 human genome using TopHat2, allowing 2 mismatches
Unique reads were identified with the NH:i:1 flag and summarized using featureCounts of the Subread package
Assembly: GRCh38
Supplementary files format and content: text file containing quantification of transcript mapped to each Ensembl gene (row) for each sample (column)

source_name: Human iPSCs

growth_protocol: Human ESC line (H9 from WiCell) was maintained on Matrigel-coated dishes (BD Biosciences) in mTeSR1 medium (STEMCELL Technologies).

extract_protocol:
1 x 10^6 cells were harvested / sorted at each time- point and RNA extracted using QIAGEN miRNeasy RNA isolation kit (217004, QIAGEN) according to manufacturer’s protocol.
Each RNA sample was converted to cDNA using oligo-dT primers and was further converted into indexed sequencing libraries using Illumina TruSeq kit.

treatment_protocol: For skeletal muscle differentiation, human pluripotent stem cells were harvested using Accutase (Stem Cell Technologies) and seeded on Matrigel-coated plates (10,000 cells/cm2) in myogenic differentiation medium-I (MDM-I) and maintained for 4 days. MDM-I medium consisted of IMDM supplemented with 5% horse serum, 3 μM CHIR99021 (Selleck Chemical LLC), 2 μM SB431542 (Selleck Chemical LLC), 10 ng/ml hr-EGF (PeproTech Inc), 10 μg/ml insulin (Sigma- Aldrich), 0.4 mg/ml dexamethasone (Sigma-Aldrich) and 200 μM L- ascorbic acid (Sigma-Aldrich). After 4 days of induction, cells were harvested using Accutase and seeded on Matrigel-coated plates (10,000 cells/cm2) in myogenic differentiation medium-II (MDM-II). MDM-II medium contained IMDM supplemented with 5% horse serum, 10 μg/ml insulin (Sigma- Aldrich), 10 ng/ml hr-EGF (Pepro Tech Inc), 20 ng/ml hr-HGF (Pepro Tech Inc), 20 ng/ml hr-FGF (Pepro Tech Inc), 10 ng/ml IGF-1 (Pepro Tech Inc), 2 μM SB431542 (Selleck Chemical LLC), 0.5 μM LDN193189 (Stemgent), 200 μM L-ascorbic acid (Sigma-Aldrich). The cells were cultured in MDM-II medium for 6-11 days. At day 10 to 15 of differentiation (4 + 6-11), the cells were harvested and sorted for EGFP or surface markers. Sorted fractions were seeded on Matrigel-coated plates with similar densities (300,000/well of a 6 well plate) and expanded in MDM-II medium for an additional 2-3 days (or longer for expansion time-course experiments). For terminal myotube differentiation, once cells reached appropriate confluency (80-90%), the medium was switched to MDM-III medium. MDM-III medium consisted of IMDM (Invitrogen), 15% knockout serum replacement (Invitrogen), 100mM MEM non-essential amino acids (Invitrogen) and 10 ng/ml IGF-1.

value: Human iPSCs

tag: sample group

value: Patient corrected

tag: time point


title: PGC1a-2, scRNAseq

description: 10x Genomics


title: NC_2

description: 10x Genomics

tag: treatment


title: mESCs_scGROseq_Exp263_c04

description: Nascent RNA


title: mESCs_scGROseq_Exp261_c15

description: Nascent RNA


title: mESCs_scGROseq_Exp261_c11

description: Nascent RNA


title: HSC_Rep3

value: Immortalized human Schwann cells (HSC)

value: Schwann cell


title: B2-23

data_processing:
RNA-Seq datasets were aligned to a custom genome containing the Mus musculus genome assembly (GRCm38/mm10 Dec. 2011) and ERCC92 sequences using STAR (Dobin et al., 2013) with parameters "-outFilterMultimapNmax 300 -outMultimapperOrder Random -outSAMmultNmax 1 -alignIntronMin 20 -alignIntronMax 1000000", allowing multimappers with up to 300 matches in the genome and choosing positions for multimappers randomly
A random transcript isoform for each gene was chosen from Bioconductor annotation package TxDb.Mmusculus.UCSC.mm10.knownGene (version 3.2.2) and exonic genomic regions for the random transcript isoforms for each gene were used in further analyses.
Expression quantification for selected exonic genomic regions was done using QuasR R package (Gaidatzis et al., 2015) selecting only uniquely mapped reads (mapqMin=255).
Assembly: mm10
Supplementary files format and content: CSV file contatining read count table for exonic regions of each gene (column ENTREZID), total length of genomic regions for each gene (column width) and each single oocyte.

source_name: GRO derived from E12.5 embryonic gonad

value: GRO derived from E12.5 embryonic gonad


title: NC_1

description: 10x Genomics


title: mESCs_scGROseq_Exp260b_c11

description: Nascent RNA


title: PTC-2_D5 (Patient corrected, rep2, day5) [RD22]


title: HSC_Rep1


title: PTC-2_D0 (Patient corrected, rep2, day0) [RD21]


title: E_AS_3

description: 10x Genomics

value: early atherosclerosis


title: B2-22


title: mESCs_scGROseq_Exp260_c08

description: Nascent RNA


title: B2-21


title: E_AS_2

description: 10x Genomics


title: PT-2_D20 (Patient, rep2, day20) [RD20]

value: Patient


title: B2-20


title: NC_3

description: 10x Genomics


title: mESCs_scGROseq_Exp260_c06

description: Nascent RNA


title: E_AS_1

description: 10x Genomics


title: mESCs_scGROseq_Exp256_c09

description: Nascent RNA


title: PTC-2_D15 (Patient corrected, rep2, day15) [RD23]


title: PT-2_D15 (Patient, rep2, day15) [RD19]


title: B2-24


title: A_AS_3

description: 10x Genomics

value: advanced atherosclerosis


title: A_AS_2

description: 10x Genomics


title: PT-2_D5 (Patient, rep2, day5) [RD18]


title: B2-18


title: mESCs_scGROseq_Exp246_c04

description: Nascent RNA

value: embryonic stem cells


title: mESCs_scGROseq_Exp246_c08

description: Nascent RNA


title: B2-17


title: mESCs_scGROseq_Exp260_c10

description: Nascent RNA


title: mESCs_scGROseq_Exp246_c03

description: Nascent RNA


title: CTL-2_D20 (Control, rep2, day20) [RD16]

value: Control


title: A_AS_1

description: 10x Genomics


title: PT-2_D0 (Patient, rep2, day0) [RD17]


title: IgG_control

city: Milan

first: Davide

last: Gabellini

address: Via Olgettina 60

department: Division of Genetics and Cell Biology

country: Italy

institute: SAN RAFFAELE SCIENTIFIC INSTITUTE

data_processing:
library strategy: CUT&Tag
Sequencing adapters were removed via the Cutadapt (v4.1)
rimmed fastq files were aligned with Bowtie2 (v2.2.1)(Langmead & Salzberg, 2012) to the human genome assembly GRCh38 (hg38) from Genome Reference Consortium, using the following parameters: --local --very-sensitive --no-mixed --no-discordant --phred33 -I 10 -X 700
Bam files containing uniquely mapped reads were created using Samtools v1.15.1 (Li et al., 2009).
CUT&Tag signals were called on using MACS v2.2.7.1 (Zhang et al., 2008) as normalized to IgG control signal, using the following settings: FDR cutoff of 1e-3, BAMPE format.
The MACS --bdg setting was used to generate IgG control scaled, normalized bigwig files, which were used to visualize normalized binding signals. The same bigwig files were also used to generate profile plots using deepTools plotprofile (Ramı´rez et al., 2016).
Genes proximal to peaks were annotated against the hg38 genome using annotatePeaks.pl from HOMER v4 (Heinz et al., 2010). DUX4, DUX4-IGH and DUX4-del50 binding motifs were identified using findMotifsGenome.pl from HOMER v4.
Assembly: hg38
Supplementary files format and content: BigWig, narrowPeak, bed

source_name: B cell precursor ALL

growth_protocol: Inducible REH cells were grown in RPMI-1640 medium (Euroclone) supplemented with 20% FBS and 1% Pen-Strep

extract_protocol:
Starting from >5x10^6 REH cells induced with doxycycline for 12h, nuclei were prepared by resuspending cells in NE1 buffer (20mM HEPES-KOH pH7.9, 10mM KCl, 0,5mM Spermidine, 1% Triton X-100) at a cellular density of 10^6 cells/mL, followed by 10’ incubation in ice. Samples were spun for 4’ at 600xg at 4°C and the nuclear pellet was resuspended in 1 volume (V) of PBS. A light cross-linking was performed by addition formaldehyde to 0,1% final concentration for 2’, followed by quenching with a double molar concentration of glycine. Nuclei were spun and resuspended in ½ V of Wash Buffer 150 (20mM HEPES-KOH pH7.9, 150mM NaCl, 0,5mM Spermidine). Nuclei were then manually counted using the trypan blue exclusion assay, as described (Kaya-Okur et al, 2019). For each CUT&Tag assay against different DUX4 versions and GTF2I, 100000 nuclei were immobilized to 4uL of Concanavalin A (ConA-Epycypher-21-1401)-conjugated beads for 10’ at RT. For each CUTAC and CUT&Tag against H3K27Ac, 50000 nuclei were immobilized to 4uL of Concanavalin A. The primary (1st) antibody (Ab) was diluted 1:100 in antibody buffer (20mM HEPES-KOH pH7.9, 150mM NaCl, 0,5mM Spermidine, 0,5mg/mL BSA, 0,4mM EDTA pH7.5) and incubated with the ConA-bound nuclei ON at 4°C while rotating. The following day,ConA-nuclei-1stAb were spun 100xg, 1’’, the supernatant was removed, nuclei were resuspended in 100ul of Wash Buffer 150 and incubated with a secondary antibody (2nd) diluted 1:100 in Wash Buffer 150 for 1h at RT. After removal of the 2nd Ab, nuclei were washed once with X ul of Wash Buffer 150 and incubated with the protein AG-conjugated- Transposase (pAG-Tn5, Epycypher- 23615-1117) preloaded with sequencing adapters, diluted 1:20 in Wash Buffer 300 (20mM HEPES-KOH pH7.9, 300mM NaCl, 0,5mM Spermidine) for 1h at RT. Nuclei were then washed twice in Wash Buffer 300 and tagmentation reaction was performed by addition of 10mM MgCl2 following incubation at 37°C for 1h. After a wash with TAPS buffer (10mM TAPS in H2O), tagmented DNA was released by addition of 5uL 0.1% SDS release buffer (0,1% SDS, 10mM TAPS in H2O) following incubation at 58°C for 1h. Finally, 15uL of 0.67% Triton-X-100 were added to neutralize SDS and allow PCR for library preparation
2uL of 10uM i5 universal adapter primer (Nextera) and 2uL of 10uM i7 uniquely barcoded adapter primers were added to released, tagmented DNA, followed by addition of 25uL of the Q5 High-Fidelity 2X Master Mix (New England Biolabs, M0492L). PCR was performed as follows: Cycle 1: 72 °C for 5’ (gap filling), Cycle 2: 98 °C for 30’’, Cycle 3: 98 °C for 10’’ Cycle 4: 63 °C for 10’’. 72°C for 1’ and hold at 8°C. For H3K27Ac CUT&Tag and CUTAC, repeat Cycles 3-4 13 times, for CUT&Tag against different DUX4 versions and GTF2I, repeat cycles 3-4 19 times. Amplified DNA was then recovered through size exclusion purification by addition of 1.3V of AMPureXP magnetic beads (Beckman-A63881), followed by two washes in 80% ethanol and elution in 22uL of 10mM Tris-HCl (pH8). Quantification and average fragment size of libraries was performed via the 4200 TapeStation system (Agilent), using the D5000 high sensitivity kit (Agilent 5067-5592/5067-5593). 450bp of average library fragment size was selected for library quantification. A 5nM pool of the libraries was then run with the NovaSeq instrument and sequenced in paired end (PE), generating 2x150bp reads.

treatment_protocol: Nearly 5x10^6 inducible REH cells were induced with 1ug/mL of Doxycycline in fully-supplemented RPMI

value: B cell precursor ALL

value: REH

tag: antibody

value: Anti-Rabbit Secondary Antibody (EpiCypher #13-1047)


title: CTL-2_D15 (Control, rep2, day15) [RD15]


title: B2-16

first: Antoine

last: Peters

address: Maulbeerstrasse 66


title: B2-15


title: mESCs_scGROseq_Exp236_c16

description: Nascent RNA


title: mESCs_scGROseq_Exp236_c15

description: Nascent RNA


title: CTL-2_D5 (Control, rep2, day5) [RD14]


title: CUT&Tag REH DUX4-IGH +SAHA rep2

value: DUX4/DUX4c (R&D MAB95351)


title: B2-14


title: CUT&Tag REH DUX4-IGH +SAHA rep1


title: B2-19


title: CTL-2_D0 (Control, rep2, day0) [RD13]


title: B2-12


title: B2-13


title: mESCs_scGROseq_Exp236_c13

description: Nascent RNA


title: CUT&Tag REH DUX4-IGH +DMSO rep2


title: mESCs_scGROseq_Exp236_c14

description: Nascent RNA


title: PTC-1_D20 (Patient corrected, rep1, day20) [RD12]


title: mESCs_scGROseq_Exp236_c03

description: Nascent RNA


title: B2-11


title: CUT&Tag REH DUX4-IGH +DMSO rep1


title: CUT&Tag HEK DUX4-IGH +GTF2I rep2

source_name: Human embryonic kidney

value: Human embryonic kidney


title: PTC-1_D5 (Patient corrected, rep1, day5) [RD10]


title: mESCs_scGROseq_Exp236_c02

description: Nascent RNA


title: PTC-1_D15 (Patient corrected, rep1, day15) [RD11]


title: B2-10


title: mESCs_scGROseq_Exp234_c09

description: Nascent RNA


title: B2-9


title: PTC-1_D0 (Patient corrected, rep1, day0) [RD09]


title: CUT&Tag HEK DUX4-IGH +GTF2I rep1


title: CUT&Tag HEK DUX4-IGH +EV rep2


title: CUT&Tag HEK DUX4-IGH +EV rep1


title: PT-1_D20 (Patient, rep1, day20) [RD08]


title: B2-8


title: mESCs_scGROseq_Exp230_c04

description: Nascent RNA


title: PT-1_D15 (Patient, rep1, day15) [RD07]


title: B2-7


title: Flt3_WT_3

description:
10x Genomics
SLX17001_SIGAE4

data_processing:
Barcode processing, mapping and gene counting were performed using the Cell Ranger pipeline v6.0.1
Assembly: mm10

source_name: bone marrow

value: bone marrow

value: LK

tag: Sex

value: F

tag: age

value: 13 weeks

value: WT


title: Flt3_WT_2

description:
10x Genomics
SLX17001_SIGAD4


title: CUT&Tag HEK DUX4 +GTF2I rep2


title: PT-1_D5 (Patient, rep1, day5) [RD06]

value: Human iPSCs


title: CUT&Tag HEK DUX4 +GTF2I rep1


title: CTX_old_neun_9

city: La Jolla

first: Nicola

middle: J

last: Allen

state: CA

address: 10010 N Torrey Pines Rd

department: Molecular Neurobiology

institute: Salk Institute for Biological Studies

data_processing:
Quality control of sequenced reads was performed with FastQC. No trimming was performed in any of the samples
Sequencing reads were mapped with STAR using Mus musculus genome version mm10, and with comprehensive gene annotation file from Gencode (GRCm38 assembly). Mapping results were observed with STAR (output log)
Reads per gene were counted with HOMER analyzeRepeats command. We used '-count genes' option to count reads that map to introns and exons.
Assembly: mm10
Supplementary files format and content: tab-delimited text files include raw sequencing counts for each Sample.
Supplementary files format and content: tab-delimited text files include Transcripts per Million (TPM) counts for each Sample.

molecule: nuclear RNA

source_name: NeuN positive neurons in cortex of aged (2 year old) WT C57BL/6 male mouse

extract_protocol:
Adult (4mo) and aged (2yo) male C57BL/6 mice (3 mice/group) were obtained from Jackson laboratories (adult) or the NIA (aged) and housed for 3 weeks. Mice were anesthetized with ketamine (10mg/ml) and xylazine (2mg/ml) solution delivered intraperitoneally, decapitated and cortex and cerebella were dissected. Dissected tissues were placed in 4 mL of 3% glyoxal solution for 15 min on ice. After that, samples were washed 3 times in PBS1X and nuclei were obtained after tissue homogenization with a dounce homogenizer followed by iodixanol gradient. Nuclei suspensions were stained with Hoetsch, anti-Sox9 and anti-NeuN antibodies, and with fluorescently-conjugated secondary antibodies. Sox9+NeuN- astrocyte and NeuN+ neuron nuclei were FACS-purified with an Aria II Fusion (BD).
RNA was extracted with Rnaeasy Micro Kit (Qiagen) following manufacturer's instructions. RNA quality and quantity were determined with TapeStation (High sensitivity RNA tape) following manufacturer's instructions. 2 ng per sample were used to construct libraries with the Illumina Stranded Total RNA prep (with Ribo-zero plus) sequencing kit, following manufactuter's instructions.

value: Brain, whole cortex

value: neuron

value: WT C57BL/6

value: male


title: PT-1_D0 (Patient, rep1, day0) [RD05]

value: Patient


title: B2-6

institute: Friedrich Miescher Institute

value: GRO derived from E12.5 embryonic gonad


title: CUT&Tag HEK DUX4 +EV rep2


title: CTX_old_neun_8


title: CTL-1_D20 (Control, rep1, day20) [RD04]

value: Control


title: B2-5


title: CUT&Tag HEK DUX4 +EV rep1


title: CTL-1_D15 (Control, rep1, day15) [RD03]


title: B2-3


title: B2-4


title: CTL-1_D5 (Control, rep1, day5) [RD02]


title: CTX_old_neun_7


title: CTX_old_ast_12

source_name: Sox9 positive astrocytes in cortex of aged (2 year old) WT C57BL/6 male mouse

value: astrocyte


title: CUT&Tag GTF2I REH DUX4-IGH rep1

value: B cell precursor ALL

value: REH

value: GTF2I/TFII-I (Bethyl # A301-331A)


title: CUT&Tag GTF2I REH DUX4-IGH rep2


title: CTL-1_D0 (Control, rep1, day0) [RD01]


title: B2-1


title: B2-2


title: CTX_old_ast_11


title: CTX_old_ast_10


title: CUT&Tag GTF2I REH EV rep2


title: A2-24

source_name: GRO derived from BVSC-iPSC

value: GRO derived from BVSC-iPSC


title: CUT&Tag GTF2I REH EV rep1


title: CTX_adult_neun_3

source_name: NeuN positive neurons in cortex of adult (4 month-old) WT C57BL/6 male mouse


title: A2-23


title: retinal organoids derived from Homo sapiens fetal stem cells DRAM2 KO, rep3

description:
SAM24352895
NGS1894_counts.csv
NGS1894_meta.csv

data_processing:
The demultiplexing, barcoded processing, gene counting and aggregation were made using the Cell Ranger software v3.1.0 (https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/what-is-cell-ranger)
Assembly: GRCh38
Supplementary files format and content: Comma-separated values files

source_name: retinal organoid

extract_protocol:
Single-cell RNAseq libraries prepared using 10x protocol from eye organoids derived from DRAM2 WT (1 sample) and DRAM2 KO stem cells (1 sample). N=2 samples.
Library was performed according to the manufacter’s instructions (single cell 3’ v2 protocol, 10x Genomics). Briefly, GCs were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the apropiated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.

value: retinal organoid

value: hPSC H1

value: DRAM2 KO

tag: clone

value: DRAM2 KO2

value: 12 months


title: CUT&Tag GTF2I HEK rep2

value: Human embryonic kidney


title: A2-22


title: retinal organoids derived from Homo sapiens fetal stem cells DRAM2 KO, rep1

description:
SAM24352893
NGS1894_counts.csv
NGS1894_meta.csv

value: DRAM2 KO1


title: CTX_adult_neun_2

value: Brain, whole cortex

value: neuron

value: WT C57BL/6


title: CUT&Tag GTF2I HEK rep1


title: CUT&Tag H3K27Ac REH DUX4-IGH +Dox rep2

value: Anti-Histone H3 (acetyl K27) antibody (ab4729)


title: retinal organoids derived from Homo sapiens fetal stem cells DRAM2 KO, rep2

description:
SAM24352894
NGS1894_counts.csv
NGS1894_meta.csv

value: male


title: A2-20


title: CUT&Tag H3K27Ac REH DUX4-IGH +Dox rep1


title: CTX_adult_neun_1


title: retinal organoids derived from Homo sapiens fetal stem cells DRAM2 WT, rep3

description:
SAM24352892
NGS1894_counts.csv
NGS1894_meta.csv

value: DRAM2 WT

value: WT


title: A2-19


title: A2-18


title: retinal organoids derived from Homo sapiens fetal stem cells DRAM2 WT, rep2

description:
SAM24352891
NGS1894_counts.csv
NGS1894_meta.csv


title: retinal organoids derived from Homo sapiens fetal stem cells DRAM2 KO, rep4

description:
SAM24352896
NGS1894_counts.csv
NGS1894_meta.csv


title: CTX_adult_ast_4

source_name: Sox9 positive astrocytes in cortex of adult (4 month-old) WT C57BL/6 male mouse

value: astrocyte


title: CUT&Tag H3K27Ac REH DUX4-IGH -Dox rep2

value: B cell precursor ALL

value: REH


title: CTX_adult_ast_5


title: CTX_adult_ast_6


title: CUT&Tag H3K27Ac REH DUX4-IGH -Dox rep1


title: A2-17

value: GRO derived from BVSC-iPSC


title: CB_old_neun_21

source_name: NeuN positive neurons in cerebellum of aged (2 year old) WT C57BL/6 male mouse

value: Brain, cerebellum


title: retinal organoids derived from Homo sapiens fetal stem cells DRAM2 WT, rep1

description:
SAM24352890
NGS1894_counts.csv
NGS1894_meta.csv

value: retinal organoid

value: hPSC H1


title: Human skin mast cell, Lesional Psoriasis, Sample 6

city: Manchester

first: Silvia

last: Bulfone-Paus

address: 46 Grafton Street

department: Lydia Becker Institute of  Immunology & Inflammation

institute: University Of Manchester

data_processing:
Raw files trimmed with Trimmomatic (v0.36) using defualt settings
Timmed Fastqs aligned with STAR (v2.5.3) using default settings
Aligned reads were sorted and filtered with samtools (v1.3)
Ordered BAM files were uploaded to Qlucore Omics Explorer (v3.3) to obtain normalised counts
Assembly: GRCh37
Supplementary files format and content: Qlucore Normalized Counts, .txt

source_name: Skin

growth_protocol: Skin biopsies were cut into small pieces in digestion solution containing RPMI 1640 medium (Sigma-Aldrich), 0.5 % BSA (Invitrogen), 100 U .ml-1 penicillin and 100 µg.ml-1 streptomycin, 0.5  Wunch units.ml-1 of Liberase TM (Roche) and 100 ng.ml-1 of SCF (Genscript) Skin was incubated overnight at 37°C, 5% CO2. Digested skin was passed through a 40 μm mesh cell strainer and cell concentration was adjusted to 50 x 10^6 cells.ml-1. Cell suspension was stained for 15 min in PBS 0.5% BSA, 2 mM EDTA with CD45 (2D1), human Lineage cocktail (CD3 (UCHT1), CD14 (HCD14), CD19 (HIB19), CD20 (2H7) and CD56 (HCD56)), CD117 (104D2), FcɛRIα (AER-37) antibodies (all from BioLegend, San Diego, CA) and a viability dye (Fixable Blue Dead Cell Stain Kit, ThermoFisher Scientific). CD45+, Lin-, CD117+, FcɛRIα+, cells were sorted using the BD Influx (BD Biosciences), MCs were collected in 1000 cell aliquots and stored at -80⁰C until required.

extract_protocol: SMART-Seq® Ultra® Low Input RNA Kit (Takara Bio, London, UK) was used to synthesize the full cDNA from 1000 mast cell according to the manufacturer’s instructions

value: Skin

value: Human Mast Cell

value: Female

value: Psoriasis

tag: pooling

value: not pooled


title: CUT&Tag H3K27Ac REH DUX4 +Dox rep2


title: Human skin mast cell, Lesional Psoriasis, Sample 5


title: A2-15

city: Basel


title: CB_old_neun_20

value: neuron

value: WT C57BL/6


title: CUT&Tag H3K27Ac REH DUX4 +Dox rep1

value: Anti-Histone H3 (acetyl K27) antibody (ab4729)


title: Human skin mast cell, Lesional Psoriasis, Sample 4

value: Male


title: A2-14


title: CB_old_neun_19

value: male


title: Human skin mast cell, Lesional Psoriasis, Sample 3


title: CUT&Tag H3K27Ac REH DUX4 -Dox rep2


title: CB_old_ast_24

source_name: Sox9 positive astrocytes in cerebellum of aged (2 year old) WT C57BL/6 male mouse

value: astrocyte


title: A2-13


title: A2-12


title: CUT&Tag H3K27Ac REH DUX4 -Dox rep1

value: B cell precursor ALL


title: A2-11


title: CB_old_ast_22


title: CB_adult_neun_15

source_name: NeuN positive neurons in cerebellum of adult (4 month-old) WT C57BL/6 male mouse


title: Human skin mast cell, Healthy Volunteer, Sample 7

description: re-analysis of GSM5869929

value: Healthy


title: A2-16

value: GRO derived from BVSC-iPSC


title: CUTAC REH DUX4-IGH +Dox rep2

value: REH

value: Phospho_Rbp1 CTD (Ser5) (D9N5I) (CST #13523)


title: CUTAC REH DUX4-IGH -Dox rep2


title: Human skin mast cell, Lesional Psoriasis, Sample 1


title: A2-9


title: CB_adult_neun_14

value: Brain, cerebellum


title: CUTAC REH DUX4-IGH +Dox rep1


title: Human skin mast cell, Healthy Volunteer, Sample 6

description: re-analysis of GSM5869928

value: Skin


title: A2-10


title: CUTAC REH DUX4-IGH -Dox rep1


title: Human skin mast cell, Healthy Volunteer, Sample 5

description: re-analysis of GSM5869930

value: Human Mast Cell


title: CB_adult_neun_13


title: A2-8


title: Human skin mast cell, Healthy Volunteer, Sample 4


title: CUTAC REH DUX4 +Dox rep2


title: CB_adult_ast_18

source_name: Sox9 positive astrocytes in cerebellum of adult (4 month-old) WT C57BL/6 male mouse


title: Human skin mast cell, Healthy Volunteer, Sample 3


title: CB_adult_ast_17


title: Human skin mast cell, Lesional Psoriasis, Sample 2

value: Female

value: pooled patients


title: A2-7


title: CB_old_ast_23


title: CUTAC REH DUX4 -Dox rep2


title: Human skin mast cell, Healthy Volunteer, Sample 2

value: not pooled


title: A2-6


title: CB_adult_ast_16


title: CUTAC REH DUX4 -Dox rep1


title: Human skin mast cell, Healthy Volunteer, Sample 1


title: A2-4


title: A2-5


title: A2-3


title: A2-2


title: iKAP_DOX / KRAS WT, replicate 3

first: Wen-Hao

last: Hsu

address: 1901 East Rd

institute: MD Anderson

description:
10x Genomics
SC220_6

data_processing:
raw count matrices were converted to Seurat objects for further analysis. The quality of scRNA-seq data was measured by the percentage of mitochondrial genes (percent.mt) per cell and the number of unique molecular identifiers (UMI count). Low-quality cells with a UMI count less than 800, a percent.mt greater than 25%, and doublets expressing both cell type markers were removed. The batch effect was counteracted using the reciprocal PCA-based integration method in Seurat. Then we performed unsupervised dimension reduction and clustering for cell identification. The annotation of cell identities in each cluster was based on both the canonical marker genes and the cluster differential genes.
Assembly: mm10
Supplementary files format and content: comma-separated values files and matrix files

source_name: CRC, Distal colon

extract_protocol:
Three independent samples each from iKAP, iAP, and iKAP-DOXoff (KRAS*-off) CRC tumors were collected for tumor dissociation and single cell sequencing analyses. All tumors were collected in size of 1 cm3 at 2 months after TAM/DOX tumor induction following the procedure described above. For the iKAP-DOXoff (KRAS*-off) CRC tumors, Doxycycline was retrieved for 7 days after 7 weeks of TAM/DOX tumor induction.
Single-cell capture, barcoding and library preparation were performed by following the 10X Genomics Single-Cell Chromium 3′ (PN-120237) protocol using V3 chemistry reagents (10X Genomics). The final libraries containing barcoded single-cell transcriptomes were sequenced at 100 cycles on an S2 flowcell on the Novoseq 6000 system (Illumina). Data were processed using the CASAVA 1.8.1 pipeline (Illumina), and sequence reads were converted to FASTQ files and UMI read counts using the CellRanger software (10X Genomics).

value: CRC, Distal colon

value: 6-8 weeks old


title: iKAP_DOX / KRAS WT, replicate 2

description:
10x Genomics
SC220_5


title: CUT&Tag REH DUX4-IGH rep2

value: DUX4/DUX4c (R&D MAB95351)


title: CUT&Tag REH DUX4-del50 rep2


title: CUT&Tag REH DUX4-del50 rep1


title: iKAP / KRAS G12D, replicate 1 [SC220_4]

description:
10x Genomics
SC220_4


title: TL-21-UQS82IRR

data_processing:
Libraries were sequenced to obtain at least 50 million reads on an Illumina HiSeq 4000 System.
RNA-seq reads were aligned to the GRCh38 assembly with STAR version 2.7.2b (Parameters:--genomeDir Ensembl_GRCh38.fa --genomeLoad NoSharedMemory --outSAMmapqUnique 60 --outSAMunmapped Within KeepPairs --outFilterIntronMotifs RemoveNoncanonicalUnannotated --outSAMstrandField intronMotif --runThreadN 8 --outStd BAM_Unsorted --outSAMtype BAM Unsorted --alignTranscriptsPerReadNmax 100000 --outFilterMismatchNoverLmax 0.1 --sjdbGTFfile Ensembl_GRCh38_genes.gtf > genome_accepted_hits.bam).
Gene counts were derived from the number of uniquely aligned unambiguous reads by Picard version 2.6.0.
All gene counts were imported into the R (3.2.3). Bioconductor (3.2) package EdgeR and TMM normalization size factors were calculated to adjust for samples for differences in library size.
Assembly: GRCh38
Supplementary files format and content: A single tab-delimited text file including non-normalized transcript counts for all samples. Column names correspond to sample names and rows correspond to genes.

source_name: sporadic vestibular schwannoma

extract_protocol:
RNA extraction was performed by Tempus, Inc. as per their protocol detailed here: https://doi.org/10.18632%2Foncotarget.25381. Briefly, FFPE tissue was digested with proteinase K and total nucleic extracted using a Chemagic360 instrument. RNA was isolated using DNAse digestion followed by magnetic bead purification.
RNA library creation was performed by Tempus, Inc. as per their protocol detailed here: https://doi.org/10.18632%2Foncotarget.25381. Briefly, RNA was fragmented with heat and cDNA amplified using random primers. Next, A-tailing, adapter ligation, bead-based cleanup, and library amplification was performed. Samples were then hybridized with the IDT xGEN Exome Research Panel and recovered using Streptavidin-coated beads, followed by amplification using the KAPA HiFi Library Amplification Kit.

value: sporadic vestibular schwannoma


title: TL-21-RP9QX42R


title: A2-1


title: CUTAC REH DUX4 +Dox rep1


title: TL-21-AC312E


title: CUT&Tag REH DUX4-IGH rep1


title: iAP / KRAS WT, replicate 3

description:
10x Genomics
SC220_2


title: CUT&Tag REH DUX4 rep2


title: mESCs_AGTuC_Exp125c_c02

description: Nascent RNA

source_name: embryonic stem cells

value: embryonic stem cells


title: mESCs_AGTuC_Exp125c_c01

description: Nascent RNA


title: iAP / KRAS WT, replicate 2

description:
10x Genomics
SC220_1


title: TL-21-4E4896


title: iKAP / KRAS G12D, replicate 1 [SC220_3]

description:
10x Genomics
SC220_3


title: CUT&Tag REH DUX4 rep1

source_name: B cell precursor ALL


title: iAP / KRAS WT, replicate 1

description:
10x Genomics
SC180_3


title: 20230124_CGATAC138_HCC827GR6_Rep2

city: Boston

first: Cigall

last: Kadoch

address: 44 Binney St

institute: Dana-Farber Cancer Institute

data_processing:
Illumina NextSeq output data were demultiplexed and converted to FASTQ format using the bcl2fastq software tool.
RNAseq reads were aligned to the hg19 genome with STAR v2.5.2b, which output gene count tables to the hg19 refFlat annotation. For ATACseq data, quality read trimming was performed by Trimmmomatic v0.36, followed by alignment, duplicate read removal, and read quality filtering, using Bowtie2 v2.29, Picard v2.8.0, and SAMtools v0.1.19, respectively. For Cut & Run libraries, the CutRunTools pipeline was used to perform read trimming, quality filtering, alignment, and peak calling using default parameters.
For Cut&Run and ATACseq data, tracks were generated using deepTools bamCoverage (--normalizeUsing CPM -bs 50 --smoothLength 600 --ignoreDuplicates --samFlagInclude 64). For RNAseq data, tracks were generated using deepTools bamCoverage (--normalizeUsingRPKM).
Assembly: hg19
Supplementary files format and content: Cut&Run and ATACseq bigWig files give the normalized coverage of DNA fragments across the genome in RPM, and gene count tables give raw RNA read counts mapped to exons of given genes. RNAseq bigWig files give the give the normalized coverage of DNA fragments across the genome in RPKM.

source_name: cancer cell line

growth_protocol: PC-9, PC-9_OR, HCC4006, HCC4006_OR, HCC827, and HCC827GR6 were maintained in RMPI-1640 (Gibco) supplemented with 10% fetal bovine serum (GeminiBio) and 1% penicillin-streptomycin (Gibco). PC-9_OR and HCC4006_OR cell lines were generated by continuously culturing the respective parental cell lines in 100 nM osimertinib for at least 2 months before being characterized. Unless stated otherwise, OR lines were maintained in 100 nM osimertinib and the drug was removed for 1 week before use in Cmp14 experiments.

extract_protocol:
ATAC-Seq: The Omni-ATAC protocol (Corces et al., 2017) was used with slight modifications as follows. 100,000 cells per condition were used and washed with PBS before lysis. Cell pellets were lysed in 50μl of cold ATAC-seq resuspension buffer containing 10mM Tris–HCl pH 7.4, 10mM NaCl and 3mM MgCl2, supplemented with fresh NP-40 (final 0.1% v/v), Tween-20 (final 0.1% v/v) and digitonin (final 0.01% v/v) for 3–5min on ice. Next cells were washed with 1ml of resuspension buffer supplemented with Tween-20 (final 0.1% v/v) and pelleted at 500g for 10min at 4 °C. Cell pellets were resuspended in 50μl of transposition reaction mix containing 25μl of 2× TD buffer, 2.5μl of transposase, 16.5μl of 1× PBS, 0.5μl of 1% digitonin (final 0.01% v/v), 0.5μl of 10% Tween-20 (final 0.1% v/v) and 5μl of nuclease-free water. The transposition reaction was incubated at 37 °C for 30min with constant shaking on a thermomixer. The Qiagen MinElute Reaction Cleanup Kit was used for DNA purification. A standard ATAC-seq amplification protocol with 5-7 cycles of amplification was used to amplify the tagmented library (Buenrostro et al., 2015).
RNA-Seq: One million cells were harvested from from PC9*, HCC4006 and HCC827* parental and osi- or gefitinib- resistant cell line pairs and washed with cold PBS to remove trypsin and stored in RLT buffer (Qiagen) until further processing. RNA was purified using the Qiagen RNeasy kit, and further processed using the Illumina NEBNext Ultra II Directional RNA Library Prep Kit.
CUT&RUN: 500,000 cells per sample were harvested from PC9*, HCC4006 and HCC827* parental and osi- or gefitinib- resistant cell line pairs. CUT&RUN was performed following EpiCypher’s protocol within their CUTANA ChIC/CUT&RUN kit with slight modifications to day 1. Prior to wash steps, cells were lysed using a nuclear extraction buffer 20mM HEPES–KOH, pH 7.9, 10mM KCl, 0.1% Triton X-100 and 20% glycerol, supplemented with fresh 0.5mM spermidine, and cOmplete Mini, EDTA-free protease inhibitor. Next, cells were pelleted at 500g for 3 mins. Cell pellets were then washed using protocol’s permeabilization buffer and subsequently incubated with activated Concanavalin A beads at room temperature for 15 mins. Next, beads were mixed in Antibody Buffer and incubated with appropriate primary antibodies overnight (antibodies in key resource table).
CUT&RUN libraries were prepared with Epicypher's CUTANA ChIC/CUT&RUN Library Prep Kit and sequenced on Illumina Next-seq 500 using 37 bp paired-end sequencing parameters.     ATAC-seq samples were sequenced on Illumina NovaSeq 6000 Next Gen Sequencing using 37 bp paired-end sequencing parameters.    RNA-seq libraries were prepared using NEBnext Ultra II Directional RNA Library Prep Kit for Illumina using standard protocols. RNA was sequenced on Illumina Next-seq 500 using 75 bp single-end sequencing parameters.

treatment_protocol: PC9* and PC9-OR* cells were treated with DMSO, 1uM Compound 14 (Cmp14), 100nM Osimertinib (Osi) and 100nM Osimertinib + 30nM Trametinib (OT) for 24hrs before harvesting for ATAC-seq and RNA-seq. The PC9-OR* cells were washed to remove continuous Osimertinib growth media for 7 days prior to experimental setup and then re-challenged with Osi or OT as appropriate.

value: EGFR E746-A750del; METamp


title: iKAP / KRAS G12D

description:
10x Genomics
SC180_2


title: mESCs_inAGTuC_Exp208_c14

description: Nascent RNA


title: TL-21-25586F


title: 20230124_CGATAC137_HCC827GR6_Rep1


title: TL-21-3C0F8E


title: TL-21-FDHQB2HP


title: TL-21-1D0112


title: mESCs_inAGTuC_Exp208_c12

description: Nascent RNA


title: 20230124_CGATAC136_HCC827_Rep2

value: EGFR E746-A750del


title: TL-20-C3E601


title: 20230124_CGATAC135_HCC827_Rep1


title: mESCs_scGROseq_Exp260b_c15

description: Nascent RNA


title: e17.5B: green and red_more diffrentiated-α cell_4

city: Tokyo

first: MIWA

last: HIMURO

address: Hongo 2-1-1, Bunkyo-ku

department: Department of Metabolism and Endocrinology

institute: Juntendo University Graduate School of Medicine

data_processing:
gene_expression e17.5 GcgTimer.xlsx:
Genedata Profiler Genome v10.1.14b
Sequence reads were aligned and mapped to GRCm38/mm10 using STAR v2.5.2b and Genedata Profiler Genome.
Read count extraction and normalization were performed using Genedata Profiler Genome.
Assembly: GRCm38/mm10
Supplementary files format and content: Excel files include TPM values and raw gene counts for each sample.
*********************
gene_expression e17.5 GcgTimer_IRES-EGFP-mRFP added.xlsx:
DRAGEN Bio-IT Platform (3.7.5).
Sequence reads were aligned and mapped to genome GRCm39 and sequence of EGFP, mRFP1 and IRES by using DRAGEN Bio-IT Platform.
Read count extraction and normalization were performed using DRAGEN Bio-IT Platform.
Assembly: GRCm39/mm39
Supplementary files format and content: Excel files include TPM values and raw gene counts for each sample.

source_name: pancreas

extract_protocol:
RNA was extracted using Rneasy mini plus kit (Qiagen).
RNA libraries for RNA-seq were prepared using SMARTER mRNA-Seq Library Prep Kit following manufacturer's protocols.

value: pancreas

value: Gcg-Timer

tag: time


title: e17.5B: green and red_more diffrentiated-α cell_3


title: mESCs_scGROseq_Exp260b_c14

description: Nascent RNA

tag: cell line


title: 2023013_CGATAC134_HCC4006_OR_Rep2


title: TL-20-6FB8A3


title: e17.5B: green and red_more diffrentiated-α cell_2


title: mESCs_scGROseq_Exp260b_c13

description: Nascent RNA


title: 2023013_CGATAC133_HCC4006_OR_Rep1


title: TL-20-636F86


title: e17.5B: green and red_more diffrentiated-α cell_1


title: mESCs_scGROseq_Exp260b_c12

description: Nascent RNA


title: TL-20-257BE9