title: AgRP neurons, leptin, ATAC, rep2

description:
atac_cov_ffl.tsv
atac_cpms.tsv

data_processing:
RNA-seq data was aligned using HISAT2.
Reads were assigned to genes using featureCounts (subread) and an mm10 genome annotation modified to minimize overlapping transcripts.
Differential expression analysis (DEA) of the data was performed with edgeR using the recommended quasi-likelihood GLM method. Significantly different genes were required to have an average expression, across group, of > 1 cpm, a fold change (FC) > 0.5, an adjusted p-value, false discovery rate (fdr), value of < 0.05.
Gene set enrichment analysis (GSEA) gene ontology (GO) for biological process was carried out with WebGestalt (WEB-based GEne SeT AnaLysis Toolkit) 49. Genes with cpm < 1 were omitted from the GSEA analysis.
Assembly: mm10
Supplementary files format and content: tab-separated-values containing raw gene expression counts from each sample
Supplementary files format and content: tab-separated-values containing normalized gene expression counts (cpms) from each sample; filtered to only genes in the DEA analysis
Library reads were mapped to the UCSC build mm10 assembly of the mouse genome using Bowtie 2.
Chromatin accessibility regions (peaks) were valled using MACS2.
Coverage summation and additional data parsing were carried out using bedtools.
Differential binding analysis including normalization and quantification were done in EdgeR. Differential peaks were defined at log2 fold change > 0.5 and a false discovery rate of < 0.05
Assembly: mm10
Supplementary files format and content: tab-separated-values containing raw reads-per-peak (coverage) from each sample
Supplementary files format and content: tab-separated-values containing normalized peak abundance (cpms) from each sample; filtered to only regions in the diff. analysis
Library reads were mapped to the mm10 mouse assembly using Bowtie 2 operating under the `--very-sensitive-local` regimen.
IRF3 peaks were called using SEACR (using IgG control) running in “relaxed” mode. CTCF peaks were called using MACS2, then filtered for reproducible peaks (per-sample) via pseudoreplication (n = 2).
Motif analysis (de novo identification, comparison with known motifs, etc.) was performed using MEME-Suite by taking a radius of 100 bp around the summit of each peak. HOCOMOCOv11 was used as the database for known motifs.
Assembly: mm10
Supplementary files format and content: tab-separated-values containing IRF3 peak information (chrom, start, end, AUC, etc.) identified via SEACR w/ relaxed thresholds

value: fasted overnight

value: leptin


title: AgRP neurons, CTCF CUT&RUN, 3K nuclei

status: Public on Apr 12 2024

type: SRA

city: Boston

first: NORC

last: Boston

state: MA

address: 3 Blackfan Circle, CLS0733

country: USA

institute: Beth Israel Deaconess Medical Center

description: CUT-RUN.CTCF.Mm_ARC_AgRP-pos_003K_01-1.filt.narrowPeak

library_source: genomic

data_processing:
RNA-seq data was aligned using HISAT2.
Reads were assigned to genes using featureCounts (subread) and an mm10 genome annotation modified to minimize overlapping transcripts.
Differential expression analysis (DEA) of the data was performed with edgeR using the recommended quasi-likelihood GLM method. Significantly different genes were required to have an average expression, across group, of > 1 cpm, a fold change (FC) > 0.5, an adjusted p-value, false discovery rate (fdr), value of < 0.05.
Gene set enrichment analysis (GSEA) gene ontology (GO) for biological process was carried out with WebGestalt (WEB-based GEne SeT AnaLysis Toolkit) 49. Genes with cpm < 1 were omitted from the GSEA analysis.
Assembly: mm10
Supplementary files format and content: tab-separated-values containing raw gene expression counts from each sample
Supplementary files format and content: tab-separated-values containing normalized gene expression counts (cpms) from each sample; filtered to only genes in the DEA analysis
Library reads were mapped to the UCSC build mm10 assembly of the mouse genome using Bowtie 2.
Chromatin accessibility regions (peaks) were valled using MACS2.
Coverage summation and additional data parsing were carried out using bedtools.
Differential binding analysis including normalization and quantification were done in EdgeR. Differential peaks were defined at log2 fold change > 0.5 and a false discovery rate of < 0.05
Assembly: mm10
Supplementary files format and content: tab-separated-values containing raw reads-per-peak (coverage) from each sample
Supplementary files format and content: tab-separated-values containing normalized peak abundance (cpms) from each sample; filtered to only regions in the diff. analysis
Library reads were mapped to the mm10 mouse assembly using Bowtie 2 operating under the `--very-sensitive-local` regimen.
IRF3 peaks were called using SEACR (using IgG control) running in “relaxed” mode. CTCF peaks were called using MACS2, then filtered for reproducible peaks (per-sample) via pseudoreplication (n = 2).
Motif analysis (de novo identification, comparison with known motifs, etc.) was performed using MEME-Suite by taking a radius of 100 bp around the summit of each peak. HOCOMOCOv11 was used as the database for known motifs.
Assembly: mm10
Supplementary files format and content: tab-separated-values containing IRF3 peak information (chrom, start, end, AUC, etc.) identified via SEACR w/ relaxed thresholds
Library strategy: Cut & Run

molecule: genomic DNA

organism: Mus musculus

source_name: arcuate nucleus

extract_protocol:
TRAP IP was performed for each sample thus: 4 frozen ARC tissue samples from NuTRAPAgRP mice between 6 and 11 weeks of age were pooled and lysed with Dounce homogenizers in low-salt IP buffer (50mM Tris [pH7.5], 12mM MgCl2, 100mM KCl, 1% NP-40; 100 µg/ml cycloheximide, 2mM DTT, 0.2units/ml RNasin, 1x Roche Complete EDTA-free protease inhibitor). After centrifugation, the supernatant was incubated with GFP ab290 antibody (Abcam) on a rotor, then incubated again with Dynabeads Protein G (Thermo Fisher). RNase-inhibitors were used throughout the protocol to mitigate potential RNA degradation. RNA was isolated with the RNeasy Micro Kit (Qiagen) according to manufacturer's instructions, and then reverse-transcribed and analyzed by qRT-PCR as described above.
TRAP-isolated RNA (<100ng) was treated with the Ribo-Zero rRNA removal kit (Epicentre) to deplete ribosomal RNA and converted into double stranded cDNA using NEBNext mRNA Second Strand Synthesis Module (E6111L). cDNA was subsequently tagmented and amplified for 12 cycles by using Nextera XT DNA Library Preparation Kit (Illumina FC-131). Sequencing libraries were analyzed by Qubit and Agilent Bioanalyzer, pooled at a final concentration of 12pM, and sequenced on a NextSeq500.
AgRP neuronal nuclei were isolated from dissected ARCs from 6-11-week-old mice and were snap frozen and stored at -80C. Isolated ARCs were dounce homogenized in nuclear preparation buffer (NPB; 10 mM HEPES [pH 7.5], 1.5mM MgCl2, 10 mM KCl, 250 mM sucrose, 0.1% NP-40, and 0.2 mM DTT), and homogenates were filtered through a 100 μM strainer and centrifuged to pellet the nuclei. Nuclei were washed with NPB, re-suspended in nuclear sorting buffer (10 mM Tris [pH 7.5], 40 mM NaCl, 90 mM KCl, 2 mM EDTA, 0.5 mM EGTA, 0.1% NP-40, 0.2 mM DTT), and filtered again through a 40 μM strainer. Isolated nuclei were sorted by flow cytometry based on AgRP neuron-specific GFP expression. With this approach we routinely obtain ~2000 AgRP neuronal nuclei per mouse. Nuclei from 4 pooled mice (per sample), were isolated, subjected to FANS, and 10,000 nuclei were collected into 500-750uL PBS (0.1% NP40) in 1.5 mL microcentrifuge tubes and stored on ice. Tubes were spun at 1000 rpm for 10 mins, at 4°C. Supernatant was removed via gentle decantation. Nuclei pellets were subjected to tagmentation after resuspension in 50ul of transposase reaction mix: 25 ul TD buffer, 2.5 ul TN5 transposase, 22.5 ul dH20. Nuclei were incubated at 37°C with shaking at 600 rpm for 30 mins, then placed on ice. DNA was purified using a Qiagen minelute PCR purification kit and eluted in 10 ul of Qiagen Elution Buffer
Library construction was conducted using an initial PCR amplification using 10 ul of transposed DNA, 11ul H2O, 2ul of 25uM Primer 1, 2ul of Primer 2, and 25 ul of NEBNext HF 2X PCR mix. The following initial amplification cycle was used: 1 cycle or 72ºC (5min), 98ºC (30s), 5 cycles of 98ºC (10s), 63C (30s), 72ºC (1 min), hold at 4ºC. A side PCR reaction was conducted to determine the optimal number of amplification cycles used during the library construction, as previously described50,  using 5 ul PCR amplified DNA, 4.41 ul H2O, 0.25ul Primer 1 Adaptor, 0.25ul Primer 2 Adaptor 2 (barcoded), 0.09 ul 100x SYBR Green (Diluted from 10,000X stock with H2O), 5 ul NEBNext HF 2X PCR mix. The following initial amplification cycle was used: 1 cycle of 98ºC (30s), 20 cycles of 98ºC (10s), 63ºC (30s), 72ºC (1 min). Amplification curves were assessed to determine the number of additional cycles needed during library construction50. The requested additional number of cycles was completed by adding the sample back to the thermocycler in its same tube and master mix. Amplified libraries were purified with a Qiagen Minlute PCR purification kit, eluted in 20ul, and the volume was brought up to 100 ul with additional EB. The eluent was subjected to a two-phase size selection using Ampure Beads, initially .55X volume of elutant (55ul) was used, followed by 1.5X volume of initial sample volume (100ul x 1.5 = 150ul) minus the existing PEG that is already in the sample (150ul – 55ul = 95 ul). Finally, the library was eluted using 50ul of EB. Libraries were sequenced on a Next-seq 500 (Illumina)
~4,000 ARC nuclei, from two mice, were isolated and subjected to FANS, and collected into 0.75 mL of C&R Wash Buffer (low salt) buffer (20 mM HEPES-KOH [pH 7.5], 75 mM NaCl, 0.5 mM Spermidine, 0.1% BSA and 1x protease inhibitor cocktail from Sigma). 180 μL BioMagPlus Concanavalin A beads slurry was washed twice and re-suspended in 180 μL Binding Buffer (20 mM HEPES pH 7.5, 10 mM KCl, 1 mM CaCl2, 1 mM MnCl2), and added to the nuclear suspension. The mixture was thoroughly mixed on a rocker for 10 minutes to allow binding of nuclei to the beads. The nuclei were pelleted with a magnet stand and blocked with Wash Buffer (20 mM HEPES-NaOH pH 7.5, 75 mM NaCl, 0.5 mM Spermidine, 0.1% BSA and 1x protease inhibitor cocktail from Sigma) supplemented with 2 mM EDTA. After washing once with Wash Buffer, nuclei were re-suspended in 50 μL Wash Buffer containing 2 μg of anti-IRF3 antibody (Proteintech, 11312-1-AP), and incubated overnight to allow antibody binding. The next day, nuclei were washed once on a magnet stand with Wash Buffer and incubated with 1:1000 pA-MN in 200 μL Wash Buffer. After 1 hour, nuclei were washed once to remove unbound pA-MN, re-suspended in 150 μL Wash Buffer, and then chilled on a 0°C metal block in a water-ice mixture. 3 μL of 100 mM CaCl2 was added to activate pA-MN, and the mixture was incubated at 0°C for 60 min. The reaction was stopped by adding 150 μL 2XSTOP buffer (200 mM NaCl, 20 mM EDTA, 4 mM EGTA, 50 μg/mL RNase A and 40 μg/mL glycogen). The protein-DNA complexes were released by centrifugation and then digested by proteinase K at 50°C overnight. DNA was extracted by ethanol precipitation, followed by Qubit fluorometer and bioanalyzer quality control. CTCF optimization experiments were performed using DAPI-treated nuclei isolated from whole mouse cerebellum, and a CTCF antibody from (Millipore, 07-729). Peri-hypothalamus IRF3-2D IgG (IgG C&R, Figures 6, S6E, S6F) sample was generated by stereotaxically injecting a IRF3LSL-2D mouse with a AAV2-Cre-GFP in the peri-hypothalmus and ~200K GFP+ nuclei were isolated by FANS and used for CUT&RUN on the same day the IRF3Ag C&R sample was generated. IRF3 CUT&RUN from hepatocytes was conducted using primary hepatocytes isolated from IRF3LSL-2D mice as described, and transiently transduced ex vivo with adenovirus expressing Cre recombinase (Ad-Cre). IRF3 and IgG CUT&RUN from immortalized AgRP neurons (IRF3 C&R AgRPin vitro and IgG C&R AgRPin vitro, Figures S6E, S6F) was conducted using those cells transiently transduced with Ad-CMV-IRF3-2D-hPGK-eGFP (Vector Builder).
CUT&RUN libraries were prepared using the NEB Ultra II library preparation kit with important modifications35. Briefly, less than 30 ng DNA was used as input. End prep was performed at 20°C for 30 minutes and then 50°C for 60 minutes, as reduced temperature prevents melting of short DNA fragments (Liu et al., 2018). 5 pmol adaptor was added and ligated to end prep products at 20°C for 15 minutes. USERTM enzyme was then used to cleave the uracil in the loop. 1.7x volume of AMPure beads were used to purify the ligation product, which aids in the recovery of short fragments. To amplify the library, the ligation product was mixed with 2x Ultra II Q5 mix, universal primer and index primers. PCR was carried out as follows: 98°C 30 seconds, 12 cycles of 98°C 10 seconds and 65°C 10 seconds, and final extension 65°C 5 minutes. The PCR product was subjected to double size selection with 0.8x and then 1.2x AMPure beads. The purified library was quantified with Qubit and Tapestation. Libraries were pooled at similar molar amounts and sequenced using Nextseq500 platform.

tag: tissue

value: arcuate nucleus

tag: cell type

value: AgRP neuron

tag: Sex

value: male

tag: condition

value: fed (ad libitum)

tag: treatment

tag: antibody

value: CTCF (Millipore, 07-729)


title: ctrl BR IP biol rep1

city: Houston

first: Matthew

middle: Neil

last: Rasband

state: Texas

address: 1 Baylor Plaza

department: Neuroscience

institute: Baylor College of Medicine

description:
RAS_61487
rasband_mm10_raw_count_expressions.csv

library_source: transcriptomic

data_processing:
Quality trimming and filtering was performed using Trimmomatic v0.39
Reads were then aligned to UCSC mm10 using STAR v2.7.10b
Gene quantification was generated using quantMode GeneCounts
Assembly: mm10
Supplementary files format and content: text file includes raw counts for each sample

molecule: total RNA

source_name: oligodendrocytes

growth_protocol: NG2CreERTM;Ank3F/F;Rpl22HA/+(AnkG icKO RiboTag) and NG2CreERTM;Rpl22HA/+ (control RiboTag) mice were kept on a 12 h/12 h light/dark cycle (lights on at 7:00 am) and had access to food and water ad libitum. All animal care and experimental procedures were conducted in compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Baylor College of Medicine Institutional Animal Care and Use Committee

extract_protocol:
Mice were anesthetized with isoflurane and perfused with ice-cold PBS, and the whole brain homogenates were immediately processed for IP-enrichment of ribosomes from oligodendrocytes. Three animals were pooled together for one RNA-seq sample. 10% of the cleared homogenate was collected as input. IP was performed overnight with HA-antibody (Biolegend Cat# 901505; RRID:AB_2565023) and Pierce protein A/G Magnetic Beads (Thermo Scientific Cat# 88803). RNA was isolated from both input and IP samples using RNeasy Mini Kit (Qiagen Cat# 74104) following manufacturer's protocols.
RNAseq libraries were prepared with the Takara SMARTer® Stranded Total RNA-Seq v3 - Pico Input Mammalian kit (Takara, p/n 634485) kit following manufacturer's protocols.

treatment_protocol: Genotyping for AnkG icKO RiboTag and control RiboTag mice was carried out by standard PCR on tail clips for Cre, Ank3, and Rpl22 alleles at weaning age. Tamoxifen (20 mg/ml in corn coil) was administered intraperitoneally for 5 consecutive days (100 mg/kg of body weight per day) to 8-week-old mice, which were analyzed after one month.

value: oligodendrocytes

tag: genotype

value: control


title: MCF7, H3K4me3, shCtrl, rep2

city: Cleveland

first: Hung-Ying

last: Kao

state: OH

address: 10900 Euclid Avenue

department: Biochemistry

institute: Case Western Reserve University

description:
instrument model: Illumina NovaSeq X Plus 10B
Incorporate 10% MEF cells into the sample as a spike control.

data_processing:
Basecalls were performed using fastQC v0.12.0 for fastq files from Admera
ChIP-seq reads were trimmed using TrimGalore v0.6.10 with default settings, and reads with a length less than 20bp were excluded.
ChIP-seq reads were aligned to the UCSC hg38 and mm10 genome using Bowtie version 1.1.2. Only uniquely mapping reads with at most two mismatches were retained.
Peaks were called using homer v4.11
SPIKER v1.0.0 was employed to derive scaling factors from input and spike-in control in BAM files. Subsequently, the bigWig files were scaled using these factors through deepTools 3.5.4.
Assembly: hg38 and mm10
Supplementary files format and content: bigWig

organism: Homo sapiens

source_name: Breast; Mammary gland

growth_protocol: MCF7 cells were cultured in DMEM with 10% FBS and 1x penicillin/streptomycin, while ZR-75-1 cells were cultured in RPMI-1640 medium with 10% FBS and 1x penicillin/streptomycin. Both cell lines were maintained at 37°C with 5% CO2.

extract_protocol:
MCF-7/ZR-75-1 cells were fixed with 1% formaldehyde and sheared with E220 focused ultrasonicator (Covaris), respectively. Sheared chromatins were mixed with 20% of lysate from MEF cells processed identically as spike-in for normalization. Mixed chromatin were incubated at 4℃ overnight with antibodies. Protein A beads were then incubated with the chromatin and antibody mixture for 2 hours at 4℃. Beads were washed, reverse-crosslinked, and DNA were purified with Qiaquick PCR purification kit (Qiagen).
KAPA HyperPrep Kit (Roche) was used to prepare ChIP-Rx libraries.

value: Breast; Mammary gland

tag: cell line

value: epithelial cell

value: shRNA-Contrl


title: NMP-negative4

city: Baltimore

first: Eric

last: Villalon

state: Maryland

address: 725 N. Wolfe St

department: Biological Chemistry

institute: Johns Hopkins University

description:
10X Genomics
488-Minus_S31_L004

library_source: transcriptomic single cell

data_processing:
Automatically called cells were further filtered to ensure usage of high-quality droplets with captured cells. RNA barcodes were then filtered on total UMI count (> 500 UMIs), feature counts (> 250 features), and percentage of mitochondrial genes (< 25%). Seurat v4.3.0142 was used for handling of normalization, identification of variable genes, scaling, principal component analysis, UMAP dimensional reduction, and SNN generation followed by Leiden clustering. Batch effect correction was performed using Harmony.143 Clusters were identified using a combination of marker genes and differential expression comparing each cluster to all other cells in the data set. Differential expression analyses were performed using Mann-Whitney U test. Fgsea v1.24.0144 was used to run gene set enrichment analysis (GSEA) with gene sets obtained from the Molecular Signatures Database.144 Features were ranked by -log(pvalue) * sign (foldchange).
Supplementary files format and content: transcript expression p-value per gene name per cluster. "pct.1" = NMP-positive samples. "pct.2" = NMP-negative samples

source_name: DRG neurons

growth_protocol: DRGs were collected and transferred to Complete Saline Solution (CSS) (137 mM NaCl, 5.3 mM KCl, 1 mM MgCl2-6H2O, 25 mM Sorbitol, 10 mM HEPES, and 3 mM CaCl2-2H2O, pH 7.2). The DRGs were digested for 20 minutes at 37 °C with 0.35 U/ml of Liberase TM followed by 15 minutes at 37 °C in 0.25 U/ml Liberase TM and 30 U/ml papain in CSS containing 0.5 mM EDTA. The cells were then triturated using a fire polished pasetur pipette to obtain single cell supsension. Cells where then supplemented with DMEM/F12 with 10% fetal calf serum, 1% penicillin/streptomycin.

extract_protocol:
Extraction was performed according to CG000124_Demonstrated_Protocol_Nuclei_isolation_RevE.pdf (ctfassets.net)
libraries were constructed according to 1000350_1000372 Chromium Next GEM Single Cell 3' HT Gel Bead Kit v3.1

treatment_protocol: Immediately after dissociation, antibody feeding labeling of the NMP was performed as follows.All steps were done at room temperature. Cells were spun down at 800rpm, the media was removed and replaced with media containing primary antibody against proteasome subunit-B5. Cells were incubated in primary antibodies at room temperature for 30 min. Then the cells pelleted by spinnng at 800prm and the media replaced with PBS to rinse. The cells where rinsed 3 times.  were rinsed 3 times with PBS. Then the cells were inclubated in media containing fluorescently labeled secondary antibodies for 40 min at room temperature. This was followed by 3 PBS rinses. The live cells were then FACS sorted to separate NMP-positive vs NMP-negative cell populations.

value: DRG neurons

value: somatosensory neuron

value: NMP labeling

tag: cell population

value: NMP-negative


title: MCF7, H3K4me3, shCtrl, rep1


title: AgRP neurons, leptin, ATAC, rep1

description:
atac_cov_ffl.tsv
atac_cpms.tsv


title: AgRP neurons, fed, ATAC, rep2

description:
atac_cov_ffl.tsv
atac_cpms.tsv

value: PBS (vehicle)


title: MCF7, H3K4me3, shCtrl, rep3


title: NMP-negative3

description:
10X Genomics
488-Minus_S31_L003


title: NMP-negative2

description:
10X Genomics
488-Minus_S31_L002


title: NMP-negative1

description:
10X Genomics
488-Minus_S31_L001


title: Hypsibius 1mg/mL Bleomycin biol rep 1

city: Chapel Hill

first: Courtney

middle: M

last: Clark-Hachtel

state: NC

address: 120 South Rd

department: Biology

institute: UNC Chapel Hill

data_processing:
Reads were adapter trimmed then mapped to the most recent genome for H. exemplaris (v3.1.5) using BBduk and BBmap (ver 39.01), and counts were assigned with featureCounts (ver 2.0.6) using the annotation file associated with this genome
Reads were aggregated at the level of genes and only genes with more than one count in at least two samples were kept for differential expression analysis.
Transcript abundance, fold changes, and FDR values were determined using EdgeR
Assembly: nHd_3.1.5
Supplementary files format and content: tab-deliminated text file including raw counts for each samples
Supplementary files format and content: tab-delimiated text files including edgeR output (logFC, CPM, FDR) for 0 mg/mL, 10 µg/mL, 100 µg/mL, and 1 mg/mL Bleomycin

molecule: polyA RNA

organism: Hypsibius exemplaris

source_name: whole body

extract_protocol:
RNA was isolated immediately from each replicate using the PicoPure RNA isolation kit (Applied Biosystems) following slightly modified manufacturer instructions.
Libraries were constructed using the KAPA mRNA stranded library prep kit and fragmented to ~300bp

treatment_protocol: Approximately 200 adult animals were used for each replicate (3 replicates each of untreated (0 mg/mL), 10 µg/mL, 100 µg/mL, and 1 mg/mL Bleomycin). Animals were exposed to an appropriate dose of Bleomycin for 24 hours (or or spring water, untreated (0 mg/mL)).

value: whole body

value: 1mg/mL Bleomycin


title: carotid_21, scRNA-seq

city: New York

first: Chenyi

last: Xue

state: NY

institute: Columbia University

data_processing:
Fastq files were pre-processed by 10x Genomics Cell Ranger 5.0.1 to generate count matrices. Reads were mapped to Cell Ranger reference version 2020-A with GENCODE v32 annotation. Duplicate gene symbols were suffixed with numbers.
Cells expressing less than 200 genes were first removed, and then genes expressed in less than 3 of the remaining cells were removed.
Cells with total UMI (unique molecular identifier) counts over 40,000, or total mitochondrial content over 30%, or total unique genes over 6,000 were filtered out.
Assembly: GRCh38
Supplementary files format and content: 10x Genomics output files: barcodes.tsv.gz, features.tsv.gz, matrix.mtx.gz

source_name: Carotid artery

extract_protocol:
RNA was extracted using 10x Genomics Chromium Single Cell 3' v3 protocol.
Library was constructed using 10x Genomics Chromium Single Cell 3' v3 protocol.

value: Carotid artery

tag: race

value: Unknown

value: Male

tag: age

tag: symptom presence

value: Symptomatic


title: carotid_20, scRNA-seq

value: Caucacian

value: Asymptomatic


title: NMP-positive4

description:
10X Genomics
488-Plus_S30_L004

value: NMP-positive


title: AgRP neurons, fasted, ATAC, rep2

description:
atac_cov_ffl.tsv
atac_cpms.tsv

value: arcuate nucleus

value: AgRP neuron

value: male

value: fasted overnight


title: HEK293T, SRα siRNA, eIF4E IPed1

city: Seoul

first: Min-kyung

last: Shin

address: Anam-ro

country: South Korea

institute: Korea University

data_processing:
Sequenced reads were processed with CutAdapt.
Ribosomal RNA reads were removed riboPicker.
Filtered reads were mapped to STAR 2.5.2b
RPM values were calculated via the htseq-count Python code
Assembly: hg19
Supplementary files format and content: Tab-separated values with count files

extract_protocol:
RNA was extracted using TRIzol reagent.
RNA libraries for RNA-seq were prepared using TruSeq Stranded mRNA Sample Prep Kit (Illumina) following manufacturer's protocols.

value: Human embryonic kidney

value: SRα siRNA


title: AgRP neurons, fasted, ATAC, rep1

description:
atac_cov_ffl.tsv
atac_cpms.tsv


title: NMP-positive3

description:
10X Genomics
488-Plus_S30_L003

value: DRG neurons

value: somatosensory neuron

value: NMP labeling


title: carotid_19, scRNA-seq


title: HEK293T, SRα siRNA, CBP80 IPed1


title: AgRP(+) neurons, rep2

value: fed (ad libitum)


title: NMP-positive2

description:
10X Genomics
488-Plus_S30_L002


title: NMP-positive1

description:
10X Genomics
488-Plus_S30_L001


title: HEK293T, control siRNA, eIF4E IPed1

value: control siRNA


title: AgRP(-) neurons, rep2

value: non-AgRP neuron


title: AgRP(+) neurons, rep1


title: HEK293T, control siRNA, CBP80 IPed1


title: liver, WT, fasted, biol rep 3

city: San Antonio

first: Yidong

last: Chen

address: 8403 Floyd Curl Drive, MSC 7784

department: Population Health Sciences

institute: UT Health Science Center at San Antonio

data_processing:
Illumina bcl2fastq software used for base-calling
All RNA-seq FastQ reads were aligned with the reference genome (UCSC human genome build hg19) using STAR-RSEM default settings to obtain the counts per gene
Gene count measurements were obtained directly from RSEM algorithm
Assembly: mm10
Supplementary files format and content: The processed data files are in tabular format, which contains gene_id, transcript_id(s), gene length, effective_length, expected_count, TPM, and FPKM

source_name: liver

extract_protocol:
Qiagen RNEasy Kit
Total RNA samples were used for sequencing library preparation with KAPA RNA HyperPrep kit with RiboErase (HMR) following KAPA RNA library preparation protocol. Each library was bar-coded and then pooled for sequencing run paired-end sequencing protocol.

treatment_protocol: Mice in the fasted group were food denied for 48 hours, but all mice were given free access to water.

value: liver

tag: strain

value: WT

value: fasted


title: HEK293T, SRα siRNA1

data_processing:
Sequenced reads were processed with CutAdapt.
Ribosomal RNA reads were removed riboPicker.
Filtered reads were mapped to STAR 2.5.2b
Assembly: hg19
Supplementary files format and content: Tab-separated values with count files


title: carotid_17, scRNA-seq

value: Carotid artery

value: Caucacian

value: Male

value: Asymptomatic


title: HEK293T, control siRNA1


title: AgRP(-) neurons, rep1

value: arcuate nucleus

value: male


title: AgRP neurons, fed, ATAC, rep1

description:
atac_cov_ffl.tsv
atac_cpms.tsv

value: AgRP neuron

value: PBS (vehicle)


title: carotid_14, scRNA-seq

value: Symptomatic


title: carotid_15, scRNA-seq

value: Female


title: liver, WT, fasted, biol rep 2


title: D04-194

city: Woodbury

first: Ashraf

middle: Sameh

last: Shabaneh

address: 2768 Liberty Trail

department: Institute of Health Informatics

institute: University of Minnesota

description: Skin, forearm, left_x000D_ ¬†¬†¬†¬†¬†Graft versus host disease, grade II- see description._x000D_

data_processing:
Unaligned reads alignment via Pathseq - microbiome analysis.
Aligned and assembled using hisat2 and Cufflinks. Single gene analysis performed and pathway analysis via Enrichr.
Assembly: GRCh38_canon
Supplementary files format and content: comma delimited FPKM counts
Supplementary files format and content: comma delimited microbiome counts

source_name: Skin

extract_protocol:
extracted RNA from the FFPE biopsies using PureLink FFPE RNA Isolation Kit. FFPE samples yielded an adequate quantity of RNA for sequencing (median = 98717524.5 reads, range = 74316820 to 229178960).
After extraction, we prepared the libraries with the SMARTer Stranded Total RNA Kit v2.
Performed sequencing on a single lane of a NovaSeq S4 2x150-bp.

value: Skin

tag: day 28_agvhd_response

value: CR


title: liver, ACSS1K635Q, control diet, biol rep 3

value: control diet


title: arcuate nucleus, leptin, RNA, rep11

value: fasted overnight

value: leptin


title: arcuate nucleus, leptin, RNA, rep14


title: carotid_13, scRNA-seq

value: Unknown


title: liver, ACSS1K635Q, fasted, biol rep 3

value: liver

value: fasted


title: carotid_18, scRNA-seq

value: Carotid artery


title: CG4 oligodendroglial cells, Phf8oe, CAG-Phf8-IRES-GFP, biological replicate 1

city: Erlangen

first: Michael

last: Wegner

state: Bavaria

address: Fahrstr. 17

department: Institut of Biochemistry

country: Germany

description:
2x 150bp paired
raw_counts.txt

data_processing:
RNA samples were quantified using Qubit 4.0 Fluorometer (Life Technologies, Carlsbad, CA, USA) and RNA integrity was checked with RNA Kit on Agilent 5300 Fragment Analyzer (Agilent Technologies, Palo Alto, CA, USA).
The sequencing libraries were multiplexed and loaded on the flow cell on the Illumina NovaSeq 6000 instrument according to manufacturer’s instructions. The samples were sequenced using a 2x150 Pair-End (PE) configuration.
Demultiplexing of the sequencing reads was performed with Illumina bcl2fastq (2.20). Adapters were trimmed with Trimmomatic (version 0.36). Quality trimming of the reads has not been performed.
Trimmed raw reads were aligned to rn6 using STAR (version 2.5.2b)
DeSeq2 was used to test for differentially expressed genes. GFP-transduced cells (ctrl) were compared versus Phf8-GFP-transduced cells (Phf8oe) with n=2 for each condition.
Assembly: Rnor_6.0
Supplementary files format and content: tab-delimited text files include raw counts for each of the samples

organism: Rattus norvegicus

growth_protocol: CG4 cells were cultured in basal medium and maintained under proliferating conditions by the addition of growth factors PDGF-AA and bFGF. Differentiation was induced by switching to SATO-containing medium. Incubation took place in a humidified atmosphere with 5% CO2 at 37°C.

extract_protocol:
RNA was extracted with the Qiagen RNeasy Micro-RNA kit. Briefly, cells were lysed with RLT buffer and lysates flash frozen and stored at -80°C until further use. RNA of lysates was extracted according to manufacturers instructions.
RNA-libraries for RNA-seq were prepared with NEBNext Ultra II RNA Library Prep Kit.

treatment_protocol: CG4 cells were ransduced with CAG-IRES-GFP or CAG-Phf8-IRES-GFP containing retroviruses. After two days, GFP+ cells were FAC-sorted and seeded into Poly-L-ornithin coated 10cm dishes.  Transduced cells were expanded and separately seeded at defined density on Poly-L-ornithin coated 6cm dishes and kept under differentiating conditons for 3 days.

value: oligodendrocyte progenitor cell

value: WT, Phf8+ GFP+

value: oligodendrocyte differentiation


title: CG4 oligodendroglial cells, Phf8oe, CAG-Phf8-IRES-GFP, biological replicate 2

description:
2x 150bp paired
raw_counts.txt


title: arcuate nucleus, fed, RNA, rep14

value: arcuate nucleus

value: male

value: fed (ad libitum)


title: CG4 oligodendroglial cells, ctrl, CAG-IRES-GFP, biological replicate 2

description:
2x 150bp paired
raw_counts.txt

value: WT, GFP+


title: carotid_6, scRNA-seq

value: Caucacian

value: Male

value: Asymptomatic


title: carotid_16, scRNA-seq


title: carotid_9, scRNA-seq

value: Symptomatic


title: CG4 oligodendroglial cells, ctrl, CAG-IRES-GFP, biological replicate 1

description:
2x 150bp paired
raw_counts.txt


title: arcuate nucleus, fed, RNA, rep11

value: PBS (vehicle)


title: liver, ACSS1K635Q, fasted, biol rep 2


title: liver, WT, control diet, biol rep 2

value: WT

value: control diet


title: CG4 oligodendroglial cells, CRISPR, Phf8 knockout, seq2, biological replicate 4

description:
second round of sequencing, 2x 150bp paired
raw_counts.txt

data_processing:
RNA samples were quantified using Qubit 4.0 Fluorometer (Life Technologies, Carlsbad, CA, USA) and RNA integrity was checked with RNA Kit on Agilent 5300 Fragment Analyzer (Agilent Technologies, Palo Alto, CA, USA).
The sequencing libraries were multiplexed and loaded on the flow cell on the Illumina NovaSeq 6000 instrument according to manufacturer’s instructions. The samples were sequenced using a 2x100 Pair-End (PE) configuration (seq1) or 2x150 Pair-End configuration (seq2).
Demultiplexing of the sequencing reads was performed with Illumina bcl2fastq (2.20). Adapters were trimmed with Skewer (version 0.2.2) for sequencing round 1 (seq1) or Trimmomatic (version 0.36) for sequencing round 2 (seq2). Quality trimming of the reads has not been performed.
Trimmed raw reads were aligned to rn7 (mRatBN7) using STAR (version 2.7.3 (seq1) / version 2.5.2b (seq2))
To account for differences between the two sample libraries and their preparation, raw counts were normalized by factor analysis using RUVseq with k=1 using residuals (Risso et al. 2014).
The normalized counts were used as input in DeSeq2 to test for differentially expressed genes. Knockouts were compared versus wildtypes with n=4 for each condition.
Assembly: mRatBN7
Supplementary files format and content: tab-delimited text files include raw counts for each of the samples

growth_protocol: CG4 cells were cultured in basal medium and maintained under proliferating conditions by the addition of growth factors PDGF-AA and bFGF. Incubation took place in a humidified atmosphere with 5% CO2 at 37°C.

extract_protocol:
RNA was extracted with the Qiagen RNeasy Micro-RNA kit. Briefly, cells were lysed with RLT buffer and lysates flash frozen and stored at -80°C until further use. RNA of lysates was extracted according to manufacturers instructions.
RNA-libraries for RNA-seq were prepared with the TruSeq Stranded mRNA kit (Illumina) for sequencing round 1 (seq1) or with NEBNext Ultra II RNA Library Prep Kit for sequencing round 2 (seq2).

treatment_protocol: CG4 cells were co-transfected with GFP-containing and SpCas9-Phf8_gRNA-containing plasmids. After two days, cells were FAC-sorted into single wells of 96-well plates and expanded. Positive clones were validated by PCR on genomic DNA.

value: KO

value: CRISPR


title: arcuate nucleus, fasted, RNA, rep14

value: fasted overnight


title: carotid_4, scRNA-seq

value: Carotid artery


title: arcuate nucleus, fasted, RNA, rep11


title: liver, WT, control diet, biol rep 1

value: liver


title: CG4 oligodendroglial cells, CRISPR, Phf8 knockout, seq1, biological replicate 3

description:
first round of sequencing, 2x 100bp paired
raw_counts.txt

value: oligodendrocyte progenitor cell


title: carotid_12, ADT

description: processed data linked to GEX sample

library_source: other

data_processing:
Fastq files were pre-processed by 10x Genomics Cell Ranger 5.0.1 to generate count matrices. Reads were mapped to Cell Ranger reference version 2020-A with GENCODE v32 annotation. An additional feature barcode reference of 283 antibody-derived tags was included. Duplicate gene symbols were suffixed with numbers.
Cells expressing less than 200 genes were first removed, and then genes expressed in less than 3 of the remaining cells were removed.
Cells with total UMI (unique molecular identifier) counts over 40,000, or total mitochondrial content over 30%, or total unique genes over 6,000 were filtered out.
Assembly: GRCh38
Supplementary files format and content: 10x Genomics output files: barcodes.tsv.gz, features.tsv.gz, matrix.mtx.gz
Library strategy: CITE-seq

molecule: protein

tag: library type

value: ADT

tag: adt

value: TotalSeq-A panel of 274 ADTs and 9 control ADTs


title: POMC neurons, leptin, RNA, rep2

description:
rna_counts.tsv
rna_cpms_pomc-ffl.tsv

value: arcuate nucleus

value: POMC neuron

value: male

value: leptin


title: liver, WT, fasted, biol rep 1

value: fasted


title: CG4 oligodendroglial cells, CRISPR, Phf8 knockout, seq2, biological replicate 2


title: carotid_12, GEX

value: Caucacian

value: Male

value: Symptomatic

value: mRNA


title: carotid_11, ADT

description: processed data linked to GEX sample


title: POMC neurons, leptin, RNA, rep1

description:
rna_counts.tsv
rna_cpms_pomc-ffl.tsv


title: CG4 oligodendroglial cells, CRISPR, Phf8 knockout, seq1,biological replicate 1

value: KO


title: liver, ACSS1K635Q, control diet, biol rep 2

value: control diet


title: carotid_11, GEX


title: liver, ACSS1K635Q, control diet, biol rep 1


title: carotid_10, ADT

description: processed data linked to GEX sample

value: Female


title: POMC neurons, fed, RNA, rep2

description:
rna_counts.tsv
rna_cpms_pomc-ffl.tsv

value: fed (ad libitum)

value: PBS (vehicle)


title: liver, ACSS1K635Q, fasted, biol rep 1

value: liver


title: CG4 oligodendroglial cells, wildtype, seq1, biological replicate 3

value: oligodendrocyte progenitor cell

value: WT

value: no treatment


title: CG4 oligodendroglial cells, wildtype, seq2, biological replicate 4


title: liver, WT, control diet, biol rep 3


title: POMC neurons, fasted, RNA, rep2

description:
rna_counts.tsv
rna_cpms_pomc-ffl.tsv

value: arcuate nucleus

value: POMC neuron

value: fasted overnight


title: POMC neurons, fed, RNA, rep1

description:
rna_counts.tsv
rna_cpms_pomc-ffl.tsv

value: male


title: carotid_8, ADT

description: processed data linked to GEX sample

value: Carotid artery

value: African American

value: ADT

value: TotalSeq-A panel of 274 ADTs and 9 control ADTs


title: CG4 oligodendroglial cells, wildtype, seq2, biological replicate 2


title: RNA__Trim33_CKO2_4OHT_d3_S4

first: Ioanna

last: Tiniakou

address: 435 E 30th St

institute: NYU Langone

data_processing:
Adapters and low quality bases trimmed using Trimmomatic
reads aligned to reference mm10 genome using STAR
Generate counts matrix using featureCounts
Assembly: mm10
Supplementary files format and content: csv; raw counts

source_name: Bone marrow

extract_protocol:
Cells were sorted and treated as described in the manuscript and RNA was isolated using RNeasy mini kit (QIAGEN) according to the manufacturer’s instructions.
Libraries were prepared for sequencing using standard Illumina protocols and Low input Clontech SMART-Seq HT with Nxt HT following manufacturer's protocols.

treatment_protocol: Lin- Flt3+ progenitors were sorted separately from bone marrow of individual mice (two mice per genotype), cultured in full DMEM supplemented with Flt3L and treated with 4-OHT for 3 days.

value: Bone marrow

value: dendritic cell progenitor

value: Trim33fl/fl R26CreER/+

value: treated with 4-OHT


title: RNA__Trim33_CKO1_4OHT_d3_S3


title: POMC neurons, fasted, RNA, rep1

description:
rna_counts.tsv
rna_cpms_pomc-ffl.tsv

value: PBS (vehicle)


title: carotid_10, GEX

value: Caucacian

value: Female

value: Symptomatic

value: mRNA


title: CG4 oligodendroglial cells, CRISPR-control, seq1, biological replicate 1

value: oligodendrocyte progenitor cell

value: CRISPR ctrl (WT)

value: CRISPR


title: arcuate nucleus, leptin, RNA, rep4

value: arcuate nucleus

value: male

value: fasted overnight

value: leptin


title: carotid_8, GEX

value: Carotid artery

value: African American

value: Male


title: carotid_7, ADT

description: processed data linked to GEX sample

value: Asymptomatic

value: ADT

value: TotalSeq-A panel of 274 ADTs and 9 control ADTs


title: RNA__Trim33_WT2_4OHT_d3_S2

value: Bone marrow

value: dendritic cell progenitor

value: treated with 4-OHT


title: RNA__Trim33_WT1_4OHT_d3_S1


title: arcuate nucleus, leptin, RNA, rep1


title: EdUseq_Exp7_HeLa_ThymRel_120_siPARP2_1

city: Geneva

first: Vasilis

last: Dionellis

address: 30, quai Ernest-Ansermet

country: Switzerland

institute: University of Geneva

data_processing:
100-bp single end reads were aligned to the reference human genome Human GRCh37/hg19 using bwa v.0.7.17, retaining only the reads with the highest quality score.
CSV processed files have been generated as described in the section "Sequence alignment and calculation of sigma values" of Methods published in https://doi.org/10.1038/nature25507
Assembly: Human GRCh37/hg19

source_name: Human cell line

growth_protocol: HeLa cells were cultured in DMEM supplemented with 10% FBS , 1% penicillin/streptomycin and 1% glutamine.

extract_protocol:
After fixation, the cells were permeabilized with 0.2% Triton-X in PBS and EdU was coupled to a cleavable biotin-azide linker (Azide-PEG(3+3)-S-S-biotin) using the Click-it Kit. Purification of genomic DNA was then performed by phenol-chloroform extraction and ethanol precipitation and subjected to EdU-labelled DNA isolation.
Purified EdU-labeled DNA was used for library preparation with TrueSeq ChIP Library Preparation Kit (Illumina) and high-throughput 100-bp single-end sequencing was performed on Illumina Hi-Seq 4000 sequencer.

treatment_protocol: To deplete TIMELESS or TIPIN expression, cells were transfected with siRNAs against TIMELESS or TIPIN. To synchronise cells in the G1-S transition, a thymidine block for 18hrs was performed. Cells were then released in S phase for 120min, in the presence of 25μΜ EdU for the last 30min. Cells were then collected and fixed with 90% methanol overnight.

value: Human cell line

value: HeLa

tag: disease state

value: Human cervical cancer

value: siRNA


title: arcuate nucleus, fed, RNA, rep4

tag: tissue

value: arcuate nucleus

value: fed (ad libitum)

value: PBS (vehicle)


title: EdUseq_Exp7_HeLa_ThymRel_120_siPARP2_2


title: carotid_7, GEX

value: Caucacian

value: mRNA


title: Monocytes, WT3 (ATAC-Seq)

city: Rochester

first: Tyler

last: Stahl

address: 601 Elmwood Ave

institute: University of Rochester Medical Center School of Medicine and Dentistry

data_processing:
Data Formatting bcltofastq-2.19.1
Data Cleaning FastP  0.23.2, --trim_poly_g -x --cut_window_size 4 -3 -l 35 -i ${SAMPLE}_R1.fastq.gz -I ${SAMPLE}_R2.fastq.gz -o synced_clt_${SAMPLE}_R1.fastq.gz -O synced_clt_${SAMPLE}_R2.fastq.gz -h ${SAMPLE}_fastp.html
Genome Alignment bowtie2 version 2.4.4, -x ${REF} -X 2000 -p ${CPUS} -1 synced_clt_${SAMPLE}_R1.fastq.gz -2 synced_clt_${SAMPLE}_R2.fastq.gz -S synced_clt_${SAMPLE}_bowtie.sam
Assembly: mg38 gencode 25
Supplementary files format and content: MACS2 broad peak file containing chromosome, peak start, and peak end

source_name: Monocytes from Control Mice

extract_protocol: ATAC-seq libraries were prepared from aliquots of 50,000 cells using the method described in Corces et al. Briefly, cells were incubated in hypotonic buffer in the presence of digitonin, washed, centrifuged to collect permeabilized samples, and resuspended in tagmentation buffer with Tn5 transposase (Illumina, San Diego, CA). Tagmented DNA was isolated with the Qiagen MinElute Reaction Cleanup kit and a dual-indexed library was prepared by subsequent PCR with Illumina-compatible primers and purified with AMPure XP beads (Beckman Coulter, Indianapolis, IN). Library quantity and quality were assessed by Qubit and Bioanalyzer (Agilent, Santa Clara, CA) assays, respectively, and paired-end sequencing performed on an Illumina NovaSeq 6000.

value: Blood Cells

tag: cell type

value: Monocyte

value: WT

value: Diptheria Toxin


title: EdUseq_Exp7_HeLa_ThymRel_120_siPARP1_2


title: Monocytes, WT2 (ATAC-Seq)


title: EdUseq_Exp7_HeLa_ThymRel_120_siPARP1_1


title: arcuate nucleus, fasted, RNA, rep4

data_processing:
RNA-seq data was aligned using HISAT2.
Reads were assigned to genes using featureCounts (subread) and an mm10 genome annotation modified to minimize overlapping transcripts.
Differential expression analysis (DEA) of the data was performed with edgeR using the recommended quasi-likelihood GLM method. Significantly different genes were required to have an average expression, across group, of > 1 cpm, a fold change (FC) > 0.5, an adjusted p-value, false discovery rate (fdr), value of < 0.05.
Gene set enrichment analysis (GSEA) gene ontology (GO) for biological process was carried out with WebGestalt (WEB-based GEne SeT AnaLysis Toolkit) 49. Genes with cpm < 1 were omitted from the GSEA analysis.
Assembly: mm10
Supplementary files format and content: tab-separated-values containing raw gene expression counts from each sample
Supplementary files format and content: tab-separated-values containing normalized gene expression counts (cpms) from each sample; filtered to only genes in the DEA analysis
Library reads were mapped to the UCSC build mm10 assembly of the mouse genome using Bowtie 2.
Chromatin accessibility regions (peaks) were valled using MACS2.
Coverage summation and additional data parsing were carried out using bedtools.
Differential binding analysis including normalization and quantification were done in EdgeR. Differential peaks were defined at log2 fold change > 0.5 and a false discovery rate of < 0.05
Assembly: mm10
Supplementary files format and content: tab-separated-values containing raw reads-per-peak (coverage) from each sample
Supplementary files format and content: tab-separated-values containing normalized peak abundance (cpms) from each sample; filtered to only regions in the diff. analysis
Library reads were mapped to the mm10 mouse assembly using Bowtie 2 operating under the `--very-sensitive-local` regimen.
IRF3 peaks were called using SEACR (using IgG control) running in “relaxed” mode. CTCF peaks were called using MACS2, then filtered for reproducible peaks (per-sample) via pseudoreplication (n = 2).
Motif analysis (de novo identification, comparison with known motifs, etc.) was performed using MEME-Suite by taking a radius of 100 bp around the summit of each peak. HOCOMOCOv11 was used as the database for known motifs.
Assembly: mm10
Supplementary files format and content: tab-separated-values containing IRF3 peak information (chrom, start, end, AUC, etc.) identified via SEACR w/ relaxed thresholds

tag: Sex

value: male

value: fasted overnight


title: arcuate nucleus, fasted, RNA, rep1

tag: condition


title: Monocytes, WT1 (ATAC-Seq)

tag: treatment


title: IgG, DIPG4, AZD_with_radiation, replicate 3

city: AURORA

first: Etienne

last: Danis

state: CO

department: Cancer Center

institute: University of Colorado, Denver - Anschutz Medical Campus

data_processing:
The quality of fastq files was accessed using FastQC (v.0.11.8), FastQ Screen (v.0.13.0) and MultiQC (v.1.8).
The Illumina adapters and low quality reads were filtered using BBDuk with the parameters for paired-end reads.
The ChIP-seq reads were aligned to the hg38 genome assembly using bowtie2 (v.2.3.4.3).
Samtools (v. 1.11) was used to select the mapped reads (samtools view -b - q 30) and sort the bam files.
PCR duplicates were removed using Picard MarkDuplicates tool (v.2.21.1).
Bedtools genomecov (v.2.27.1) was used to create bedgraph files from the bam files.
Bigwig files were created using deepTools bamCoverage (v.3.2.1) and visualized using IGV (v.2.8.2).
Peaks were called using MACS2 (v2.1.2) (Ref. 10) using ENCODE recommendations.
IDR (v.2. 0.3) was used to identify the reproducible peaks between the replicates.
Further processing of the peak data was performed in R, using in particular the following tools: valR (v.0.6.4), and DiffBind (v.3.6.2).
Metagene plots and heatmaps were generated using ngs.plot (v.2.61).
Assembly: hg38
Supplementary files format and content: bigwig
Library strategy: CUT & RUN

growth_protocol: DIPG4 cell lines were cultured in 50% Neurobasal-A medium (Invitrogen), 50% DMEM/F12(Invitrogen). supplemented with, 1 mM sodium pyruvate (Gibco), 10 mM HEPES Buffer Solution, 1 mM MEM Non-Essencial Amino Acids, GlutaMAX-I Supplement. 1× penicillin/streptomycin solution, 1x B-27 Supplement Minus Vitamin A, H-EGF, H-FGF basic 154, H-PDGFAA, H-PGF -BBa and Heparin Solution 0.25%. Cells were cultured at 37 °C with 95% air and 5% CO2.

extract_protocol:
Beads were prepared using 10 ul/sample of CUTANA Concavalin A Conjugated Paramagnetic Beads (EpiCypher, SKU:21-1401). Beads were transferred to a 1.5 ml tube and placed in a magnetic separation rack. Supernatant was removed and beads were washed 2 times with 100 ul/sample of cold Bead Activation Buffer (20 mM HEPES pH7.9, 10 mM Kcl, 1 mM CaCl2, 1 mM MnCl2). Beads were then resuspended in 10 ul/sample of cold Bead Activation Buffer and aliquot 10 ul/sample of activated bead slurry into 8-strip tube and kept on ice.  Cells were collected using 0.05% trypsin and washed with 100 ul/sample of Wash Buffer (20 mM HEPES pH7.5, 150 mM NaCl, 0.5 mM Spermidine, 1x Roche cOmplete EDTA-free Protease Inhibitor (1187358001)) at room temperature and centrifuged at RT, 600 g for 3min, for a total of two washes. Cells were resuspended in 100 ul/sample RT Wash Buffer and then aliquot of 100ul washed cells to each 8-strip tube containing 10 ul of activated beads and mixed by pipetting. Cells were incubated with bead slurry for 10 min at RT. Tube strip was placed on a magnetic separation rack until slurry clears and the supernatant was removed. 50 ul of cold Antibody Buffer was added to each sample and mixed by pipetting; 1 ul of antibody, IgG (Rabbit IgG Negative Control, EpiCypher, 13-0042K), PolI (Rpb1 CTD (4H8), Cell Signaling, #2629S), pPolI (pRpb1 CTD (S2), Cell Signaling, #13499S), Brd4 (Brd4, EpiCypher, 13-2003), ENL (MLLT1/ENL (D9M4B);Cell Signaling, #14893) was added to each sample, mixed and incubated on a nutator overnight at 4oC. Tube was placed on a magnet until slurry clears and the supernatant was removed. While beads were on magnet, 250 ul of cold Digitonin Buffer was added directly onto beads of each sample and then pipet to remove supernatant, for a total of 2-washes. 50 ul of cold Digitonin Buffer was added to each sample and mixed. 2.5 ul of CUTANA pAG-MNase (20Xstock) was added to each sample, mixed and incubated for 10min at RT. Tube was placed on a magnet and supernatant was removed. Beads were washed 2 times with cold Digitonin Buffer. Supernatant and 50 ul of cold Digitonin Buffer was added to each sample and mixed. Tubes were placed on ice and 1 ul of 100 mM CaCl2 was added to each sample, mixed, and then incubated on nutator 2 hrs at 4oC. 33 ul/sample of Stop buffer, to stop the MNase activity by chelating Ca++ ions, containing 0.5 ng/sample of E.coli Spike-in DNA, was added to the samples, mixed by pipetting and incubated for 10 min at 37oC in a thermocycler. Tubes were placed on a magnet stand until slurry cleared and the supernatant was transferred to a 1.5ml tube. Finally, the DNA was purified using CUTANA DNA Purification Kit (EpiCypher, 14-0050) according to the manufactory instructions and 1ul of DNA was used to be quantified using Qubit and library preparation.
The NEBNext Ultra DNA Library Prep Kit for Illumina (NEB #E7645S) with Dual Index Primers (NEB #E7600) were used to library preparation for ChiPseq and Cut and Run samples sequencing.  For the NEBNext end prep, 1x TE was added to the DNA to get final volume of 50 ul and mixed with 3ul of NEBNext Ultra II End Prep Enzyme Mix and 7ul of NEBNext Ultra II End Prep Reaction Buffer, placed in thermocycler with heated lid set to 75oC and run the program 30 minutes at 20oC, 60 minutes at 50oC and hold at 4oC. The adaptor ligation reaction was performed with 60u l of Prep Reaction Mixture, 2.5ul of NEBNext Adaptor for Illumina diluted following the manufacture recommendations, 30 ul of NEBNext Ultra Ligation Master Mix and 1ul of NEBNext Ligation Enhancer, incubated at 20oC for 15 min with heated lid off. 3 ul of USER Enzyme was added to the reaction and incubated at 37oC for 15 min with heated led set to 47oC. For the cleanup of the adaptor-ligated DNA without size selection, 1.1x of AMPure XP Beads were added to the Adaptor Ligation reaction and samples were incubated for 5min at room temperature. Tubes were placed on a magnetic stand to separate beads from supernatant, after 5 min, supernatant was removed, and beads were washed 2 times with 200ul of 80% freshy prepared ethanol. Beads were air dried for up 5 min while tubes were on magnet with lid open. DNA was eluted from the beads by adding 17 ul of 0.1XTE buffer.  For the PCR enrichment of adaptor-ligated DNA step, to 15 ul of the Adaptor Ligated DNA fragments was added 25ul of NEBNext Ultra Q5 Master Mix, 5 ul of Index primer i7 and 5ul Universal PCR Primer i5, and PCR amplification was performed following the cycling conditions: 98oC for 45s, 98oC 15s and 60oC 10s for 16 cycles, 72oC for 1 min and hold at 4oC. Followed by a Clean Up of the PCR reaction using AMPure XP Beads.  The DNA was quantified using Qubit and the quality and size of libraries were checked with the Bioanalyzer and sent for sequencing at the Genomics Shared Resource (sequencing core) of CU Anschutz Medical Campus.

treatment_protocol: Cells were treated with DMSO or 40 nM AZD. After 2 hrs, cells were irradiated at 6 Gy and collected after 4 hrs.

value: Diffuse intrinsic pontine glioma

value: AZD and 6 Gy radiation

tag: chip antibody

value: IgG Negative Control; EpiCypher Cat. #13-0042


title: EdUseq_Exp7_HeLa_ThymRel_120_siCTRL_2

value: Human cell line

value: HeLa


title: EdUseq_Exp7_HeLa_ThymRel_120_siCTRL_1

value: Human cervical cancer

value: siRNA


title: ENL, DIPG4, AZD_with_radiation, replicate 1

value: MLLT1/ENL (D9M4B);Cell Signaling Cat.#14893


title: EdUseq_Exp6_HeLa_ThymRel_120_siPARP2


title: ENL, DIPG4, AZD_with_radiation, replicate 2


title: AgRP neurons, leptin, RNA, rep4

description:
rna_counts.tsv
rna_cpms_agrp-ffl.tsv

value: arcuate nucleus

value: AgRP neuron

value: leptin


title: Monocytes, DTR2 (ATAC-Seq)

source_name: Monocytes from thombocytopenic mice

value: Blood Cells

tag: genotype


title: EdUseq_Exp6_HeLa_ThymRel_120_siPARP1

tag: cell line


title: Monocytes, DTR1 (ATAC-Seq)

value: Monocyte


title: carotid_5, ADT

description: processed data linked to GEX sample

value: Carotid artery

value: Caucacian

value: Male

value: Symptomatic

value: ADT

value: TotalSeq-A panel of 274 ADTs and 9 control ADTs


title: AgRP neurons, leptin, RNA, rep2

description:
rna_counts.tsv
rna_cpms_agrp-ffl.tsv

value: male

value: fasted overnight


title: AgRP neurons, leptin, RNA, rep3

description:
rna_counts.tsv
rna_cpms_agrp-ffl.tsv


title: Brd4, DIPG4, AZD_with_radiation, replicate 2

value: Diffuse intrinsic pontine glioma

value: AZD and 6 Gy radiation


title: AgRP neurons, leptin, RNA, rep1

description:
rna_counts.tsv
rna_cpms_agrp-ffl.tsv


title: Monocytes, PLT_LPS_3 (RNA-Seq)

description: Platelet Exposed+LPS

data_processing:
Data Formatting bcltofastq-2.19.1
Data Cleaning fastp 0.23.1, --in1 ../${SAMPLE}_R1.fastq.gz --out1 clt_${SAMPLE}_R1.fastq.gz --length_required 35 --cut_front_window_size 1 --cut_front_mean_quality 13 --cut_front --cut_tail_window_size 1 --cut_tail_mean_quality 13 --cut_tail -w 8 -y -r -j ${SAMPLE}_fastp.json
Genome Alignment STAR_2.7.9a, --twopassMode Basic --runMode alignReads --genomeDir ${GENOME} --readFilesIn ${SAMPLE} --outSAMtype BAM Unsorted --outSAMstrandField intronMotif --outFilterIntronMotifs RemoveNoncanonical
Read Quantification1 subread-2.0.1, featurecounts, -s 2 -t exon -g gene_name
Read Quantification3 salmon-1.5.2, --seqBias --gcBias --posBias
Assembly: Mouse m39, annotation M27
Supplementary files format and content: gene counts

source_name: Bone Marrow

extract_protocol:
Total RNA was isolated using the RNeasy Plus Micro Kit (Qiagen, Valencia, CA). RNA concentration was determined with the NanopDrop 1000 spectrophotometer (NanoDrop, Wilmington, DE) and RNA quality assessed with the Agilent Bioanalyzer 2100 (Agilent, Santa Clara, CA).
1ng of total RNA was pre-amplified with the SMARTer Ultra Low Input kit v4 (Clontech, Mountain View, CA) per manufacturer’s recommendations. The quantity and quality of the subsequent cDNA was determined using the Qubit Flourometer (Life Technologies, Carlsbad, CA) and the Agilent Bioanalyzer 2100 (Agilent, Santa Clara, CA). 150pg of cDNA was used to generate Illumina compatible sequencing libraries with the NexteraXT library preparation kit (Illumina, San Diego, CA) per manufacturer’s protocols.

value: Bone Marrow

value: WT

value: Platelet Overnight +LPS


title: Brd4, DIPG4, AZD_with_radiation, replicate 1


title: EdUseq_Exp5_HeLa_ThymRel_120_siTIPI

value: Human cell line

value: HeLa

value: Human cervical cancer

value: siRNA


title: IgG, DIPG4, DMSO_with_radiation, replicate 3

value: DMSO and 6 Gy radiation

value: IgG Negative Control; EpiCypher Cat. #13-0042


title: arcuate nucleus, fed, RNA, rep1

value: arcuate nucleus

value: fed (ad libitum)

value: PBS (vehicle)


title: Monocytes, PLT_LPS_2 (RNA-Seq)

description: Platelet Exposed+LPS

value: Monocyte


title: Monocytes, DTR3 (ATAC-Seq)

value: Blood Cells

value: Diptheria Toxin


title: Monocytes, PLT_LPS_1 (RNA-Seq)

description: Platelet Exposed+LPS


title: AgRP neurons, fed, RNA, rep4

description:
rna_counts.tsv
rna_cpms_agrp-ffl.tsv

value: AgRP neuron

value: male


title: carotid_5, GEX

value: Carotid artery

value: Caucacian

value: Male

value: Symptomatic

value: mRNA


title: Monocytes, PLT_3 (RNA-Seq)

description: Platelet Exposed

value: Bone Marrow

value: WT

value: Platelet Overnight


title: AgRP neurons, fed, RNA, rep3

description:
rna_counts.tsv
rna_cpms_agrp-ffl.tsv


title: EdUseq_Exp5_HeLa_ThymRel_120_siCTRL

value: Human cell line

value: HeLa

value: Human cervical cancer

value: siRNA


title: ENL, DIPG4, DMSO_with_radiation, replicate 1

value: Diffuse intrinsic pontine glioma

value: DMSO and 6 Gy radiation

value: MLLT1/ENL (D9M4B);Cell Signaling Cat.#14893


title: EdUseq_Exp6_HeLa_ThymRel_120_siCTRL


title: AgRP neurons, fed, RNA, rep1

description:
rna_counts.tsv
rna_cpms_agrp-ffl.tsv

value: arcuate nucleus

value: AgRP neuron

value: fed (ad libitum)

value: PBS (vehicle)


title: EdUseq_Exp5_HeLa_ThymRel_120_siTIME


title: Monocytes, PLT_2 (RNA-Seq)

description: Platelet Exposed

value: Monocyte


title: EdUseq_Exp5_HeLa_ThymRel_090_siTIPI


title: AgRP neurons, fasted, RNA, rep4

description:
rna_counts.tsv
rna_cpms_agrp-ffl.tsv

value: male

value: fasted overnight


title: Monocytes, PLT_1 (RNA-Seq)

description: Control

value: Bone Marrow


title: Brd4, DIPG4, DMSO_with_radiation, replicate 2


title: EdUseq_Exp5_HeLa_ThymRel_090_siTIME


title: Brd4, DIPG4, DMSO_with_radiation, replicate 1


title: AgRP neurons, fasted, RNA, rep3

description:
rna_counts.tsv
rna_cpms_agrp-ffl.tsv


title: IgG, DIPG4, DMSO, replicate 3

value: DMSO

value: IgG Negative Control; EpiCypher Cat. #13-0042


title: Monocytes, NC_3 (RNA-Seq)

description: Control

value: WT


title: Monocytes, NC_2 (RNA-Seq)

description: Control


title: EdUseq_Exp5_HeLa_ThymRel_090_siCTRL

value: Human cell line

value: HeLa


title: ENL, DIPG4, DMSO, replicate 2


title: MEG3 KD 10nM EZH2 replicate 2, ChIPseq

city: Edinburgh

first: Hywel

last: Dunn-Davies

address: Max Born Crescent

department: Wellcome Trust Centre for Cell Biology

country: United Kingdom

institute: University of Edinburgh

description: sample 6 MG3

data_processing:
ChIP sequencing data was filtered remove low quality reads, N reads and adaptor sequences to obtain clean data.
ChIP-seq data processing and alignment onto human genome (hg38) was performed by using SOAP2, initial fastq filtering done using SOAPnuke.
Clean Parameter: SOAPnuke filter -l 10 -q 0.5 -n 0.1 -Q 2 -c 55 -f AAGTCGGAGGCCAAGCGGTCTTAGGAAGACAA -r GAACGACATGGCTACGATCCGACTT -t 0,50,0,50
ChIP-seq  peak calling was done using MACS 1.4.2 p-value < le-05. Dynamic Possion Distribution was used to calculated p-value of the specific region based on the unique mapped reads.
Assembly: hg38
Supplementary files format and content: called peaks in bed format for all samples except input control

source_name: HUVECs

growth_protocol: Human Umbilical Vein Endothelial Cells (HUVECs) (C-12203) were purchased from Promocell, Germany, and cultured in the recommended growth media (EGM2) according to the manufacturer’s instructions under 5% CO2 at 37 °C.

extract_protocol:
Bound DNA was purified by Monarch kit (T1030L) and used for sequencing.
(1) DNA-end repair, 3'-dA overhang and ligation of methylated sequencing adaptor. (2) PCR amplification and size selection (usually 100-300bp, including adaptor sequence).

treatment_protocol: HUVECs (2 x 10^6)  were transfected with MEG3 GapmeRs (10 nM, 48 h) or a scrambled control GapmeRs (Ctr). For transfection, we used 10 nM scrambled LNA (locked nucleic acids) GapmeR control (Cat. No. 339515) or phosphorothioate antisense standard GapmeRs MEG3-lncRNA (Cat No. 339511, Qiagen). We immunoprecipitated EZH2, together with associated chromatin isolated from sonicated chromatin. DNA was fragmented into 200-700 bp pieces using chilling Bioruptor sonicator (6X 2 min ON/30 sec OFF/30 sec, 4°C) and lysates centrifuged (15 min, 1000 rcf, 4°C). Aliquots of lysates containing 200 μg of protein were used for each immunoprecipitation reaction with EZH2 (D2C9) XP(R) Rabbit mAb, (5246S Cell signalling technology), Tri-Methyl-Histone H3 (H3K27me3) (C36B11) Rabbit mAb (9733S, CST) antibodies or IgG control (Normal Rabbit IgG, 2729S, CST) and captured on beads using Protein G Dyneabeads (10003D, Life Technologies). The immune complexes were eluted with the elution buffer (1% SDS, 100 mM NaCO3) supplemented with proteinase K (10 µg/mL) and with shaking at 65°C 1000 rpm for 3-6 h. DNA was purified using Monarch kit (T1030L).

value: HUVECs

value: MEG3 KD 10nM EZH2


title: AgRP neurons, fasted, RNA, rep1

description:
rna_counts.tsv
rna_cpms_agrp-ffl.tsv

extract_protocol:
TRAP IP was performed for each sample thus: 4 frozen ARC tissue samples from NuTRAPAgRP mice between 6 and 11 weeks of age were pooled and lysed with Dounce homogenizers in low-salt IP buffer (50mM Tris [pH7.5], 12mM MgCl2, 100mM KCl, 1% NP-40; 100 µg/ml cycloheximide, 2mM DTT, 0.2units/ml RNasin, 1x Roche Complete EDTA-free protease inhibitor). After centrifugation, the supernatant was incubated with GFP ab290 antibody (Abcam) on a rotor, then incubated again with Dynabeads Protein G (Thermo Fisher). RNase-inhibitors were used throughout the protocol to mitigate potential RNA degradation. RNA was isolated with the RNeasy Micro Kit (Qiagen) according to manufacturer's instructions, and then reverse-transcribed and analyzed by qRT-PCR as described above.
TRAP-isolated RNA (<100ng) was treated with the Ribo-Zero rRNA removal kit (Epicentre) to deplete ribosomal RNA and converted into double stranded cDNA using NEBNext mRNA Second Strand Synthesis Module (E6111L). cDNA was subsequently tagmented and amplified for 12 cycles by using Nextera XT DNA Library Preparation Kit (Illumina FC-131). Sequencing libraries were analyzed by Qubit and Agilent Bioanalyzer, pooled at a final concentration of 12pM, and sequenced on a NextSeq500.
AgRP neuronal nuclei were isolated from dissected ARCs from 6-11-week-old mice and were snap frozen and stored at -80C. Isolated ARCs were dounce homogenized in nuclear preparation buffer (NPB; 10 mM HEPES [pH 7.5], 1.5mM MgCl2, 10 mM KCl, 250 mM sucrose, 0.1% NP-40, and 0.2 mM DTT), and homogenates were filtered through a 100 μM strainer and centrifuged to pellet the nuclei. Nuclei were washed with NPB, re-suspended in nuclear sorting buffer (10 mM Tris [pH 7.5], 40 mM NaCl, 90 mM KCl, 2 mM EDTA, 0.5 mM EGTA, 0.1% NP-40, 0.2 mM DTT), and filtered again through a 40 μM strainer. Isolated nuclei were sorted by flow cytometry based on AgRP neuron-specific GFP expression. With this approach we routinely obtain ~2000 AgRP neuronal nuclei per mouse. Nuclei from 4 pooled mice (per sample), were isolated, subjected to FANS, and 10,000 nuclei were collected into 500-750uL PBS (0.1% NP40) in 1.5 mL microcentrifuge tubes and stored on ice. Tubes were spun at 1000 rpm for 10 mins, at 4°C. Supernatant was removed via gentle decantation. Nuclei pellets were subjected to tagmentation after resuspension in 50ul of transposase reaction mix: 25 ul TD buffer, 2.5 ul TN5 transposase, 22.5 ul dH20. Nuclei were incubated at 37°C with shaking at 600 rpm for 30 mins, then placed on ice. DNA was purified using a Qiagen minelute PCR purification kit and eluted in 10 ul of Qiagen Elution Buffer
Library construction was conducted using an initial PCR amplification using 10 ul of transposed DNA, 11ul H2O, 2ul of 25uM Primer 1, 2ul of Primer 2, and 25 ul of NEBNext HF 2X PCR mix. The following initial amplification cycle was used: 1 cycle or 72ºC (5min), 98ºC (30s), 5 cycles of 98ºC (10s), 63C (30s), 72ºC (1 min), hold at 4ºC. A side PCR reaction was conducted to determine the optimal number of amplification cycles used during the library construction, as previously described50,  using 5 ul PCR amplified DNA, 4.41 ul H2O, 0.25ul Primer 1 Adaptor, 0.25ul Primer 2 Adaptor 2 (barcoded), 0.09 ul 100x SYBR Green (Diluted from 10,000X stock with H2O), 5 ul NEBNext HF 2X PCR mix. The following initial amplification cycle was used: 1 cycle of 98ºC (30s), 20 cycles of 98ºC (10s), 63ºC (30s), 72ºC (1 min). Amplification curves were assessed to determine the number of additional cycles needed during library construction50. The requested additional number of cycles was completed by adding the sample back to the thermocycler in its same tube and master mix. Amplified libraries were purified with a Qiagen Minlute PCR purification kit, eluted in 20ul, and the volume was brought up to 100 ul with additional EB. The eluent was subjected to a two-phase size selection using Ampure Beads, initially .55X volume of elutant (55ul) was used, followed by 1.5X volume of initial sample volume (100ul x 1.5 = 150ul) minus the existing PEG that is already in the sample (150ul – 55ul = 95 ul). Finally, the library was eluted using 50ul of EB. Libraries were sequenced on a Next-seq 500 (Illumina)
~4,000 ARC nuclei, from two mice, were isolated and subjected to FANS, and collected into 0.75 mL of C&R Wash Buffer (low salt) buffer (20 mM HEPES-KOH [pH 7.5], 75 mM NaCl, 0.5 mM Spermidine, 0.1% BSA and 1x protease inhibitor cocktail from Sigma). 180 μL BioMagPlus Concanavalin A beads slurry was washed twice and re-suspended in 180 μL Binding Buffer (20 mM HEPES pH 7.5, 10 mM KCl, 1 mM CaCl2, 1 mM MnCl2), and added to the nuclear suspension. The mixture was thoroughly mixed on a rocker for 10 minutes to allow binding of nuclei to the beads. The nuclei were pelleted with a magnet stand and blocked with Wash Buffer (20 mM HEPES-NaOH pH 7.5, 75 mM NaCl, 0.5 mM Spermidine, 0.1% BSA and 1x protease inhibitor cocktail from Sigma) supplemented with 2 mM EDTA. After washing once with Wash Buffer, nuclei were re-suspended in 50 μL Wash Buffer containing 2 μg of anti-IRF3 antibody (Proteintech, 11312-1-AP), and incubated overnight to allow antibody binding. The next day, nuclei were washed once on a magnet stand with Wash Buffer and incubated with 1:1000 pA-MN in 200 μL Wash Buffer. After 1 hour, nuclei were washed once to remove unbound pA-MN, re-suspended in 150 μL Wash Buffer, and then chilled on a 0°C metal block in a water-ice mixture. 3 μL of 100 mM CaCl2 was added to activate pA-MN, and the mixture was incubated at 0°C for 60 min. The reaction was stopped by adding 150 μL 2XSTOP buffer (200 mM NaCl, 20 mM EDTA, 4 mM EGTA, 50 μg/mL RNase A and 40 μg/mL glycogen). The protein-DNA complexes were released by centrifugation and then digested by proteinase K at 50°C overnight. DNA was extracted by ethanol precipitation, followed by Qubit fluorometer and bioanalyzer quality control. CTCF optimization experiments were performed using DAPI-treated nuclei isolated from whole mouse cerebellum, and a CTCF antibody from (Millipore, 07-729). Peri-hypothalamus IRF3-2D IgG (IgG C&R, Figures 6, S6E, S6F) sample was generated by stereotaxically injecting a IRF3LSL-2D mouse with a AAV2-Cre-GFP in the peri-hypothalmus and ~200K GFP+ nuclei were isolated by FANS and used for CUT&RUN on the same day the IRF3Ag C&R sample was generated. IRF3 CUT&RUN from hepatocytes was conducted using primary hepatocytes isolated from IRF3LSL-2D mice as described, and transiently transduced ex vivo with adenovirus expressing Cre recombinase (Ad-Cre). IRF3 and IgG CUT&RUN from immortalized AgRP neurons (IRF3 C&R AgRPin vitro and IgG C&R AgRPin vitro, Figures S6E, S6F) was conducted using those cells transiently transduced with Ad-CMV-IRF3-2D-hPGK-eGFP (Vector Builder).
CUT&RUN libraries were prepared using the NEB Ultra II library preparation kit with important modifications35. Briefly, less than 30 ng DNA was used as input. End prep was performed at 20°C for 30 minutes and then 50°C for 60 minutes, as reduced temperature prevents melting of short DNA fragments (Liu et al., 2018). 5 pmol adaptor was added and ligated to end prep products at 20°C for 15 minutes. USERTM enzyme was then used to cleave the uracil in the loop. 1.7x volume of AMPure beads were used to purify the ligation product, which aids in the recovery of short fragments. To amplify the library, the ligation product was mixed with 2x Ultra II Q5 mix, universal primer and index primers. PCR was carried out as follows: 98°C 30 seconds, 12 cycles of 98°C 10 seconds and 65°C 10 seconds, and final extension 65°C 5 minutes. The PCR product was subjected to double size selection with 0.8x and then 1.2x AMPure beads. The purified library was quantified with Qubit and Tapestation. Libraries were pooled at similar molar amounts and sequenced using Nextseq500 platform.


title: ENL, DIPG4, DMSO, replicate 1

value: Diffuse intrinsic pontine glioma


title: ENL, DIPG4, DMSO_with_radiation, replicate 2

value: DMSO and 6 Gy radiation

value: MLLT1/ENL (D9M4B);Cell Signaling Cat.#14893


title: Monocytes, LPS_3 (RNA-Seq)

description: Treatment

value: LPS only


title: MEG3 KD 10nM EZH2 replicate 1, ChIPseq

description: sample 5 MG2

value: Human Umbilical Vein Endothelial Cells (HUVECs)


title: 56_ENDO14_t2_PB_CD8T

city: Huddinge

first: Dan

last: Sun

address: Alfred Nobels allé 8

department: MedH

country: Sweden

institute: Karolinska Institutet

description: Longitudinal ind3 t2

data_processing:
Adaptor and quality trimming (-q 30) of RNAseq data was performed using Trim Galore! 0.6.1.
Processed RNA-seq reads were then aligned to the human genome (GRCh38 build) using HISAT2 2.2.1 (Kim et al., 2019), and non-uniquely and non-paired alignments were filtered out by the 'view’ command of SAMTools 1.12 (-f 2 -q 60).
Non-uniquely and non-paired alignments were filtered out by the 'view’ command of SAMTools 1.12 (-f 2 -q 60).
StringTie 2.1.4 was used to count the number of reads per gene per sample, and DESeq2 was used to normalize counts across libraries.
Assembly: GRCh38
Supplementary files format and content: Comma-separated values

source_name: Peripheral blood

extract_protocol:
PB CD56dim and MB uNK cells as well as PB CD8+ TEM and MB CD8+ TRM cells were pre-gated on alive CD45+CD14-CD15-CD19- cells and sorted directly into 300 ul of RLT plus buffer (QIAGEN) in a sterile ribonuclease- and DNase-free 1.5 ml Eppendorf tube and frozen on dry ice immediately after sorting. RNA was isolated with the RNeasy Plus Micro Kit (QIAGEN).
Extracted RNA was quality tested with a Bioanalyzer Pico kit (Agilent), before cDNA was generated with the Smart-seq v4 (Clontech). Next, indexed cDNA libraries were normalized, combined, and pool-sequenced on the Illumina NextSeq 550 for a 75-cycle v2.5 sequencing generating 150–base pair paired-end reads.

value: Peripheral blood

value: CD8+ Tem

tag: sample type

value: longitudinal

tag: cmv

value: pos


title: Brd4, DIPG4, DMSO, replicate 2


title: Monocytes, NC_1 (RNA-Seq)

description: Control

value: Bone Marrow

value: Monocyte


title: input samples ChIPseq

description:
sample 4 MG11
NOTE: Called peaks are not provided as this sample is the input control for the peak calling algorithm.

value: input


title: Brd4, DIPG4, DMSO, replicate 1

value: DMSO


title: Monocytes, LPS_2 (RNA-Seq)

description: Treatment

value: WT


title: 55_ENDO14_t2_PB_NK

description: Longitudinal ind3 t2

value: CD56dim NK


title: Monocytes, LPS_1 (RNA-Seq)

description: Treatment


title: Subject_4_pre_NeoVax_region_2

city: Columbus

first: Katherine

middle: E

last: Miller

address: 700 Children's Dr.

department: Institute for Genomic Medicine

institute: Nationwide Children's Hospital

data_processing:
bcl to fastq was performed using Illumina bcl2fastq conversion software version 2.20.0.422
paired-end FASTQ files were used as input for salmon version 0.9.1 (https://combine-lab.github.io/salmon/getting_started/)
the salmon transcriptome index was prepared using the following command: "salmon index -t GRCh38_latest_rna.fna -i GRCh38_latest_rna.fna_transcripts_index -k 31"; the GRCh38_latest_rna.fna was obtained from NCBI via ftp.ncbi.nlm.nih.gov
the following parameters were used with salmon processing:
libType: A
numBootstraps: 100
index: GRCh38_latest_rna.fna_transcripts_index
Assembly: GRCh38
Supplementary files format and content: tab-delimited text files including transcript per million (TPM) values; each sample has a separate file labeled "Sample_name.quant.sf"

source_name: Glioblastoma brain tumor

extract_protocol:
High Pure isolation kit (Roche Life Science)
Paired-end 2 x 151 bp reads, NovaSeq6000
Illuminia NEBNext Ultra II Directional RNA library kit

value: Glioblastoma brain tumor

tag: brain tumor region

tag: neovax status

tag: age (years)

value: male

tag: overall surviva; (days)


title: 53_ENDO14_t1_PB_NK

description: Longitudinal ind3 t1


title: Monocytes, WT3 (RNA-Seq)

data_processing:
Data Formatting bcltofastq-2.19.1
Data Cleaning fastp 0.23.1, --in1 ../${SAMPLE}_R1.fastq.gz --out1 clt_${SAMPLE}_R1.fastq.gz --length_required 35 --cut_front_window_size 1 --cut_front_mean_quality 13 --cut_front --cut_tail_window_size 1 --cut_tail_mean_quality 13 --cut_tail -w 8 -y -r -j ${SAMPLE}_fastp.json
Genome Alignment STAR_2.7.9a, --twopassMode Basic --runMode alignReads --genomeDir ${GENOME} --readFilesIn ${SAMPLE} --outSAMtype BAM Unsorted --outSAMstrandField intronMotif --outFilterIntronMotifs RemoveNoncanonical
Read Quantification1 subread-2.0.1, featurecounts, -s 2 -t exon -g gene_name
Read Quantification3 salmon-1.5.2, --seqBias --gcBias --posBias
Assembly: Mouse m39, annotation M31
Supplementary files format and content: gene counts

source_name: isolated monocytes from control mice

extract_protocol:
Total RNA was isolated using the RNeasy Plus Micro Kit (Qiagen, Valencia, CA) per manufacturers recommendations. RNA concentration was determined with the NanoDrop 1000 spectrophotometer (NanoDrop, Wilmington, DE) and RNA quality assessed with the Agilent Bioanalyzer 2100 (Agilent, Santa Clara, CA).
The TruSeq Stranded mRNA Sample Preparation Kit (Illumina, San Diego, CA) was used for next generation sequencing library construction per manufacturer’s protocols. Briefly, mRNA was purified from 200ng total RNA with oligo-dT magnetic beads and fragmented. First-strand cDNA synthesis was performed with random hexamer priming followed by second-strand cDNA synthesis using dUTP incorporation for strand marking. End repair and 3` adenylation was then performed on the double stranded cDNA. Illumina adaptors were ligated to both ends of the cDNA and amplified with PCR primers specific to the adaptor sequences to generate cDNA amplicons of approximately 200-500bp in size. The amplified libraries were hybridized to the Illumina flow cell and sequenced using the NovaSeq6000 sequencer (Illumina, San Diego, CA) SP flow cell.

value: Peripheral Blood

value: Diptheria Toxin


title: 54_ENDO14_t1_PB_CD8T

description: Longitudinal ind3 t1


title: Scr control EZH2 replicate 2, ChIPseq

description: sample 2 MG10

value: HUVECs

value: Scr control EZH2


title: Scr control EZH2 replicate 1, ChIPseq

description: sample 1 MG1

value: Human Umbilical Vein Endothelial Cells (HUVECs)


title: AgRP neurons, fasted, RNA, rep2

first: NORC

last: Boston

description:
rna_counts.tsv
rna_cpms_agrp-ffl.tsv

source_name: arcuate nucleus

value: arcuate nucleus

value: AgRP neuron

value: fasted overnight

value: PBS (vehicle)


title: MEG3_Even probes replicate 2, ChIRPseq [TM_N2]

description: sample 8 TM_N2

data_processing:
library strategy: ChIRP-seq
The data were processed using the nf-core chip-seq pipeline (https://zenodo.org/record/7139814, version 2.0.0).
Adapter trimming was performed using Trim Galore! (https://www.bioinformatics.babraham.ac.uk/projects/trim_galore/).
Paired-end reads were aligned to the human genome (hg38) using bwa.
normalised bigWig files were produced scaled to 1 million mapped reads using bedtools (https://github.com/arq5x/bedtools2/) and bedGraphToBigWig (http://hgdownload.soe.ucsc.edu/admin/exe/).
Assembly: hg38
Supplementary files format and content: bigWig

extract_protocol:
DNA was eluted with a cocktail of 100 mg/mL RNase A (Sigma-Aldrich) and 0.1 U/mL RNase H (Ambion) and recovered using Monarch kit (T1030L) and  DNA used for library construction. RNA was TRIzol-extracted and purified to test specificity of probes against MEG3 and LacZ
(1) DNA-end repair, 3'-dA overhang and ligation of methylated sequencing adaptor. (2) PCR amplification and size selection (usually 100-300bp, including adaptor sequence).

treatment_protocol: Genome binding/occupancy profiling by high throughput sequencing was performed. Glutaraldehyde crosslinked (1%, 10 min, 22°C) HUVECs were quenched with glycine (0.125 mM),  lysed and sonicated at Covaris E220 Evolution Focused-Ultrasonicator System using a water at temp range 5-7 °C and following protocol: peak power 140 W, duty factor 10%, 200 Cycles per burst for 60 sec. Total of 30-35 cycles were used for cell lysates prepared from 150 mg of crosslinked HUVEC pellet. Two sets of biotin-labelled antisense oligonucleotide DNA probes targeting MEG3 and lacZ were then used for hybridisation. Exact protocol was used as here https://www.sigmaaldrich.com/GB/en/technical-documents/protocol/protein-biology/protein-and-nucleic-acid-interactions/chromatin-isolation-by-rna-purification.

value: Human umbilical vein endothelial cells

value: MEG3_Even probes


title: Subject_4_pre_NeoVax_region_1


title: 49_ENDO14_t2_MB_NK

description: Longitudinal ind3 t2

source_name: Menstrual blood

value: Menstrual blood

value: uNK

value: longitudinal

value: pos


title: 50_ENDO14_t2_MB_CD8T

description: Longitudinal ind3 t2

value: CD8+ Trm


title: MEG3_Odd probes replicate 1, ChIRPseq [TM_N1]

description: sample 7 TM_N1

value: MEG3_Odd probes


title: Subject_3_post_NeoVax

value: Glioblastoma brain tumor

value: Post-NeoVax

value: female


title: Scr control EZH2 replicate 3, ChIPseq

description: sample 3 MG9

value: HUVECs

value: Scr control EZH2


title: Monocytes, WT1 (RNA-Seq)

value: Peripheral Blood

value: Monocyte

value: WT

value: Diptheria Toxin


title: 48_ENDO14_t1_MB_CD8T

description: Longitudinal ind3 t1


title: Monocytes, DTR2 (RNA-Seq)

source_name: isolated monocytes from thrombocytopenic mice


title: Subject_3_pre_NeoVax_region_3


title: Monocytes, WT2 (RNA-Seq)


title: Monocytes, DTR3 (RNA-Seq)


title: Subject 3_pre_NeoVax_region_1


title: Subject_3_pre_NeoVax_region_2


title: input samples ChIRPseq [TM_input]

description: sample 6 TM input

value: Human umbilical vein endothelial cells

value: input samples ChIRPseq


title: 47_ENDO14_t1_MB_NK

description: Longitudinal ind3 t1

value: Menstrual blood


title: LacZ_probes replicate 3, ChIRPseq [TM_IgG]

description: sample 5 TM_IgG


title: 44_MB056_t2_PB_CD8T

description: Longitudinal ind2 t2

value: Peripheral blood

value: CD8+ Tem


title: 42_MB056_t1_PB_CD8T

description: Longitudinal ind2 t1

value: longitudinal

value: pos


title: Monocytes, DTR1 (RNA-Seq)


title: Subject_2_pre_NeoVax_region_3


title: LacZ_probes replicate 2, ChIRPseq [BR3a]

description: sample 4 BR3a


title: Subject_2_pre_NeoVax_region_2


title: 40_MB056_t2_MB_CD8T

description: Longitudinal ind2 t2

value: CD8+ Trm


title: KO rep1

city: Xuzhou

first: Jianlin

last: Qiao

state: --- Select One ---

address: 84 West Huaihai Road

country: China

institute: Xuzhou Medical University

description: Hepatocyte Notch1 knockout mice

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.
Reference genome and gene model annotation files were downloaded from genome website directly. 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.
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.
Assembly: GRCm38
Supplementary files format and content: tab-delimited text files include FPKM values for each Sample

source_name: Liver

extract_protocol:
Total RNA was isolated from liver using Trizol reagent and mRNA was purified from total RNA using poly-T oligo-attached magnetic beads followed by fragmentation and subsequent cDNA synthesis.
RNA sequencing libraries were constructed using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer's instructions.

treatment_protocol: No treatment

value: Liver

value: Hepatocyte Notch1 KO

value: No treatment


title: 191227_Vec_L5Ec_IgG

data_processing:
Mircat33 adapters were trimmed from the read pairs using cutadapt version 1.18: 'cutadapt -j 5 -e 0.1 -q 16 -O 3 --trim-n --minimum-length 15 -a TGGAATTCTCGGGTGCCAAGGC -A GCCTTGGCACCCGAGAATTCCA'
PCR duplicates were collapsed using pyDuplicateRemover.py from the pyCRAC suite (https://git.ecdf.ed.ac.uk/sgrannem/pycrac).
Read 1 from each read pair in the collapsed dataset was aligned to the human genome (hg38) using novoalign 2.07: novoalign -f [READS] -c 48 -s 1 -r Random -o Native -l 17 -d [GENOME INDEX], and bed files were generated using pyReadCounters.py and pyGTF2bed.py from the pyCRAC suite.
RNA-RNA hybrids were obtained from the same files using the hyb pipeline (https://github.com/gkudla/hyb): 'hyb align=bowtie2 word=11 format=fasta type=all pref=none'
Assembly: hg38
Supplementary files format and content: Files ending in .hyb.txt.gz describe RNA-RNA hybrids, and are generated using the hyb pipeline.
Supplementary files format and content: Files ending in .bed.gz describe the genomic location of FLASH reads, and are in bed6 format.  The fourth field in each read describes the genome features overlapped by the read.
Library strategy: FLASH-seq

source_name: HUVEC

growth_protocol:
EGM2 C-22011 Promocell medium Normoxia
Human Umbilical Vein Endothelial Cells (HUVECs) (C-12203), and Human Dermal Lymphatic Endothelial cells (HDLECs) (C-12217) were purchased from Promocell, Germany, and cultured in the recommended growth media (EGM2) according to the manufacturer’s instructions under 5% CO2 at 37 °C. Hypoxia was stimulated in Coy chamber with a gas mixture of 1% O2, 5% CO2 and 94% N2 (Coy Laboratory Products).

extract_protocol:
Formaldehyde crosslinking was used during purification step, to stabilize binding of the covalent bait protein-RNA complex to the protein A beads. This step allows column washes under highly denaturing conditions, hence assuring reliable downstream hits. Subsequent linker ligation reactions were performed at 16°C, largely preserving RNA stem structures and allowing formation of chimeras.
RNA-protein complexes were resolved on a NuPage gel, RNA was isolated, the cDNA library was amplified and samples pooled before submitting for sequencing.

treatment_protocol: Intact cells were used to capture RNA-protein interactions using UV crosslinking (400 mJoules/cm2) and antibodies for affinity purification of endogenous RNA-protein complexes.

value: HUVEC

value: Human Umbilical Vein Endothelial Cells

tag: bait protein

value: IgG control


title: Subject_1_pre_NeoVax_region_3

value: Glioblastoma brain tumor

value: male


title: MEG3_Even probes replicate 2, ChIRPseq [KN2_8]

description: sample 2 KN2

value: HUVECs

value: MEG3_Even probes


title: Control rep3

description: Notch1fl/oxflox mice


title: 39_MB056_t2_MB_NK

description: Longitudinal ind2 t2

value: uNK


title: KO rep2

description: Hepatocyte Notch1 knockout mice


title: Control rep2

description: Notch1fl/oxflox mice


title: Subject_2_pre_NeoVax_region_1

value: female


title: 191227_Vec_L5De_Hypox

growth_protocol:
EGM2 C-22011 Promocell medium [1% Hypoxia, gas mixture of 1%O2, 5%CO2 and 94%N2 (Coy Laboratory Products)]
Human Umbilical Vein Endothelial Cells (HUVECs) (C-12203), and Human Dermal Lymphatic Endothelial cells (HDLECs) (C-12217) were purchased from Promocell, Germany, and cultured in the recommended growth media (EGM2) according to the manufacturer’s instructions under 5% CO2 at 37 °C. Hypoxia was stimulated in Coy chamber with a gas mixture of 1% O2, 5% CO2 and 94% N2 (Coy Laboratory Products).


title: 191227_Vec_L5Eb_EZH2 (2)


title: Subject_1_pre_NeoVax_region_1


title: 38_MB056_t1_MB_CD8T

description: Longitudinal ind2 t1

value: Menstrual blood


title: KO rep3

description: Hepatocyte Notch1 knockout mice


title: Control rep1

description: Notch1fl/oxflox mice


title: MDA-MB-453, DUSP6 Knockdown, Sample ID 6, rep3

city: Turku

first: Teemu

middle: Daniel

last: Laajala

address: Turun yliopisto

department: Department of Mathematics and Statistics

country: Finland

institute: University of Turku

data_processing:
Reads were called using the manufacturer's bcl2fastq2 base calling software.
Reads were then aligned the reference genome (hg38) using R-package Rsubread (v2.0.0) function align, with the reference genome along with annotations downloaded from Illumina iGenomes web site.
Reads were then associated with known genes and the number of reads associated with each gene was counted using the Rsubreads package v2.0.0, with non-default parameters for isPairedEnd="yes" and strandSpecific=2.
Gene-wise read counts were normalised using the TMM normalization method of the edgeR R/Bioconductor package with log(x+1) transformation for downstream applications.
The data processing pipeline was run using default parameters when possible.
Assembly: hg38
Supplementary files format and content: Tab-separated gene-wise expression (rows) per samples (columns) normalized using TMM.

growth_protocol: MDA-MB-453 cells were maintained in RPMI supplemented with 10% fetal bovine serum (FBS) and antibiotics in humidified atmosphere with 5% CO2 at 37C

extract_protocol:
Illumina TruSeq Stranded Total mRNA kit
RNA was harvested using the NucleoSpin RNA purification kit (Macherey‐Nagel). 300 ng of total RNA was used for the construction of sequencing libraries.
TruSeq® Stranded mRNA Sample Preparation, Illumina

value: HER2+ breast cancer cell line

value: DUSP6 KD


title: 41_MB056_t1_PB_NK

description: Longitudinal ind2 t1

value: Peripheral blood

value: CD56dim NK

value: longitudinal

value: pos


title: LacZ_probes replicate 1, ChIRPseq [KN3_9]

description: sample 3 KN3

value: Human umbilical vein endothelial cells


title: QZH2

city: Dallas

first: Philipp

last: Scherer

state: TX

address: 5323 Harry Hines Boulevard

department: Touchstone Diabetes Center, Internal Medicine

institute: UT Southwestern Medical Center

description: 10X Genomics

data_processing:
Data was analyzed using Cell Ranger (v7.0.0) to perform demultiplexing, aligning reads, filtering, clustering, and gene expression analyses, using default parameters.
Assembly: mm10
Supplementary files format and content: The processed data files contain the unfiltered barcodes, matrix, and genes.

source_name: white adipose tissue

extract_protocol:
Subcutaneous adipose tissues were dissected, minced, and digested with 1X Hank's balanced slated solution contained 1mg/ml Collagenase D (Roche, cat# 11088882001) and 1.5% BSA at 37°C for 45 min within a shaking water bath. Subsequently, 10mM EDTA was added and incubated for another 5 min. The digested mixture was then filtered through 100 μm cell strainer and then 40 μm cell strainer. Red blood cells were lysed with 1X RBC lysis buffer. Cells were resuspended with FACS buffer (2% FBS/PBS) and incubated with anti-mouse CD16/CD32 Fc Block (1:200) for 15 min on ice, and then stained with antibodies for 30 min on ice. Cells were washed twice and sorting was conducted by using a BD Biosciences FACSAria cytometer.
Library prepared followed by 10X Genomics standard protocol.

value: white adipose tissue

value: Stromal vascular fraction

value: Paqr4 transgenic


title: 43_MB056_t2_PB_NK

description: Longitudinal ind2 t2


title: MDA-MB-453, DUSP6 Knockdown, Sample ID 5, rep2


title: 191227_Vec_L5Bb_EZH2 (1)

value: HUVEC

value: Human Umbilical Vein Endothelial Cells


title: 191227_Lec_L5Ea_hnRNPAB

source_name: HDLEC

growth_protocol:
MV2 C22022 Promocell medium Normoxia
Human Umbilical Vein Endothelial Cells (HUVECs) (C-12203), and Human Dermal Lymphatic Endothelial cells (HDLECs) (C-12217) were purchased from Promocell, Germany, and cultured in the recommended growth media (EGM2) according to the manufacturer’s instructions under 5% CO2 at 37 °C. Hypoxia was stimulated in Coy chamber with a gas mixture of 1% O2, 5% CO2 and 94% N2 (Coy Laboratory Products).

value: HDLEC

value: hnRNPAB


title: Subject_1_pre_NeoVax_region_2

value: Glioblastoma brain tumor

value: male


title: QZH1

description: 10X Genomics

value: WT


title: 36_ENDO5_t2_PB_CD8T

description: Longitudinal ind1 t2

value: CD8+ Tem


title: MDA-MB-453, DUSP6 Knockdown, Sample ID 3, rep1

value: HER2+ breast cancer cell line

value: DUSP6 KD


title: MEG3_Odd probes replicate 1, ChIRPseq [KN1_1]

description: sample 1 KN1

value: HUVECs

value: MEG3_Odd probes


title: 35_ENDO5_t2_PB_NK

description: Longitudinal ind1 t2

value: Peripheral blood

value: CD56dim NK

value: longitudinal

value: pos


title: 191227_Lec_L5Dc_EZH2 (2)

value: Human Dermal Lymphatic Endothelial cells


title: MDA-MB-453, Scrambled, Sample ID 4, rep3


title: MDA-MB-453, Scrambled, Sample ID 2, rep2


title: 31_ENDO5_t1_PB_NK

description: Longitudinal ind1 t1


title: 37_MB056_t1_MB_NK

description: Longitudinal ind2 t1

value: Menstrual blood

value: uNK


title: 191227_Lec_L5Db_IgG2

value: HDLEC

value: IgG control


title: mESC Srrm2 KD 72h Replicate 1

city: Berlin

first: Alexander

last: Kukalev

address: Hannoversche Str., 28

department: Berlin Institute for Medical Systems Biology

institute: Max-Delbrueck Center for Molecular Medicine

data_processing:
Expression levels from RNA-seq profiles were pseudo-aligned using Kallisto v0.46.1 (Bray et al., 2016, Nat. Biot. 34, p.525).
Assembly: mm10 (M24)
Supplementary files format and content: Tab-delimited text file includes raw counts for each sample

growth_protocol: mESCs E14tg2a.4 were grown as previously described (Ferrai et al. 2017 Mol. Syst. Biol. 13, p. 946) on 0.1% v/v gelatin-coated flasks in GMEM medium with LIF and additional supplements. Prior to collection of material for experiments, cells were grown for 24h or 72h in serum-free ESGRO medium supplemented with LIF.

extract_protocol:
Total RNA  from three replicates of each condition was isolated with TRIzol (Invitrogen) and DNA was degraded with TURBO DNase (Invitrogen), according to the manufacturer’s protocol.
RNA spike-ins (Invitrogen) were added to each purified RNA sample. PolyA RNA-seq libraries were produced from 1 ug of RNA using the TruSeq® Stranded mRNA Library Prep kit (Illumina), according to the manufacturer's instructions. The libraries were paired-end sequenced using the NovaSeq 6000 system at 100-bp length, according to the manufacturer's protocol.

treatment_protocol: mESC were transfected using OptiMEM (Invitrogen), Lipofectamine RNAiMAX (Invitrogen) and 50 nM synthetic siRNA duplexes (Eurogentec) targeting either Srrm2 or firefly luciferase (GL2), which was used as a control. RNA interference was done according to the manufacturer’s instructions. Cells were harvested at 24h or re-transfected with the same siRNA duplexes and transfection reagents and harvested 72h after seeding.

value: mouse embryonic stem cells

value: Srrm2 knockdown


title: 191227_Lec_L5Bc_EZH2 (1)


title: 30_ENDO5_t2_MB_CD8T

description: Longitudinal ind1 t2

value: CD8+ Trm


title: 29_ENDO5_t2_MB_NK

description: Longitudinal ind1 t2


title: Histone_3_K27_acetylation, DIPG4, AZD4573, radiation, replicate 2

data_processing:
The quality of fastq files was accessed using FastQC (v.0.11.8), FastQ Screen (v.0.13.0) and MultiQC (v.1.8).
The Illumina adapters and low quality reads were filtered using BBDuk with the parameters for paired-end reads.
The ATAC-seq reads were aligned to the hg38 genome assembly using bowtie2 (v.2.3.4.3).
Samtools (v. 1.11) was used to select the mapped reads (samtools view -b - q 30) and sort the bam files.
PCR duplicates were removed using Picard MarkDuplicates tool (v.2.21.1).
Bedtools genomecov (v.2.27.1) was used to create bedgraph files from the bam files.
Bigwig files were created using deepTools bamCoverage (v.3.2.1) and visualized using IGV (v.2.8.2).
Peaks were called using MACS2 (v2.1.2) using ENCODE recommendations.
IDR (v.2. 0.3) was used to identify the reproducible peaks between the replicates.
Further processing of the peak data was performed in R, using in particular the following tools: valR  (v.0.6.4), and DiffBind (v.3.6.2).
Metagene plots and heatmaps were generated using ngs.plot (v.2.61).
Assembly: hg38
Supplementary files format and content: bigwig

growth_protocol: DIPG4 cell lines were cultured in 50% Neurobasal-A medium (Invitrogen), 50% DMEM/F12(Invitrogen) supplemented with 1 mM sodium pyruvate (Gibco), 10mM HEPES Buffer Solution, 1mM MEM Non-Essencial Amino Acids, GlutaMAX-I Supplement. 1× penicillin/streptomycin solution, 1x B-27 Supplement Minus Vitamin A, H-EGF, H-FGF basic 154, H-PDGFAA, H-PGF -BBa and Heparin Solution 0.25%. Cells were cultured at 37 °C with 95% air and 5% CO2.

extract_protocol:
Cells were crosslinked with 1% formaldehyde added to the growth media for 10 min, followed by quenching with 0.125M glycine for 5 min. Cells were washed twice with cold PBS, collected in ice-cold PBS by scraping, pelleted, and resuspended in cell lysis buffer (5mM PIPES, pH 8.0; 85mM KCl; 0.5% NP-40). Following incubation on ice for 10 min, a nuclear-enriched fraction was collected by centrifugation for 5 min at 2500 xg at 4 C. Pellet was resuspended in ChIP lysis buffer (50mM Tris-HCl, pH 8.0; 10mM EDTA; 1% SDS) containing COmplete EDTA free protease inhibitors (Sigma) and quantified with BCA. 2ug/ul of lysates were sonicated with a Bioruptor Plus (Diagenode) for 20 cycles (30 seconds ON and 30 seconds OFF). The size of the sonicated DNA fragments was checked on an 1X TAE 1% agarose gel electrophoresis and ranged between 250 to 500 bp. After removing the debris via centrifugation, chromatin extracts were collected.   For immunoprecipitation, chromatin extracts were diluted with Chip Dilution Buffer (16.7mM Tris-HCl, pH8.1, 1.2Mm EDTA, 167mM NaCl, 0.01%SDS and 1.1% Triton x100) incubated with primary antibodies anti-K27Ac (#39133, Active Motif) overnight at 4 C.  After incubation with the primary antibody, 20 uL of pre-washed magnetic beads (Magna ChIP Protein A+G Magnetic Beads, Millipore Sigma) were added to each sample for 2 hours at 4 C. Using a magnetic rack, the immunoprecipitates were washed successively with 1 ml of low salt buffer (20 mM Tris-HCl [pH 8.0], 150 mM NaCl, 0.1% SDS, 1% triton X-100, 2 mM EDTA), high salt buffer (20 mM Tris-HCl [pH 8.0], 500 mM NaCl, 0.1% SDS, 1% triton X-100, 2 mM EDTA), LiCl washing buffer (10 mM Tris-HCl [pH 8.0], 250 mM LiCl, 1.0% NP40, 1.0% deoxycholate, 1 mM EDTA) and twice with TE buffer. The DNA-protein complexes were eluted with 300 µl of IP elution buffer (1% SDS, 0.1 M NaHCO3). The cross-links were reversed by adding NaCl (a final concentration 0.2 M) into the eluents and incubating them at 65  C overnight. The DNA was recovered by proteinase K and RNase A digestion, followed by phenol/chloroform extraction and ethanol precipitation. Pellets were resuspended in 25 ul of RNase and DNase free water, and ChIP-DNA was quantified using the Qubit dsDNA High Sensitivity Assay kit (Thermo Fisher Scientific), followed by library preparation.
The NEBNext Ultra DNA Library Prep Kit for Illumina (NEB #E7645S) with Dual Index Primers (NEB #E7600) were used to library preparation for ChIP-seq.    NEBNext End Prep  1x TE was added to the DNA to get final volume of 50 ul and mixed with 3 ul of NEBNext Ultra II End Prep Enzyme Mix and 7 ul of NEBNext Ultra II End Prep Reaction Buffer. This was placed in a thermocycler with heated lid set to 75 C and run for 30 minutes at 20 C, 60 minutes at 50 C, then held at 4 C.  Adaptor Ligation  The adaptor ligation reaction was performed with 60 ul of Prep Reaction Mixture, 2.5 ul of NEBNext Adaptor for Illumina diluted following the manufacture recommendations, 30 ul of NEBNext Ultra Ligation Master Mix, and 1 ul of NEBNext Ligation Enhancer, incubated at 20 C for 15 min with heated lid off. 3 ul of USER Enzyme was then added to the reaction and incubated at 37 C for 15 min with heated led set to 47 C.  Cleanup of Adaptor-Ligated DNA without size selection  1.1x of AMPure XP Beads were added to the Adaptor Ligation reaction, and samples were incubated for 5 min at room temperature. Tubes were placed on a magnetic stand for 5 min to separate beads from supernatant, then supernatant was removed and beads were washed 2 times with 200 ul of 80% freshly prepared ethanol. Beads were air dried for up 5 min while tubes were on magnet with lid open. DNA was eluted from the beads by adding 17 ul of 0.1XTE buffer.   PCR Enrichment of Adaptor-ligated DNA  To 15 ul of the Adaptor Ligated DNA fragments was added 25 ul of NEBNext Ultra Q5 Master Mix, 5 ul of Index primer i7 and 5 ul Universal PCR Primer i5, and PCR amplification was performed following the cycling conditions: 98 C for 45s, 98 C 15s and 60 C 10s for 16 cycles, 72 C for 1 min, and hold at 4 C. Followed by a clean up of the PCR reaction using AMPure XP Beads.   The DNA was quantified using Qubit, and the quality and size of libraries were checked at the Bioanalyzer prior to sequencing.

treatment_protocol: Cells were treated with DMSO or 40nM AZD4573. After 2 hrs, cells were irradiated at 6 Gy and collected after an additional 4 hrs.

value: Diffuse intrinsic pontine glioma

value: AZD4573 (40 nM) and 6 Gy radiation

value: H3K27Ac_histone H3_acetyl K27; Abcam Cat. #ab4729


title: Histone_3_K27_acetylation, DIPG4, AZD4573, radiation, replicate 1


title: 26_ENDO5_t1_MB_CD8T

description: Longitudinal ind1 t1