title: PRC1CPM_TAM_RNA_rep3

description:
mESC_PRC1_CPM_TotalRNAseq_TAM_mm10_mapped_sorted_rmdup_downsampled_MERGED_FORWARD.bw
mESC_PRC1_CPM_TotalRNAseq_TAM_mm10_mapped_sorted_rmdup_downsampled_MERGED_REVERSE.bw

data_processing:
For cRNA-seq, first, paired-end reads were aligned using Bowtie 2 (with “--very-fast”, “--no-mixed” and “--no-discordant” options) against the concatenated mm10 and dm6 rDNA genomic sequence (GenBank: BK000964.3 and M21017.1) and reads mapping to rDNA were discarded. All unmapped reads were then aligned against the concatenated mm10 and dm6 genome sequences using STAR (Dobin et al., 2013). Finally, reads that failed to map using STAR were aligned against the mm10+dm6 concatenated genome using Bowtie 2 (with “--sensitive-local”, “--no-mixed” and “--no-discordant” options) to improve mapping of introns. Uniquely aligned reads from the last two steps were combined for further analysis. PCR duplicates were removed using Sambamba (Tarasov et al., 2015). For the corresponding gDNA-seq experiments, paired-end reads were aligned to the concatenated mm10+dm6 genome using Bowtie 2 (Langmead and Salzberg, 2012) with the “--no-mixed” and “--no-discordant” options. Only uniquely mapped reads after removal of PCR duplicates with Sambamba (Tarasov et al., 2015) were used for downstream analysis.
For visualisation of cRNA-seq data, uniquely aligned mouse reads were normalised using dm6 spike-in as described previously (Fursova et al., 2019). Briefly, mm10 reads were randomly subsampled based on the total number of spike-in reads in each sample. To account for any minor variations in spike-in cell mixing between replicates, we used the ratio of spike-in/mouse total read counts in the corresponding Input/gDNA-seq samples to correct the subsampling factors. After normalisation, read densities for individual biological replicates were compared across gene bodies using multiBamSummary and plotCorrelation from deepTools (Ramirez et al., 2014). For each experimental condition, biological replicates correlated well (Pearson correlation coefficient > 0.96, see Table S3) and were merged for downstream analysis.
For differential gene expression analysis, read counts from individual biological replicates prior to spike-in normalisation were obtained. A custom Perl script utilising SAMtools was used to annotate a custom-built non-redundant mm10 gene set (n = 20633) with read counts. The non-redundant gene set was derived from mm10 refGene genes by removing very short genes with poor sequence mappability and highly similar transcripts.
To identify significant changes, we used a custom R script that incorporates spike-in calibration into DESeq2 analysis (Love et al. 2014). In order to do this, we used read counts from the spike-in genome at a control set of unique dm6 refGene genes to calculate DESeq2 size factors for normalisation of raw mm10 read counts (as has been previously described in Taruttis et al., 2017 and Fursova et al., 2019). Prior to quantification, spike-in reads were pre-normalised using the spike-in/mouse read ratio derived from the corresponding genomic DNA-seq sample in order to account for minor variations in mixing of mouse and spike-in cells. For a change to be called significant, we applied a threshold of p-adj < 0.05 and fold change > 1.5, unless otherwise specified. To identify genes that were differentially affected by the removal of PRC1 or specifically PRC1 catalytic activity, we used two complementary approaches. First, we isolated genes that were expressed at significantly lower levels (p-adj < 0.05 and fold change > 1.5) in PRC1CPM as compared to PRC1CKO cells following tamoxifen treatment (PRC1CPM_OHTvsPRC1CKO_OHT). In addition, we also identified genes, for which the magnitude of expression changes following tamoxifen treatment was significantly smaller in PRC1CPM cells than in PRC1CKO cells, using the DESeq2 design that included the interaction term between the cell line and treatment factors (PRC1CPM_OHTtoUNTvsPRC1CKO_OHTtoUNT).
Genome_build: mm10
Supplementary_files_format_and_content: Stranded bigWig files were generated using genomeCoverageBed from BEDTools representing genome coverage of merged spike-in normalised biological replicates.
Supplementary_files_format_and_content: DESeq2 results table for differential gene expression analysis containing normalised read counts, estimated log2 fold changes and statistical significance levels for a non-redundant set of mm10 refGene genes.
Supplementary_files_format_and_content: Table with RefSeq IDs and MGI symbols for a selected group of genes described in the paper (related to Figure 7)

source_name: Mouse embryonic stem cells with Drosophila spike-in, tamoxifen-treated (72hr OHT), RNA-seq


title: PRC1CPM_TAM_RNA_rep2

status: Public on Jan 08 2020

type: SRA

city: Oxford

first: Nadezda

middle: A

last: Fursova

address: South Parks Rd

department: Department of Biochemistry

country: United Kingdom

institute: University of Oxford

library_source: transcriptomic

molecule: total RNA

organism: Mus musculus

growth_protocol: Mouse embryonic stem cells were grown on gelatin-coated plates at 37°C and 5% CO2, in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 15% fetal bovine serum (Labtech), 2 mM L-glutamine (Life Technologies), 1x penicillin-streptomycin solution (Life Technologies), 1x non-essential amino acids (Life Technologies), 0.5 mM beta-mercaptoethanol (Life Technologies), and 10 ng/mL leukemia inhibitory factor. ). Drosophila S2 (SG4) cells were grown adhesively at 25°C in Schneider’s Drosophila Medium (Life Technologies), supplemented with 1x penicillin-streptomycin solution (Life Technologies) and 10% heat-inactivated fetal bovine serum (Labtech).

extract_protocol:
For cRNA-seq, 1 x 10^7 mouse ESCs (both untreated and following 72 hours OHT treatment) were mixed with 4 x 10^6 Drosophila SG4 cells in 600 ml PBS. For RNA extraction, 400 ml of cells was used (corresponding to 6.7 x 10^6 mouse ESCs), and for DNA extraction the remaining 200 ml of cells was used (corresponding to 3.3 x 10^6 mouse ESCs). RNA extraction was performed using RNeasy mini kit columns (Qiagen) following manufacturer’s guidelines, and 10 mg was subjected to Turbo DNase (ThermoFisher) treatment to remove any contaminating DNA. Quality of RNA was assessed using 2100 Bioanalyzer RNA 6000 Pico kit (Agilent). Next, RNA samples were depleted of rRNA using the NEBNext rRNA Depletion kit (NEB). To quantitate the consistency of spike-in cell mixing for each individual sample, a matched sample of cells was used to isolate genomic DNA using Quick-DNA miniprep kit (Zymo).
RNA-seq libraries were prepared using the NEBNext Ultra II Directional RNA Library Prep kit (NEB). Libraries from gDNA were prepared using NEBNext Ultra II FS kit (NEB) following manufacturer’s guidelines. RNA and DNA libraries were sequenced as 80 bp paired-end reads on the Illumina NextSeq 500 platform.

treatment_protocol: To induce conditional removal of PRC1 or PRC1 catalytic activity in PRC1CKO and PRC1CPM ESC lines, cells were treated with 800 nM 4-hydroxytamoxifen (TAM) for 72 hours.

tag: cell line

tag: genotype

tag: clone

tag: strain

tag: replicate

tag: treatment agent

value: tamoxifen (OHT)

tag: treatment time point

value: 72 hr

tag: spike-in reference organism

value: Drosophila melanogaster

tag: spike-in cell line


title: PRC1CPM_UNT_Input_rep1

library_source: genomic

data_processing:
Library strategy: DNA-seq
For cRNA-seq, first, paired-end reads were aligned using Bowtie 2 (with “--very-fast”, “--no-mixed” and “--no-discordant” options) against the concatenated mm10 and dm6 rDNA genomic sequence (GenBank: BK000964.3 and M21017.1) and reads mapping to rDNA were discarded. All unmapped reads were then aligned against the concatenated mm10 and dm6 genome sequences using STAR (Dobin et al., 2013). Finally, reads that failed to map using STAR were aligned against the mm10+dm6 concatenated genome using Bowtie 2 (with “--sensitive-local”, “--no-mixed” and “--no-discordant” options) to improve mapping of introns. Uniquely aligned reads from the last two steps were combined for further analysis. PCR duplicates were removed using Sambamba (Tarasov et al., 2015). For the corresponding gDNA-seq experiments, paired-end reads were aligned to the concatenated mm10+dm6 genome using Bowtie 2 (Langmead and Salzberg, 2012) with the “--no-mixed” and “--no-discordant” options. Only uniquely mapped reads after removal of PCR duplicates with Sambamba (Tarasov et al., 2015) were used for downstream analysis.
For visualisation of cRNA-seq data, uniquely aligned mouse reads were normalised using dm6 spike-in as described previously (Fursova et al., 2019). Briefly, mm10 reads were randomly subsampled based on the total number of spike-in reads in each sample. To account for any minor variations in spike-in cell mixing between replicates, we used the ratio of spike-in/mouse total read counts in the corresponding Input/gDNA-seq samples to correct the subsampling factors. After normalisation, read densities for individual biological replicates were compared across gene bodies using multiBamSummary and plotCorrelation from deepTools (Ramirez et al., 2014). For each experimental condition, biological replicates correlated well (Pearson correlation coefficient > 0.96, see Table S3) and were merged for downstream analysis.
For differential gene expression analysis, read counts from individual biological replicates prior to spike-in normalisation were obtained. A custom Perl script utilising SAMtools was used to annotate a custom-built non-redundant mm10 gene set (n = 20633) with read counts. The non-redundant gene set was derived from mm10 refGene genes by removing very short genes with poor sequence mappability and highly similar transcripts.
To identify significant changes, we used a custom R script that incorporates spike-in calibration into DESeq2 analysis (Love et al. 2014). In order to do this, we used read counts from the spike-in genome at a control set of unique dm6 refGene genes to calculate DESeq2 size factors for normalisation of raw mm10 read counts (as has been previously described in Taruttis et al., 2017 and Fursova et al., 2019). Prior to quantification, spike-in reads were pre-normalised using the spike-in/mouse read ratio derived from the corresponding genomic DNA-seq sample in order to account for minor variations in mixing of mouse and spike-in cells. For a change to be called significant, we applied a threshold of p-adj < 0.05 and fold change > 1.5, unless otherwise specified. To identify genes that were differentially affected by the removal of PRC1 or specifically PRC1 catalytic activity, we used two complementary approaches. First, we isolated genes that were expressed at significantly lower levels (p-adj < 0.05 and fold change > 1.5) in PRC1CPM as compared to PRC1CKO cells following tamoxifen treatment (PRC1CPM_OHTvsPRC1CKO_OHT). In addition, we also identified genes, for which the magnitude of expression changes following tamoxifen treatment was significantly smaller in PRC1CPM cells than in PRC1CKO cells, using the DESeq2 design that included the interaction term between the cell line and treatment factors (PRC1CPM_OHTtoUNTvsPRC1CKO_OHTtoUNT).
Genome_build: mm10
Supplementary_files_format_and_content: Stranded bigWig files were generated using genomeCoverageBed from BEDTools representing genome coverage of merged spike-in normalised biological replicates.
Supplementary_files_format_and_content: DESeq2 results table for differential gene expression analysis containing normalised read counts, estimated log2 fold changes and statistical significance levels for a non-redundant set of mm10 refGene genes.
Supplementary_files_format_and_content: Table with RefSeq IDs and MGI symbols for a selected group of genes described in the paper (related to Figure 7)

molecule: genomic DNA

source_name: Mouse embryonic stem cells with Drosophila spike-in, untreated control, gDNA-seq

value: 0 hr


title: PRC1CPM_UNT_RNA_rep3

description:
mESC_PRC1_CPM_TotalRNAseq_UNT_mm10_mapped_sorted_rmdup_downsampled_MERGED_FORWARD.bw
mESC_PRC1_CPM_TotalRNAseq_UNT_mm10_mapped_sorted_rmdup_downsampled_MERGED_REVERSE.bw

source_name: Mouse embryonic stem cells with Drosophila spike-in, untreated control, RNA-seq


title: PRC1CPM_UNT_RNA_rep2


title: PRC1CKO_UNT_Input_rep3


title: PRC1CKO_TAM_Input_rep2

source_name: Mouse embryonic stem cells with Drosophila spike-in, tamoxifen-treated (72hr OHT), gDNA-seq


title: PRC1CKO_UNT_Input_rep2


title: PRC1CPM_TAM_RNA_rep1


title: PRC1CKO_TAM_Input_rep1


title: PRC1CPM_UNT_RNA_rep1


title: PRC1CKO_TAM_RNA_rep2

description:
mESC_PRC1_CKO_TotalRNAseq_TAM_mm10_mapped_sorted_rmdup_downsampled_MERGED_FORWARD.bw
mESC_PRC1_CKO_TotalRNAseq_TAM_mm10_mapped_sorted_rmdup_downsampled_MERGED_REVERSE.bw


title: PRC1CKO_UNT_RNA_rep3

description:
mESC_PRC1_CKO_TotalRNAseq_UNT_mm10_mapped_sorted_rmdup_downsampled_MERGED_FORWARD.bw
mESC_PRC1_CKO_TotalRNAseq_UNT_mm10_mapped_sorted_rmdup_downsampled_MERGED_REVERSE.bw


title: PRC1CKO_TAM_RNA_rep1


title: PRC1CKO_UNT_RNA_rep2


title: PRC1CKO_UNT_Input_rep1


title: RA

status: Public on Jan 01 2020

city: Wuhan

first: Wenjie

last: XU

address: No. 7 Donghu South Road

country: China

institute: Institute of Hydrobiology, Chinese Academy of Sciences

data_processing:
cDNA libraries were sequenced by Illumina HiSeq2000 system.
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence.
Samples were de novo into contigs using the Trinity software (r20140717,http: //trinityrnaseq.sourceforge.net/). The longest transcript was selected as the unigenes.
Clean reads were mapped to the unigenes by bowtie (1.0.1, mismatch=2), and then gene and isoform abundances were quantified by RSEM (v1.2.17).
Genome_build: Samples were de novo into contigs using the Trinity software (r20140717,http: //trinityrnaseq.sourceforge.net/). The longest transcript was selected as the unigenes.
Supplementary_files_format_and_content: Tab-delimited text files include FPKM values for each Sample.

organism: Carassius gibelio

source_name: Liver

extract_protocol:
Liver were removed, flash frozen in liquid nitrogen, and RNA was harvested using Trizol reagent.
RNA libraries were prepared for sequencing using standard Illumina protocols

value: A strain

tag: diet

value: Rapeseed meal diet

tag: tissue

value: Liver


title: RF

value: F strain


title: PRC1CKO_TAM_RNA_rep3


title: FF

value: Fishmeal diet


title: PdgfraEGFP Single Cell RNA-seq

status: Public on Jan 17 2020

city: Boston

first: Ramesh

last: Shivdasani

state: MA

address: 450 Brookline Ave. Dana 720D

department: Medical Oncology

country: USA

institute: Dana Farber Cancer Institute

data_processing:
Cell Ranger (2.1.1, 10X Genomics) was used to generate UMI matrix. Data was analyzed using the Suerat package (ver. 2.3.3) in R. Cells with atleast 1500 detected genes and genes expressing in at least 100 single cells were kept. This resulted in 9,334 detectable genes in 2,894 singel cells. The data was then normalized and log-transformed using the "LogNormalize" function in Seurat. We additionally filtered out cells with high mitochondrial percentage (>3%) which resulted in 2,595 single cells that were used for the rest of the analysis.
All bulk RNAseq data was analyzed using the VIPER pipeline
It was aligned to the mouse reference genome Mm10 using the STAR aligner
Genome_build: mm10
Supplementary_files_format_and_content: Rawgenescounts file was generated using the VIPER pipeline, aligned to the mm10 reference genome using STAR aligner, which also produced gene counts.

source_name: PDGFRAEGFP+ cells from small intestinal mesenchyme

extract_protocol: Cells were isolated from entire small intestinal tissues, muscle stripped, epithelium denuded via 20 minutes in warm 10 mM EDTA/HBSS (at 37degrees C), and tissue was further minced by fine scalpels. Digestion of tissue was performed using 1.5mg/ml collagenase II (Worthington) in 10%FBS/DMEM at 37 degrees C. Supernatants were collected every 20 minutes for one hour, and washed twice to remove debris. Antibody-labelling was performed prior to FACS. GFP cells were FACS sorted at a ratio of 2:1 GFPlo to GFPhi. A manually estimated 8700 cells were loaded on a 10X Chromium Controller (10X Genomics) for targeting 5000 cells using the single cell 3' V2 assay. Reverse-transcription, cDNA amplification and library prep were compelted according to manufacturer's protocol. Sequencing of library was performed using a HiSeq4000 Illumina System.

tag: cell type

value: primary cells

tag: cell subtype

value: Mixed telocyte and PDGFRAlo stromal cells


title: FA


title: HapX_Venus_Fe_plus_Input_Rep1

status: Public on Dec 31 2019

city: Manchester

first: Michael

middle: J

last: Bromley

state: Greater Manchester

address: 46 Grafton Street

department: Division of Infection, Immunity, and Respiratory mediciney

institute: The University of Manchester

data_processing:
Raw sequencing reads were quality controlled with Illumina chastity filter and fastqc v0.11.3.
Illumina adapters were trimmed from them using Trimmomatic v0.32.
The resulting reads were aligned to the Aspergillus fumigatus A1163 CADRE genome from Ensemble fungi, version 28 using Bowtie2 v2.2.3.
Peak calling was carried out using a Model-based Analysis for ChIP-Sequencing (MACS2) version 2.1.0.20140616 with a q-value cutoff of 0.01.
Genome_build: Aspergillus fumigatus A1163 CADRE genome from Ensemble fungi, version 28
Supplementary_files_format_and_content: bigWig file were generated using bedGraphToBigWig v305

organism: Aspergillus fumigatus

source_name: Mycelia

growth_protocol: 1x10e6 spores/ml of the Venus-tagged HapX expressing strain were grown in 50 ml of Aspergillus minimal medium (AMM) in the presence and the absence of iron for 24 h at 37 °C with constant shaking at 180 rpm.

extract_protocol:
Cross-linked mycelia were ground to a fine powder under liquid nitrogen. Samples were suspended in a ChIP lysis buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Deoxycholate (Sigma D6750), 0.1% SDS, 1 mM PMSF, and fungal proteinase inhibitor cocktail (Sigma)), and then sonicated with a Q125 sonicator (Qsonica, USA). The sheared chromatin was immunoprecipitated with a GFP-polyclonal antibody (A-11122, Thermo Fischer Scientific) on Dynabeads Protein A magnetic beads (Thermo Fischer Scientific). Immunoprecipitated DNA was reverse cross-linked, treated with RNase A (Sigma), and then purified using a MinElute PCR purification kit (QIAGEN).
Libraries were constructed following the manufacture's instractions for Illumina TruSeq ChIP-seq samplepreparation kit.

treatment_protocol: Cross-linking was carried out by the addition of formaldehyde to a final concentration of 1.0 % followed by incubation at 37°C for 20 min. Fixation was stopped by adding glycine to a final concentration of 125 mM and continuing shaking for 10 min at 37 °C.

tag: condition

value: A1160pyrG+, {delta}hapX::ptrA, Venus-hapX, hph

tag: chip antibody

value: Mycelia


title: HapX_Venus_Fe_minus_Input_Rep2


title: PRC1CKO_TAM_Input_rep3


title: HapX_Venus_Fe_minus_Input_Rep1


title: PRC1CKO_UNT_RNA_rep1


title: HapX_Venus_Fe_minus_ChIP_Rep2

value: GFP Tag polyclonal antibody (ThermoFischerScientific, A-11122)


title: FABPi_4

status: Public on Dec 26 2019

city: Baltimore

first: Immunometabolism

last: Department

state: Maryland

address: 1650 Orleans Street

department: Immunometabolism

institute: Johns Hopkins University

description: BMS309403 treatment

data_processing:
Sequenced libraries were processed with the Galaxy platform and deepTools (Afgan et al., 2016; Ramírez et al., 2016)
STAR (Dobin et al., 2013), for trimming and mapping
featureCounts (Liao et al., 2014) to quantify mapped reads.
Raw mapped reads were processed in R (Lucent Technologies) with DESeq2 (Love et al., 2014), to determine differentially expressed genes and generate normalized read counts to visualize as heatmaps using Morpheus (Broad Institute).
Genome_build: GRCm38
Supplementary_files_format_and_content: raw read counts for every sample

source_name: BMS309403 treatment

growth_protocol: Total CD4+ T cells were positively selected by MACS isolation from spleen and peripheral LNs of C57BL/6 mice. Cells were cultured on anti-CD3 coated plates (5 Âµg/mL) at a density of 1 x 106/mL with 0.5 Âµg/mL anti-CD28, 100U/mL IL-2, 5 ng/mL TGF-β and 10 ug/mL anti-IL-4, anti-IL-12 and anti-IFN-Î³ in 1640 RPMI media supplemented with 10% Hyclone serum, 1% Penstrep and 1% L-Glut and 55 µM beta-mercaptoethanol.

extract_protocol:
total RNA was extracted with the Rneasy kit (Qiagen) and quantified using Qubit 2.0 (Thermo Fisher Scientific) following the manufacturer’s instructions.
Libraries were prepared using the TruSeq stranded mRNA kit (Illumina) and sequenced in a HISeq 3000 (Illumina)

treatment_protocol: After 72 hours, cells were split 1:2 either in the presence or absence of BMS309403 (50 µM).

value: Spleen

value: In vitro polarized Treg

value: Wild type


title: Treg_3

description: Treg control

source_name: Treg control


title: Treg_1

description: Treg control

growth_protocol: Total CD4+ T cells were positively selected by MACS isolation from spleen and peripheral LNs of C57BL/6 mice. Cells were cultured on anti-CD3 coated plates (5 µg/mL) at a density of 1 x 106/mL with 0.5 µg/mL anti-CD28, 100U/mL IL-2, 5 ng/mL TGF-β and 10 ug/mL anti-IL-4, anti-IL-12 and anti-IFN-γ in 1640 RPMI media supplemented with 10% Hyclone serum, 1% Penstrep and 1% L-Glut and 55 µM beta-mercaptoethanol.


title: FABPi_2

description: BMS309403 treatment


title: HapX_Venus_Fe_minus_ChIP_Rep1


title: in vivo BB518 RAD001 Rep1 [TS92_14_4-2]

status: Public on Dec 23 2019

first: Tim

middle: C

last: Somervaille

address: Wilmslow Rd

department: Manchester Institute

institute: Cancer Research UK

data_processing:
Paired-end reads were mapped to the human genome (GRCh38 Gencode release 25) with STAR version 2.4.2a with the settings --outFilterMultimapNmax 20, --outFilterType BySJout,  --alignSJoverhangMin 8 and --quantMode GeneCounts using the matching feature annotation GTF file for GRCh38 Gencode release 25. Differential gene expression analysis was performed using DESeq2 version 1.20.0.
Genome_build: GRCh38
Supplementary_files_format_and_content: tab-delimited text files include raw read count values per gene for each sample

organism: Homo sapiens

source_name: in vivo BB518 RAD001

growth_protocol: Primary MLL-AML patient samples were recovered by thawing cells directly into PBS supplemented with 20% human serum albumin, 4µg/ml DNase1, 2.5mM MgCl2 and 16.4mM trisodium citrate (“DAMP solution”) followed by CD3+ cell depletion (Beads) using the POSSELD program of an AutoMACS Pro device (both from Miltenyi Biotec) according to the manufacturer’s instructions. Cells were placed over night in StemSpanTM at a density of 2.5x104 /ml supplemented with 100ng/ml each of FL, IL-6, TPO and SCF.

extract_protocol:
Total RNA was extracted from DMSO or OG86-treated THP1 AML cells using QIAshredder spin columns and an RNeasy Plus Micro Kit (Qiagen, Manchester, UK) and its quality confirmed using an Agilent Bioanalyzer (Agilent Technologies, Santa Clara, CA).
RNA libraries were prepared for sequencing using standard Illumina protocols

treatment_protocol: 5-10x10^6 million cells were injected in sub-lethally irradiated (1 Gy) female NSG mice (6-8 weeks, Envigo) via the tail vein. Tail bleeds were performed 8 weeks after transplantation and blood engraftment levels and lineage contributions determined by flow cytometry analysis of leukocyte populations following ammonium chloride lysis of erythrocytes. When transplanted mice exhibited signs of ill health they were euthanized and leukaemia cells from BM and spleen cryopreserved for later use. After passaging of MLL-AML cells through NSG mice twice, 1x10^5 cells were injected in sub-lethally irradiated (50 cGy) female NSG mice (6-8 weeks, Envigo) via the tail vein. 34 days after transplantation, mice were divided in to 4 groups treated via oral gavage with either vehicle, RAD001 (5mg/kg), OG98 (3mg/kg), or RAD001 (5mg/kg) + OG98 (3mg/kg) for 5 days.

value: Primary MLL-AML patient cells

tag: host mouse strain

value: NSG

tag: treatment

value: RAD001 (40 nM) for 24 hours


title: in vivo BB518 RAD001 Rep2 [TS92_16_4-7]


title: FABPi_3

description: BMS309403 treatment

growth_protocol: Total CD4+ T cells were positively selected by MACS isolation from spleen and peripheral LNs of C57BL/6 mice. Cells were cultured on anti-CD3 coated plates (5 Âµg/mL) at a density of 1 x 106/mL with 0.5 Âµg/mL anti-CD28, 100U/mL IL-2, 5 ng/mL TGF-β and 10 ug/mL anti-IL-4, anti-IL-12 and anti-IFN-Î³ in 1640 RPMI media supplemented with 10% Hyclone serum, 1% Penstrep and 1% L-Glut and 55


title: in vivo BB518 RAD001 & OG98 Rep2 [TS92_7_2-4]

source_name: in vivo BB518 RAD001 & OG98

value: RAD001 (40 nM) and OG86 (250 nM) for 24 hours


title: in vivo BB518 OG98 Rep2 [TS92_12_3-7]

source_name: in vivo BB518 OG98

value: OG86 (250 nM) for 24 hours


title: HapX_Venus_Fe_plus_ChIP_Rep1


title: in vivo BB518 OG98 Rep1 [TS92_9_3-1]


title: in vivo BB518 DMSO Rep1 [TS92_3_1-5]

source_name: in vivo BB518 DMSO

value: DMSO (vehicle) for 24 hours


title: in vivo BB518 DMSO Rep2 [TS92_4_1-7]


title: THP1 RAD001 & OG86 Rep1

source_name: THP1 RAD001 & OG86

growth_protocol: THP1 cells were cultured in RPMI1640 supplemented with 10% FBS, 10 U/mL Penicillin, 10 µg/mL Streptomycin, 2 mM L-glutamine. Culture densities were 5x10^4 - 5x10^5 for cells in liquid culture.

treatment_protocol: Cells were treated with DMSO (vehicle), RAD001 (40 nM), OG86 (250 nM) or RAD001 (40 nM) and OG86 (250 nM) for 24 hours followed by RNA isolation

tag: cell line/sample

value: monocytic cell line derived from an acute monocytic leukemia patient


title: THP1 RAD001 & OG86 Rep2


title: THP1 RAD001 & OG86 Rep3


title: HapX_Venus_Fe_plus_Input_Rep2

tag: genotype


title: Induced cells

status: Public on Jan 11 2020

type: RNA

city: Beijing

first: Benyu

last: Liu

state: Beijing

address: 15, Datun Road, Chaoyang district

department: The CAS key Laboratory of Infection and Immunity, Institute of Biophysics

institute: Institute of Biophysics, CAS

hyb_protocol: Hybridization was performed by NimbleGen Systems Inc., Madison, WI, USA following their standard operating protocol. See www.nimblegen.com.

scan_protocol: Slides were scanned at 5 μm/pixel resolution using an Axon GenePix 4000B scanner (Molecular Devices Corporation) piloted by GenePix Pro 6.0 software (Axon).

data_processing:
 Scanned images (TIFF format) were then imported into NimbleScan software (version 2.5) for grid alignment and expression data analysis. Expression data were normalized through quantile normalization and the Robust Multichip Average (RMA) algorithm included in the NimbleScan software. The Probe level (*_norm_RMA.pair) files and Gene level (*_RMA.calls) files were generated after normalization.
gene expression analysis: high quality expression values (for example, where at least 1 out of 2 samples have values within cut-off, cut-off =100) will be selected for analysis. Then differentially expressed genes were identified through Fold change filtering between two samples or t-test filtering between two groups. Hierarchical clustering was performed using R scripts. GO analysis and Pathway analysis were performed using the standard enrichment computation methods.

source_name: Thymidine-induced MEFs by CD45 sorting

label_protocol: Labeling was performed by NimbleGen Systems Inc., Madison, WI USA, following their standard operating protocol. See www.nimblegen.com.

extract_protocol: Trizol

tag: strain background

tag: genotype/variation

value: Wild-type

value: mouse embryonic fibroblasts (MEFs)

value: thymidine for 12 days


title: THP1 OG86 Rep2

source_name: THP1 OG86


title: HapX_Venus_Fe_plus_ChIP_Rep2

tag: strain


title: THP1 OG86 Rep3


title: Treg_4

description: Treg control


title: FABPi_1

description: BMS309403 treatment


title: THP1 OG86 Rep1


title: THP1 RAD001 Rep3

source_name: THP1 RAD001


title: THP1 DMSO Rep3

source_name: THP1 DMSO


title: in vivo BB518 RAD001 & OG98 Rep1 [TS92_6_2-3]


title: THP1 DMSO Rep2


title: piwi_CRISPR_line2_F13_csr-1_RNAi_sRNA-seq

status: Public on Dec 10 2019

city: Paris

first: Germano

last: Cecere

address: Institut Pasteur, 28 Rue Du Docteur Roux, Batiment Monod, 4eme Etage

department: Development and stem cell biology

country: France

institute: Institut Pasteur

description: total sRNA

data_processing:
The 3' adaptor (TGGAATTCTCGGGTGCCAAGG) was trimmed from the raw reads using cutadapt (version 1.15)
The trimmed reads were sorted by sequence using fastq-sort (from fastq-tools version 0.8) with option -s and deduplicated using a custom haskell program, keeping the highest quality among duplicates, at any given position
The 5' and 3' 4 nt UMIs were removed from the deduplicated reads using cutadapt (version 1.15) with options -u 4 and -u -4
After removing UMIs, the reads from 18 to 24 nt were selected using bioawk version 20110810
The size-selected reads were mapped on the C. elegans genome (WBcel235) using bowtie2 (version 2.3.4.1) with options -L 6 -i S,1,0.8 -N 0
The reads that failed to map were inspected using grep -E -B 1 -A 2 "^G[ACGTN]{20,25}T+$" to detect possible reads starting with G with 20 to 25 nt followed by a poly-U tail that might have prevented the mapping, and this tail was removed from such reads using a custom haskell program before re-mapping them.
Mapped and remapped reads were used to estimate the abundance of small RNAs derived from structural RNAs using featureCounts (version 1.5.2) with options -O -s 1 --fracOverlap 1 and annotations corresponding to tRNA, snRNA, snoRNA, rRNA or RNA (as annotated in the iGenome distribution of WBcel235 obtained at ftp://igenome:G3nom3s4u@ussd-ftp.illumina.com/Caenorhabditis_elegans/Ensembl/WBcel235/Caenorhabditis_elegans_Ensembl_WBcel235.tar.gz)
The abundance of non-structural RNAs was estimated by subtracting the above counts from the number of mapped and remapped reads.
Initially mapped reads were classified using a custom python program according to their length, composition and on the annotations on which they mapped. Reads that didn't match miRNA and piRNA annotations were considered as potential endo-siRNAs.
The potential endo-siRNAs of size 21 to 23 nt that started with G were classified as “si_22G” if they mapped antisense to annotation belonging to the following categories: DNA transposons, RNA transposons, satellites, simple repeats (as annotated in http://hgdownload.cse.ucsc.edu/goldenPath/ce11/database/rmsk.txt.gz) or pseudogene or protein-coding genes (as annotated in the iGenome distribution of WBcel235 obtained at ftp://igenome:G3nom3s4u@ussd-ftp.illumina.com/Caenorhabditis_elegans/Ensembl/WBcel235/Caenorhabditis_elegans_Ensembl_WBcel235.tar.gz)
The “si_22G” reads were re-mapped on the C. elegans genome (WBcel235) using bowtie2 (version 2.3.4.1) with options -L 6 -i S,1,0.8 -N 0
The resulting alignment was used to generate the normalized bigwig file using millions of non-structural RNAs as normalizer. This was done with a custom bash script using bedtools (version 2.27.1), bedops (version 2.4.26) and bedGraphToBigWig (version 4)
Genome_build: C. elegans ce11 (WBcel235)
Supplementary_files_format_and_content: bigwig files allowing to display the normalized abundance of “si_22G” RNAs along the genome

organism: Caenorhabditis elegans

source_name: nematodes

growth_protocol: Strains were maintained at 20°C, using standard methods (Brenner, 1974). Bristol N2 was the wild-type strain used.

extract_protocol:
Synchronous populations of worms were grown at 20°C on NGM plates seeded with OP50 E. coli concentrated food at a density of maximum 40,000 animals per 15 cm Petri dish and harvested at Young Adult stage at 48 hours post hatching. The harvested animals were washed three times with M9 buffer and 40 µl of worm pellet was frozen in dry ice with TRI Reagent (MRC, Inc.). After five repetitions of freeze and thaw, total RNA was isolated according to the TRI Reagent protocol. Ten micrograms of RNA were treated with 2 U of Turbo DNase (Ambion) at 37°C for 30 minutes followed by phenol-extraction and isopropanol precipitation. Agilent 2200 TapeStation System was used to evaluate the RIN indexes of all the RNA preps, and only samples with RIN > 8 was used for downstream applications. 10 µg of total DNase treated RNAs with RIN > 8 was size seleceted from 15% UREA PAGE gel and used to generate small RNA libraries.
The library preparation was performed as essentially described by Jayaprakash et al., 2011, except that a 5’ polyphosphatase (lucigen RP8092H) treatment was performed to be able to clone tri-phosphate small RNAs, and that the PAGE gel extraction after each ligation was substitute with purification by 1.8 volumes of Agencourt RNAClean XP Beads (Beckman Coulter, NC0068576) and 3 volumes of isopropanol. The multiplexed amplified libraries were further purified using PippinPrep DNA size selection with 3% gel cassettes and the following parameters for the selection: BP start (115) and the BP end (165). The purified libraries were quantified using Qubit Fluorometer High Sensitivity dsDNA assay kit (ThermoFisher, Q32851) and sequenced either on NextSeq-500 Illumina platform using the NextSeq 500/550 High Output v2 kit 75 cycles (FC-404-2005) or Illumina MiniSeq platform using MiniSeq High Output Reagent Kit 75-cycles (FC-420-1001).

treatment_protocol: RNAi treatment was performed using worms grown on 15 cm Petri dishes seeded with concentrated RNAi food. The Arhinger RNAi library was used to pick the selected bacteria clones.

value: CRISPR-cas9 piwi mutant line 2

value: whole worm

tag: developmental stage

value: young adult, 48 hours post hatching

value: RNAi treatment csr-1


title: THP1 RAD001 Rep1


title: scRNA_SLE3_PBMC

status: Public on Dec 13 2019

city: Bethesda

first: Hong-wei

last: Sun

state: MD

address: 9000 Rockville Pile

department: Office of Science & Technology

institute: NIAMS

data_processing:
[alignment, scRNA-seq]  Raw sequencing data were processed with Cell Ranger software (version 2.2.0)
Genome_build: GRCh38
Supplementary_files_format_and_content: barcodes.tsv, genes.tsv, matrix.mtx

source_name: PBMC

extract_protocol:
Peripheral blood was collected by heparinized tubes and the PBMC fraction was obtained using Ficoll/Hypaque gradient.  LDGs were sorted by flow cytometry using  CD10-APC,CD14-FITC and CD15-PE antibodies.
Isolated PBMCs were encapsulated using microfluidics technology, and barcoded using a unique molecular identifier (UMI)
scRNAseq data were generated on Illumina HiSeq  3000 instruments according to the manufacturer's instructions

tag: healthy control or lupus

value: LUPUS Patient

value: PBMC

tag: exp id

tag: lab  data id


title: csr-1_ADH_piwi_CRISPR_line2_F6_sRNA-seq

description: total sRNA

value: CRISPR-cas9 piwi mutant line 2; CRISPR-cas9 ADH csr-1


title: piwi_CRISPR_line2_F13_control_RNAi_sRNA-seq

description: total sRNA

value: RNAi treatment control


title: csr-1_WT_piwi_CRISPR_line2_F6_sRNA-seq

description: total sRNA


title: scRNA_SLE1_PBMC


title: THP1 DMSO Rep1


title: Treg_2

description: Treg control

value: Spleen

value: In vitro polarized Treg

value: Wild type


title: Th0_TEPP_5

status: Public on Nov 26 2019

data_processing:
Sequenced libraries were analyzed with SnakePipes 1.2.0
Raw mapped reads were processed in R (Lucent Technologies) with DESeq2 (Love et al., 2014), to determine differentially expressed genes and generate normalized read counts to visualize as heatmaps using Morpheus (Broad Institute).
Genome_build: GRCm38
Supplementary_files_format_and_content: raw read counts matrix containing count for every sample

source_name: Primary murine splenocytes

growth_protocol: CD4+ T cells were obtained from total mouse splenocytes by magnetic cell sorting with the CD4 T cell isolation kit, followed by incubation with CD62L microbeads and isolation of CD4+CD62L+ resting T cells, following manufacturer's instructions (Miltenyi Biotech).

extract_protocol:
Total RNA was isolated using the PureLink RNA Mini kit (Ambion) and quantified using Qubit 2.0.
Libraries were prepared using the NEBNext Single Cell/Low Input RNA Library Prep Kit for Illumina (New England Biolabs) and sequenced using the HiSeq 3000 (Illumina).

treatment_protocol: Cells were stimulated in vitro with plate-bound anti-CD3 and anti-CD28 antibodies. Cells were pre-incubated for 15 min with TEPP-46 (TEPP; 50 - 100 μM) or DMSO (Ctrl) before activation.

value: CD4+ T cells isolated from primary murine splenocytes

value: CD4+ Th0 cells activated with CD3/28 in the presence of TEPP-46


title: Th0_Ctrl_5

value: CD4+ Th0 cells activated with CD3/28


title: Th0_Ctrl_4


title: Th0_TEPP_4


title: Th0_Ctrl_1


title: Th0_TEPP_1


title: Resting_5

value: Resting CD4+ T cells


title: piwi_CRISPR_line2_F13_cdl-1_RNAi_sRNA-seq

description: total sRNA

value: RNAi treatment cdl-1


title: Resting_1


title: estradiol rep3

status: Public on Nov 16 2019

city: Austin

first: Keiko

middle: U.

last: Torii

state: TX

address: MNS 5.104A 2506 Speedway

institute: The University of Texas at Austin

data_processing:
Sequences were aligned on TAIR10 genome using tophat2 (version 2.0.13).
Reads on each gene were counted using our in-house program.
Statisitical evaluation was performed using DeSeq2 algorithm provided by Bioconductor.
Genome_build: TAIR10
Supplementary_files_format_and_content: txt, fpkm values from each samples

organism: Arabidopsis thaliana

source_name: seedling

growth_protocol: Homozygous transgenic lines for iMUTE were grown on ½ MS agar media for 4 days.

extract_protocol:
Total RNA was isolated from the seedlings using RNeasy Plant Mini Kit (Qiagen, 74904) and DNase I digestion (Qiagen, 79254) was performed on column during the RNA extraction according to the instructions of the manufacturer.
Total RNA samples were sent to the New York Genome Center (NYGC) facility for library construction and sequencing. RNA samples were run on the Agilent 2100 bioanalyzer to access the RNA quality. mRNA libraries were prepared by using Illumina TruSeq Stranded mRNA.
RNA-seq, Libriary was read in paired-end 50bp length with 30M coverage per sample on Illumina Hiseq 2500 system.

treatment_protocol: 40-50 seedlings for each replicate were bathed in a ½ MS liquid media containing 10 μM estradiol or DMSO (mock).

value: MUTE cDNA in pER8 / Col-0

tag: age

value: 4-day old

value: estradiol 10 uM 8hrs


title: estradiol rep2


title: estradiol rep1


title: mock rep2

value: DMSO 8hrs


title: mock rep3


title: scRNA_SLE2_PBMC


title: mock rep1


title: C2-43-BL

status: Public on Oct 23 2019

city: Cambridge

first: Victor

last: Farutin

address: 301 Binney St.

department: Research

institute: Momenta Pharmaceuticals, Inc.

data_processing:
FASTQ files were mapped to UCSC hg19 human reference genome using two-pass STAR alignment software (version 2.4.0b) to generate BAM files where table of splice junctions from the first pass is used to update genome index used for the second pass alignment.  Additional STAR arguments included: --outSAMattrRGline ID:=sample identifier here= SM:CERTAIN PL:Illumina.  For the remaining STAR software parameters their default settings have been used.

RNA_seQC software (v.1.1.8.1) was used to generate quality metrics for BAM files produced by the second pass of STAR alignment
Read counts per gene were generated by featureCounts software (subread package v.1.4.5-p1) using GTF from Illumina iGenomes (archive-2014-06-02-13-47-56)  annotation for UCSC hg19 with "-O -s 1 -p -B" settings and default values for the remaining arguments to featureCounts (resulting in summarizing read counts per meta-feature, i.e. gene in GTF)
Genome_build: UCSC hg19
Supplementary_files_format_and_content: text file concatenating raw (no filtering on abundance, variance, etc.) read counts per gene in featureCounts output for all samples in the data series; the first six  columns represent gene id, chromosome, start, end, strand and summary length of each of the gene exons (from Illumina iGenomes gene annotation GTF for UCSC hg19) as in featureCounts output, followed by one column per sample in the order shown in sample annotation above with column headers matching "title" attribute in sample annotation

molecule: polyA RNA

source_name: PAXgene Blood RNA Tube

extract_protocol:
RNA extraction was performed according to the PAXGene Blood miRNA kit protocol (Cohort 1) or PAXGene Blood RNA kit protocol (Cohort 2) using the QIAcube instrument (Qiagen)
Libraries were prepared for RNA-seq analysis with the Apollo 324 system from WaferGen Biosystems using the WaferGen Prep-X Directional RNA-Seq kit (Cohort 1) or Illumina’s TruSeq Stranded mRNA Library Preparation Kit (Cohort 2) according to manufacturer’s protocols.  Both library construction methods involved polyA selection.

tag: subject status

value: naive rheumatoid arthritis patient

value: BASELINE

tag: cohort

value: Cohort 2

value: neg


title: C2-6-BL

value: Good

value: pos


title: C2-63-MO3

value: MONTH 3


title: C2-46-MO3


title: C2-94-BL


title: C2-46-BL


title: C2-64-BL


title: C2-33-BL


title: C2-62-BL


title: C2-9-BL


title: C2-100-BL


title: C2-6-MO3


title: C2-56-MO3


title: Resting_4

tag: cell type

tag: tissue

value: Spleen

tag: cell subtype


title: C2-98-BL


title: C2-77-MO3


title: C2-50-MO3


title: C2-171-BL


title: C2-77-BL


title: THP1 RAD001 Rep2

value: monocytic cell line derived from an acute monocytic leukemia patient

value: RAD001 (40 nM) for 24 hours


title: C2-76-BL


title: C2-124-BL


title: C2-136-BL


title: C2-140-BL


title: C2-81-MO3


title: C2-86-MO3


title: C2-22-BL


title: C2-8-MO3


title: C2-136-MO3


title: C2-50-BL


title: C2-173-MO3


title: C2-28-BL


title: C2-170-MO3


title: C2-24-MO3


title: C2-30-BL


title: C2-97-MO3


title: C2-121-MO3


title: C2-22-MO3


title: C2-140-MO3


title: C2-127-BL


title: C2-124-MO3


title: C2-54-MO3


title: C2-170-BL


title: C2-171-MO3


title: C2-76-MO3


title: C2-113-MO3


title: C2-113-BL


title: C2-61-MO3


title: C2-61-BL


title: C2-2-MO3


title: C2-2-BL


title: C2-41-MO3


title: C2-121-BL


title: C2-28-MO3


title: C2-24-BL


title: C2-97-BL


title: C2-33-MO3


title: C1-46-BL

value: Cohort 1


title: C1-52-MO3


title: C1-46-MO3


title: C1-66-MO3


title: C1-37-BL


title: C1-49-BL


title: C1-115-MO3


title: C1-68-MO3


title: C1-12-BL


title: C1-23-MO3


title: C1-76-MO3


title: C1-71-BL


title: C1-26-MO3


title: C1-66-BL


title: C1-94-BL


title: C1-115-BL


title: C1-29-MO3


title: C1-57-BL


title: C1-96-BL


title: C1-56-MO3


title: C1-45-BL


title: C1-88-MO3


title: C1-69-MO3


title: C1-71-MO3


title: C2-41-BL


title: C1-80-MO3


title: C1-43-BL


title: C1-23-BL


title: C1-100-MO3


title: C1-120-MO3


title: C1-6-MO3


title: C1-57-MO3


title: C1-36-BL


title: C1-25-MO3


title: C1-101-BL


title: C1-21-MO3


title: C1-69-BL


title: C1-1-BL


title: C1-36-MO3


title: C1-85-BL


title: C1-2-MO3


title: C1-21-BL


title: C1-68-BL


title: C1-25-BL


title: C1-45-MO3


title: C1-99-MO3


title: C1-12-MO3


title: C1-77-MO3


title: C1-42-BL


title: C1-26-BL


title: C1-58-BL


title: C1-58-MO3


title: C1-2-BL


title: C1-55-MO3


title: C1-49-MO3


title: C1-119-MO3


title: C1-94-MO3


title: C1-56-BL


title: C1-119-BL


title: C1-80-BL


title: C1-101-MO3


title: C1-52-BL


title: C1-63-MO3


title: C1-43-MO3


title: C1-76-BL


title: C1-55-BL


title: vector -3

status: Public on Oct 08 2019

city: West Lafayette

first: Qing

last: Deng

state: Indiana

address: 915 W. State Street

department: Department of Biological Sciences

institute: Purdue University

data_processing:
Base calling and quality scoreing were performed by Real-Time Analysis (RTA) v2 in Illumina HiSeq 2500 for Samples 1-5 in Illumina HiSeq 4000 for Samples 6-11.
The bcl2fastq2 Conversion software were used to convert base call (bcl) files to FASTQ files, and trim adapter sequence at the same time.
The reads were mapped to the zebrafish genome using STAR (v2.5) RNA-seq aligner with the following parameter:  for Samples 1-5:  “--alignEndsType EndToEnd --outFilterMismatchNmax 1 --outFilterMultimapScoreRange 0 --outFilterMultimapNmax 10 --outSAMunmapped Within --outFilterScoreMinOverLread 0 --outFilterMatchNminOverLread 0 --outFilterMatchNmin 16 --alignSJDBoverhangMin 1000”, for Samples 6-11: “--outSAMmapqUnique 60”.
Uniquely mapped sequencing reads were assigned to genes using featureCounts (from subread v1.5.1) with the following parameters: for Samples 1-5:  “-s 1 –Q 10”, for Samples 6-11, "-p -Q 10".
The data was filtered: for Samples 1-5 using read count > 10 in more than 1 of the samples;  for Samples 6-11, using read count per million (CPM) > 0.5 in more than 3 of the samples, normalized using TMM (trimmed mean of M values) method and subjected to differential expression analysis using edgeR (v3.20.8).
Genome_build: GRCz11
Supplementary_files_format_and_content: Raw counts

organism: Danio rerio

growth_protocol: zebrafish embryos were grown to 3 day post fertilization in embryonic media

extract_protocol:
tissue specific isolation: cells were labeled with fluorescent reporters and isolated from 3dpf zebrafish embryos using FACS. mRNA was extracted using Qiagen RNeasy Mini Kit  and total RNA was extracted using Invirtogen mirVANA kit for miRNA sequencing.
miRNA sequencing library were constructed using Illumina TruSeq Small RNA Preparation and 145-160bp band are excised for sequencing.  mRNA sequencing library were constructed withSMART-Seq v4 Ultra Low Input RNA Kit for Sequencing (Takara Clontech Laboratories, Inc.).

treatment_protocol: transgenic zebrafish lines were used to produce embryos. For miRNA profiling, transgenic lines expressing GFP in either neutrophils or apical keratincyes were used. For miR-199 target discovery, transgenic lines expressing miR-199 or a vector control together with a GFP reporter were used.

value: AB

tag: gentoype/variation

value: overexpressing vector

value: neutrophils


title: vector -1


title: C1-100-BL


title: C1-96-MO3


title: C1-37-MO3


title: C1-99-BL


title: C1-88-BL


title: array_CA2_DE_2

status: Public on Sep 23 2019

city: Roanoke

first: Shannon

last: Farris

state: VA

address: 2 Riverside Circle

department: Fralin Biomedical Research Institute

institute: Virginia Tech

description: run number 907

hyb_protocol: Four micrograms (4.0 μg) of amplified biotin-cDNAs were fragmented and hybridized to Affymetrix Mouse Transcriptome 1.0 arrays for 18 hours at 45°C in a rotating hybridization oven. Array slides were stained with streptavidin/phycoerythrin utilizing a double-antibody staining procedure and then washed for antibody amplification according to the GeneChip Hybridization, Wash and Stain Kit and user manual following protocol FS450-0004.

scan_protocol: Arrays were scanned in an Affymetrix Scanner 3000 and the CEL data files were obtained using the GeneChip Command Console software.

data_processing: CEL files from the transcriptome array were imported into R and RMA normalized using the Oligos package (v1.42.0). Genes present on multiple rows were collapsed to one row by taking the highest intensity value per sample. Group mean Log2 intensity values were use to correlated with the accompanied RNA-Seq data.

label: biotin

source_name: hippocampal CA2 dendritic layer

label_protocol: Approximately 5-7 ng of total RNA were amplified as directed in the WT-Ovation Pico RNA Am-plification System (Nugen, San Carlos, CA) protocol, sense-strand cDNA target was made using the Nugen Encore Exon Module, and after fragmentation the product was labelled with the Nugen Encore Biotin biotin module.

extract_protocol: Total RNA was extracted using the Arcturus PicoPure RNA Isolation kit (Thermo Fisher Scientific, Cat #KIT0204). Approximated 20 LCM caps (5 sections/cap) were acquired per region (4 regions per brain) and immediately lysed with 10ul of XB extraction buffer incubated for 30min at 42C. Lysates were stored at -80C until further processing. For RNA extraction, lysates from each subregion were pooled and RNA was isolated one at a time to minimize degradation. All samples were subjected to on-column DNase-treatment (Qiagen, cat#79254). We obtained 5-30ng of total RNA (RINs 6.9-7.5) from each subregion and compartment, measured using the Agilent RNA 6000 Pico kit and 2100 Bioanalyzer.

treatment_protocol: Three 6-8-week-old male Amigo2-EGFP mice from separate litters were used for the main study. Tissue was harvested within a 2-hour time window near the end of the light cycle (3-5pm, lights off 6pm). Mice were deeply anesthetized with Fatal Plus (50mg/kg) before swift removal of the brain (<2 min). Brains were bisected in the sagittal plane and individually flash frozen by being placed in a 22 x 22mm disposable cryomold (Polysciences, Inc.) filled with Optimal Cutting Temperature compound and submerged in isopentane cooled to -20C on a dry ice and ethanol slurry. Samples were stored at -80C until laser capture microdissection (LCM). Mouse hippocampal subregions CA1 and CA2 cell body and dendrite samples were separately captured using IR-based laser capture microdissection from eight micron fresh frozen brain sections as described in detail in Farris et al., 2017, Frontiers in Molecular Neuroscience.

value: Amigo2-GFP C57BL/6J

value: brain/hippocampus

value: 6-8 weeks

tag: Sex

value: male


title: array_CA2_CB_3

description: run number 907

source_name: hippocampal CA2 cell body layer


title: array_CA2_CB_1

description: run number 907


title: array_CA2_DE_3

description: run number 907


title: array_CA2_CB_2

description: run number 907


title: array_CA1_CB_3

description: run number 938

source_name: hippocampal CA1 cell body layer


title: array_CA2_DE_1

description: run number 907


title: array_CA1_CB_2

description: run number 938


title: C1-1-MO3

value: naive rheumatoid arthritis patient


title: array_CA1_CB_1

description: run number 938


title: array_CA1_DE_3

description: run number 938

source_name: hippocampal CA1 dendritic layer


title: array_CA1_DE_2

description: run number 938


title: array_CA1_DE_1

description: run number 938


title: Skin_Control_14

status: Public on Sep 10 2019

city: Dessau

first: Christos

last: Zouboulis

address: Auenweg 38

department: Departments of Dermatology, Venereology, Allergology and Immunology

country: Germany

institute: Dessau Medical Center, Brandenburg Medical School Theodor Fontane

hyb_protocol: cRNA was purified using the RNA Clean & Concentrator 5 kit (Zymo, Irvine, CA). Quantification of cRNA and Cy3-CTP was conducted using NanoDrop ND-1000. RNA fragmentation was performed by incubation with 10x GEX Blocking Agent and Fragmentation Buffer (Agilent) for 30 minutes at 60°C. Then, 2x GEX hybridization buffer (Agilent) was added and samples were loaded on array slides. Hybridization was performed at 65°C at 10 rpm for 17 h and then washed with GEX wash buffer 2 (Agilent).

scan_protocol: Arrays were scanned using the Agilent Scanner and images were processed using the Agilent Feature Extraction software to generate text files.

data_processing: Raw transcriptome profile data were preprocessed using the limma package in R. Background correction was performed using the normexp convolution model and corrected data were subsequently normalized using Loess normalization.

label_protocol: For complimentary RNA (cRNA) synthesis and labeling the Low Input Quick Amp Labeling Kit, one-color (Agilent) including the dye cyanine 3-labeled CTP (Cy3 CTP) was used.

extract_protocol: Human skin biopsies were homogenised in ~400 µl of RLT buffer (Qiagen) using the Polytron PT3000 with a Polytron-Aggregate® homogenizer (Kinematika, Littau, Switzerland). The homogenizer was sequentially pre-treated with 3% H2O2 (Merck, Darmstadt, Germany), 70% 191 ethanol and dH2O to remove a possible RNase contamination. Each sample was homogenised for 1 minute, then cooled on ice for 1 minute and again homogenized for 1 minute. After each sample processing the homogenizer was washed sequentially with dH2O, 70% ethanol and dH2O. RNA isolation from the homogenates was performed using the RNeasy® Mini Kit (Qiagen) including DNase I on column treatment following the manufacturer's protocol. Quality and quantity of each RNA samples were determined by analysis on a 2100 Bioanalyzer (Agilent, Santa Clara, CA) and by photometric analysis. Only samples with RIN values above 8 were considered for further processing.

tag: gender

value: F

tag: condition

value: Control

tag: location

tag: hurley stage

value: skin

tag: site

value: clear site (H)


title: Skin_Control_13


title: Skin_Control_16


title: Skin_Control_11


title: Skin_HS_LS_7

value: HS

value: Sterum

value: II

value: lesional site (LS)


title: Skin_HS_NL_6

value: non-lesional site (NL)


title: Skin_HS_LS_8

value: Inguinal


title: Skin_HS_NL_7


title: Skin_Control_9


title: Skin_HS_LS_6


title: Skin_Control_12


title: C1-98-MO3

value: MONTH 3

value: Good

value: Cohort 1

value: pos


title: Skin_HS_LS_5

value: III


title: Skin_HS_NL_8


title: Skin_HS_NL_2


title: IP Clone b, 4OHT treated for 2hrs [TBP]

status: Public on Aug 08 2019

city: Copenhagen

first: Josh

middle: M

last: Brickman

address: 3B Blegdamsvej

department: reNEW

country: Denmark

institute: Copenhagen University

data_processing:
reads were mapped uning bowtie2
duplicate reads were removed
reads were quality filtered
reads were counted using bedtools coverage
peaks were called using MACS2 (narrow)
Genome_build: mm10
Supplementary_files_format_and_content: MACS PEAK BED

source_name: mouse embryonic stem cells stably expressing a cRAF-ErT2 expression cassette

growth_protocol: mESCs were grown in serum containing medium, supplemented with 1x LIF (made in house) and 2uM Chir99021. 24hrs prior to treatment, media was changed to serum containing media supplemented with 1x LIF and 250nM PD173074.

extract_protocol:
Cells we fixed in 1% formaldehyde at RT for 10 minutes and subsequently 2mM disuccinimidyl glutarate , nuclei extracted and chromatin sonicated using a bioruptor (10 cycles, 30s on 30s off).  Cleared chromatin was incubated with anti-TBP antibody (abcam, ab28175). Input DNA serves as negative control for antibody enrichment.
Libraries were prepared using the NEB Next Ultra 2 kit.

treatment_protocol: Cells were left untreated (0h), or treated with 250nM 4OHT (2h).

tag: treatment

value: 4OHT for 2 hrs

tag: strain

tag: background strain

tag: Stage

value: embryonic


title: Skin_HS_LS_3

value: F

value: Axilliary


title: Skin_Control_15

value: Control

value: skin


title: IP Clone a, 4OHT treated for 2hrs [TBP]

value: mouse embryonic stem cells stably expressing a cRAF-ErT2 expression cassette


title: Skin_Control_10

value: clear site (H)


title: Skin_HS_NL_5

value: HS


title: Skin_HS_LS_4

value: II

value: lesional site (LS)


title: Input Clone b, 4OHT treated for 2hrs [TBP]


title: IP Clone b, untreated [TBP]

value: untreated


title: IP Clone a, untreated [TBP]


title: Skin_HS_NL_4

value: non-lesional site (NL)


title: Skin_HS_NL_3


title: Skin_HS_LS_2

value: Inguinal


title: Input Clone a, 4OHT treated for 2hrs [TBP]


title: Skin_HS_LS_1


title: Input Clone b, untreated [TBP]


title: IP_clone21

extract_protocol:
Cells we fixed in 1% formaldehyde at RT for 10 minutes and subsequently 2mM disuccinimidyl glutarate , nuclei extracted and chromatin sonicated using a bioruptor (10 cycles, 30s on 30s off).  Cleared chromatin was incubated with anti-FLAG antibody (Sigma, F3165). Input DNA serves as negative control for antibody enrichment.
Libraries were prepared using the NEB Next Ultra 2 kit.

value: 4OHT for 2hrs

tag: antibody

value: anti-FLAG


title: IP_clone2


title: Input_clone2

value: input sample


title: Skin_HS_NL_1


title: IP_clone11


title: Input_clone21


title: Input_clone11


title: repB input 8h

extract_protocol:
Cells we fixed in 1% formaldehyde at RT for 10 minutes and subsequently 2mM disuccinimidyl glutarate , nuclei extracted and chromatin sonicated using a bioruptor (10 cycles, 30s on 30s off).  Cleared chromatin was incubated with anti-ESRRB (R&D, PP-H6705-00), or anti SOX2 antibody (R&D, AF2018). Input DNA serves as negative control for antibody enrichment.
Libraries were prepared using the NEB Next Ultra 2 kit.

treatment_protocol: Cells were left untreated (0h), or treated with 250nM 4OHT for 2h, 4h or 8h

value: 8h 4OHT

value: input


title: repB input 2h

value: 2h 4OHT

value: embryonic


title: repB ESRRB 8h

value: anti-ESRRB


title: repB SOX2 0h

value: 0h 4OHT


title: repB SOX2 8h

value: mouse embryonic stem cells stably expressing a cRAF-ErT2 expression cassette


title: repA SOX2 0h


title: repB input 4h

value: 4h 4OHT


title: repA input 8h


title: repA SOX2 4h


title: repB SOX2 2h


title: repB ESRRB 0h


title: repA SOX2 8h


title: repA SOX2 2h


title: repB input 0h


title: Input Clone a, untreated [TBP]


title: repB SOX2 4h


title: repB ESRRB 2h


title: repA input 4h


title: repA input 0h


title: repA ESRRB 8h


title: repA ESRRB 2h


title: repA ESRRB 0h


title: IP Clone a, untreated  [EP300]

extract_protocol:
Cells we fixed in 1% formaldehyde at RT for 10 minutes and subsequently 2mM disuccinimidyl glutarate , nuclei extracted and chromatin sonicated using a bioruptor (10 cycles, 30s on 30s off).  Cleared chromatin was incubated with anti-TBP antibody (Abcam,ab14984). Input DNA serves as negative control for antibody enrichment.
Libraries were prepared using the NEB Next Ultra 2 kit.

value: anti-TBP


title: IP Clone b, 4OHT treated for 2hrs  [EP300]


title: Input Clone b, 4OHT treated for 2hrs  [EP300]


title: IP Clone b, untreated  [EP300]


title: repB ESRRB 4h


title: IP Clone a, 4OHT treated for 2hrs  [EP300]


title: repA input 2h


title: Input Clone a, 4OHT treated for 2hrs  [EP300]


title: Input Clone a, untreated  [EP300]


title: MED24 IP Clone a, 4OHT treated for 2hrs for Mediator ChIP

description: ChIP-seq

source_name: mouse embryonic stem cells

extract_protocol:
Cells we fixed in 1% formaldehyde at RT for 10 minutes and subsequently 2mM disuccinimidyl glutarate , nuclei extracted and chromatin sonicated using a bioruptor (10 cycles, 30s on 30s off).  Cleared chromatin was incubated with either anti-MED1  antibody (Bethyl Laboratories, A300-793A), or anti-FLAG (to IP MED24),  (Sigma, F3165) . Input DNA serves as negative control for antibody enrichment.
Libraries were prepared using the NEB Next Ultra 2 kit.

value: 4OHT treated for 2hrs

tag: cell line

tag: chip antibody

value: anti-FLAG (to IP MED24),  (Sigma, F3165)


title: MED1 IP Clone a, 4OHT treated for 2hrs for Mediator ChIP

description: ChIP-seq

value: anti-MED1  antibody (Bethyl Laboratories, A300-793A)


title: MED24 IP Clone b, 4OHT treated for 2hrs for Mediator ChIP

description: ChIP-seq


title: MED24 IP Clone b, untreated for Mediator ChIP

description: ChIP-seq

value: untreated


title: Input Clone a, 4OHT treated for 2hrs for Mediator ChIP

description: ChIP-seq


title: Input Clone b, 4OHT treated for 2hrs for Mediator ChIP

description: ChIP-seq


title: Input Clone a, untreated for Mediator ChIP

description: ChIP-seq


title: MED1 IP Clone b, 4OHT treated for 2hrs for Mediator ChIP

description: ChIP-seq


title: ChIP-seq_mal-H3K27me3

status: Public on Aug 01 2019

city: Badalona

first: Jeannine

last: Diesch

state: Barcelona

address: Ctra de Can Ruti, Camí de les Escoles s/n

country: Spain

institute: Josep Carreras Leukaemia Research Institute

description: malignant

data_processing:
Basecalls performed using CASAVA version 1.8.2
Reads were aligned to the mm9 genome assembly containing one rDNA copy using bowtie with default parameters
Read coverage was determined using Bed tools package
Genome_build: mm9
Supplementary_files_format_and_content: Read coverage across rDNA

source_name: primary preB cells_malignant

growth_protocol: Eμ-Myc mice were maintained as heterozygote on a C57BL/6 background

extract_protocol:
Primary pre-B cells were purified from bone marrow of wt, prem and mal mice on the basis of high B220, low IgM and low IgD surface marker expression by flow cytometry.
Libraries were prepared using ThruPLEX-FD Prep Kit (Rubicon Genomics Inc.)

treatment_protocol: Wt and prem mice were sacrificed at age 4-6 weeks and mal mice as soon as they established spontaneous lymphoma (age 10-20 weeks)

value: bone marrow

tag: age

value: 10-20 weeks

tag: cell type

value: primary preB cells from bone-marrow

tag: genotype

value: Eu-Myc/+


title: ChIP-seq_mal-PolR1A

description: malignant

tag: tissue


title: ChIP-seq_prem-PolR1A

description: premalignant

source_name: primary preB cells_premalignant

value: 4-6 weeks


title: MED1 IP Clone a, untreated for Mediator ChIP

description: ChIP-seq


title: MED1 IP Clone b, untreated for Mediator ChIP

description: ChIP-seq


title: repA ESRRB 4h


title: ChIP-seq_wt-H3K27me3

description: wild type

source_name: primary preB cells_wildtype

value: wild type


title: MED24 IP Clone a, untreated for Mediator ChIP

description: ChIP-seq


title: Input Clone b, untreated for Mediator ChIP

description: ChIP-seq


title: ChIP-seq_wt-PolR1A

description: wild type


title: ChIP-seq_prem-H3K27me3

description: premalignant


title: E12_PFL_delattP-Rel5_H3K27ac_ChIP-seq

status: Public on Jun 12 2019

city: Geneva

first: Eddie

last: Rodríguez-Carballo

address: 4, Boulevard d'Yvoy

department: Department of Genetics and Evolution

country: Switzerland

institute: Université de Genève

data_processing:
Analyses were performed using the facilities of the Scientific IT and Application Support Center of EPFL. Sequences were filtered and adapters were removed using cutadapt (v1.16) with parameters -m 15 -q 30 -a CTGTCTCTTATACACATCTCCGAGCCCACGAGAC for ChIPmentation and -a GATCGGAAGAGCACACGTCTGAACTCCAGTCAC for ChIP-seq. Reads were mapped on mm10 using bowtie2 (v2.3.4.1) using default parameters. Only reads with a mapping quality above 30 were kept. A profile was obtained with macs2 (version 2.1.1.20160309 option –extsize 300). Bedgraphs were normalized to their number of million tags used in the profile and replicates were merged using the tool unionbedg (bedtools v2.27). An artifactual peak, which is due to a PCR contamination, was found in region chr2:74207282-74208158 in some datasets.
Genome_build: mm10
Supplementary_files_format_and_content: mean of normalized macs2 bedgraphs

source_name: Proximal forelimb mouse E12.5 delHoxd(attP-Rel5)d9lac H3K27ac ChIP-seq

extract_protocol:
Micro-dissected proximal and distal segments of E12.5 forelimbs either from wild type or mutant embryos (delHoxD(attP-Rel5)d9lac, invHoxD(attP-Itga6), invHoxD(TgHd11lacNsi-Itga6)). Tissues were dissected, fixed in 1% formaldehyde (in PBS) for 10 min at room temperature and the reaction was quenched with Stop Solution from the ChIP-IT High sensitive kit (Active Motif). Samples were then washed 3 times with working Washing Solution (ChIP-IT, Active Motif) and then snap-frozen in liquid nitrogen and stored at -80ºC until further processing. After genotyping, samples were pooled according to the required cell number. The total amount of tissue used for each line was different due to the size variations of the limb buds. Limb tissues were disrupted with a polytron device, lysed in RIPA buffer or Prep Buffer (ChIP-IT, Active Motif) and sonicated in Diagenode Bioruptor Pico. All H3K27ac ChIP experiments were processed as ChIP-seqs using the reagents from ChIP-IT High Sensitive kit (Active Motif). IPs were performed in parallel technical duplicates with 11 to 14μg of chromatin on each. Antibody incubation was performed overnight on a final volume of 1.5-2ml dilution buffer (0.1% SDS, 50mM Tris-HCl pH8, 10mM EDTA pH8 and proteinase inhibitors), including 2μl of H3K27ac antibody (Diagenode C15410196) at 4°C on a rotating platform. Agarose beads were added for 3 to 4h at 4ºC. Washes were performed on column and DNA purification was carried out by phenol-chloroform extraction. The technical replicates were merged and yielded 1.5 to 2ng of chromatin, which were used to generate DNA libraries using the TruSeq ChIP library preparation kit. RING1B ChIP-seq experiments were processed as for ChIP-seq using 4μl of RING1B antibody (Active Motif 39664) as described in (Beccari et al. 2016; PMID:27198226). All H3K27me3 and EZH2 ChIP were performed following the ChIPmentation protocol . Around 0.1 to 0.4 million cells were used for each IP on a final volume of 800 to 1000μl of RIPA-LS buffer (10mM Tris-HCl pH8, 140mM NaCl, 1mM EDTA pH8, 0.1% SDS, 0.1% sodium deoxycholate, 1% Triton x-100 and proteinase inhibitors), to which 2μl of H3K27me3 (Millipore 17-622) or EZH2 (Diagenode C15410039) antibodies were added. Samples were incubated for at least 2 hours with Dynabeads Protein A (Invitrogen 10001D) rotating at 4ºC. Washes were performed as follows: two times RIPA-LS, two times RIPA-HS (10mM Tris-HCl pH8, 500mM NaCl, 1mM EDTA pH8, 0.1% SDS, 0.1% sodium deoxycholate, 1% Triton x-100 and proteinase inhibitors), two times RIPA-LiCl (10mM Tris-HCl pH8, 250mM LiCl, 1mM EDTA pH8, 0.5% NP-40, 0.5% sodium deoxycholate and proteinase inhibitors) and once with 10mM Tris-HCl pH8. Beads were resuspended in 24μl of tagmentation buffer (10mM Tris pH8, 5mM MgCl2, 10% dimethylformamide) and 1μl of Tn5 transposase (Illumina 15027865, from Nextera DNA Library Prep Kit 15028212) and transferred to PCR tubes, which were then incubated at 37ºC for five minutes in a thermocycler. Samples were then resuspended and washed twice in 1ml of RIPA-LS and twice in 1ml TE buffer (10mM Tris-Hcl pH8, 1mM EDTA pH8). Beads were magnetised, DNA was eluted in ChIP elution buffer (10mM Tris-HCl pH8, 5mM EDTA pH8, 300mM NaCl, 0.4% SDS) with 2μl of proteinase K (20mg/ml stock) and then incubated for 1 hour at 55ºC and 6 hours to overnight at 65ºC. After de-crosslinking, the supernatant was recovered and beads were resuspended again in 19μl ChIP elution buffer with 1μl of proteinase K and left 1 hour at 55ºC. The two supernatants were combined and purified with MinElute kit (Qiagen) in 22μl of EB buffer. Relative quantitation was performed using SYBR-green as in (Schmidl et al, 2015; PMID:26280331) using 2μl of DNA. Libraries were amplified according to the Cq values obtained in the previous step (12 to 14 cycles for both sets of samples), purified using Agentcourt AMPureXP beads (Beckman Coulter A63880) and eluted in 15μl of water. DNA sequencing was performed in HiSeq 2500 or HiSeq 4000 machine as 50bp single reads or 100bp single reads.
Libraries of ChIP experiments of H3K27me3 and EZH2 were generated following the ChIPmentation protocol (Schmidl et al, 2015; PMID:26280331). H3K27ac and RING1B ChIP-seq libraries were generated following the TrueSeq Illumina protocol.

tag: embryonic day

value: Proximal Forelimbs

value: H3K27ac (Diagenode, C15410196, 2μg )


title: Input Clone b, untreated  [EP300]

value: embryonic

value: input


title: E12_PFL_Wt_H3K27ac_ChIP-seq

source_name: Proximal forelimb mouse E12.5 Wild type H3K27ac ChIP-seq

value: Wt


title: E12_DFL_Wt_EZH2_ChIP-seq

source_name: Distal forelimb mouse E12.5 Wild type EZH2 ChIPmentation

value: Distal Forelimbs

value: EZH2 (Diagenode, C15410039, 3μg)


title: E12_DFL_delattP-Rel5_H3K27ac_ChIP-seq

source_name: Distal forelimb mouse E12.5 delHoxd(attP-Rel5)d9lac H3K27ac ChIP-seq


title: E12_PFL_invNsi-Itga6_H3K27me3_ChIP-seq

source_name: Proximal forelimb mouse E12.5 invHoxd(TgHd11lacNsi-Itga6) H3K27me3 ChIPmentation

value: H3K27me3 (Millipore, Cat 17-622, 3μg )


title: E12_DFL_Wt_H3K27ac_ChIP-seq

source_name: Distal forelimb mouse E12.5 Wild type H3K27ac ChIP-seq


title: E12_PFL_Wt_EZH2_ChIP-seq

source_name: Proximal forelimb mouse E12.5 Wild type EZH2 ChIPmentation


title: E12_DFL_invNsi-Itga6_H3K27me3_ChIP-seq

source_name: Distal forelimb mouse E12.5 invHoxd(TgHd11lacNsi-Itga6) H3K27me3 ChIPmentation


title: E12_PFL_invattP-Itga6_H3K27me3_ChIP-seq

source_name: Proximal forelimb mouse E12.5 invHoxd(attP-Itga6) H3K27me3 ChIPmentation


title: E12_DFL_Wt_RING1B_ChIP-seq

source_name: Distal forelimb mouse E12.5 Wild type RING1B ChIP-seq

value: RING1B (Active Motif ,39664, 4μg)


title: E12_PFL_delattP-Rel5_H3K27me3_ChIP-seq

source_name: Proximal forelimb mouse E12.5 delHoxd(attP-Rel5)d9lac H3K27me3 ChIPmentation


title: d9_Acetate_Input

status: Public on May 14 2019

description: d9 1mM_Glucose + 5mM Acetate

data_processing:
Sequenced samples were trimmed with Trimmomatic (Bolger et al., 2014)
Mapped using Bowtie2 (Langmead and Salzberg, 2012)
Replicate mapped files merged with SAM tools (Li et al., 2009).
Heatmaps and coverage files were generated with deepTools (Ramírez et al., 2016).
Genome_build: GRCm38
Supplementary_files_format_and_content: Coverage files in bigwig format

source_name: d9 1mM_Glucose + 5mM Acetate

growth_protocol: OVA peptide-specific T cells were isolated from spleens and peripheral lymph nodes of OT-I transgenic mice and activated with the ovalbumin peptide SIINFEKL (OVA257-264, 100 ng/ml, New England Peptide). T cells were cultured in RPMI 1640, added with 10% heat-inactivated fetal bovine serum (Gibco, lot n. 1640960), glutamine (4 mM), penicillin/streptomycin (1%), β-mercaptoethanol (55 μM) and glucose (concentrations indicated in the text). Cells were grown at 37° C, in 5% CO2, atmospheric O2, in a humidified incubator. Briefly, cells were activated on day 0 with OVA257-264, with rhIL-2 (100 U/ml, Peprotech) in medium containing 25 mM glucose.

extract_protocol: Fixed cell pellets (1% paraformaldehyde, 10 minutes, RT) were processed for multiplex RELACS (Arrigoni et al., 2018) and sequenced by the staff at the Deep-sequencing Facility at the Max-Planck-Institute for Immunobiology and Epigenetics.

treatment_protocol: After 3 days, cells were switched to 1 mM glucose for 1 or 5 days (referred to as d5 and d9 respectively). 1 day before analysis, cells maintained in 1 mM glucose were treated with or without 5 mM sodium acetate (Sigma). Starting from day 2, and every day, cultured cells were harvested, washed, counted and plated at a concentration of 10^6 cells/ml in fresh medium with rhIL-2.

value: spleens and peripheral lymph nodes

value: CD8 T cells

tag: days of glucose restriction

value: OT-I

value: none (input)


title: Prev_ENV_F2

status: Public on Jun 05 2019

city: Auzeville Tolosane

first: Sylvain

last: MERLOT

address: 24, chemin de Borde Rouge

department: Biological Sciences

institute: CNRS

description: environmental sample

data_processing:
Illumina HiSeq 2000 100bp paired-end mode
Adaptors and low quality pair-end sequences were removed from the raw reads and the ribosomal RNA was filtered using the SortMeRNA algorithm (Kopylova, E., Noé, L. & Touzet, H. SortMeRNA: Fast and accurate filtering of ribosomal RNAs in metatranscriptomic data. Bioinformatics 28, 3211–3217 (2012).
To generate transcriptome sequences, paired-end reads originating from one individual plant of Psychotria grandis (Pgra),  Psychotria costivenia (Pcos)or Psychotria revoluta (Prev) were assembled de novo using CLC Genomic Workbench 9 with Lenght fraction:0.75; Similarity fraction: 0.95; word size: 24 (Pgra), 23 (Pcos), 24 (Prev). Contig sequences were annotated using Blast2GO using BlastX with E-value≤10-6.
Paired-end reads of all independant samples were mapped to the Pgra_transcriptome_v2 using CLC Genomic Workbench 9 with Lenght fraction 0.5 and Similarity fraction 0.875. Read counts are used for abundance measurements.
Genome_build: Pgra_transcriptome_v1.fasta
Supplementary_files_format_and_content: csv file with counts

organism: Psychotria revoluta

source_name: Leaf

growth_protocol: Plant samples were collected in their natural environment (gallery forest, serpentine soil)

extract_protocol:
Total RNA were extracted from frozen leaves of one individual plant using McKenzie et al's Qiagen hybrid method  (Protocol#2, Johnson et al. 2012, PLoS ONE 7(11): e50226. doi:10.1371), and treated with DNAseI.
Libraries were constructed using 1 µg total RNA with Illumina TruSeq Stranded mRNA kit by the POPS transcriptomic plateform (IPS2, Orsay, France) following Illumina kit recommendations.

treatment_protocol: Samples were stabilized in in RNAlater (Sigma). The stabilization solution was removed at the laboratory and samples were stored at -80°C before RNA extraction

tag: phenotype

value: nickel-non-accumulator

value: Leaf

tag: geographical location

value: Mil Cumbres, La Palma, Cuba

tag: gps location

tag: collection date


title: Pcos_ENV_F2

description: environmental sample

organism: Psychotria costivenia

value: nickel hyperaccumulator

value: La Coca, La Habana del Este, Cuba


title: E12_DFL_invattP-Itga6_H3K27me3_ChIP-seq

source_name: Distal forelimb mouse E12.5 invHoxd(attP-Itga6) H3K27me3 ChIPmentation


title: d5_Acetate_Input

description: d5 1mM_Glucose + 5mM Acetate

source_name: d5 1mM_Glucose + 5mM Acetate


title: Pcos_ENV_F1

description: environmental sample


title: E12_PFL_Wt_RING1B_ChIP-seq

source_name: Proximal forelimb mouse E12.5 Wild type RING1B ChIP-seq

value: Proximal Forelimbs


title: E12_PFL_Wt_H3K27me3_ChIP-seq

data_processing:
Instrument models: HiSeq2500 and HiSeq4000
Analyses were performed using the facilities of the Scientific IT and Application Support Center of EPFL. Sequences were filtered and adapters were removed using cutadapt (v1.16) with parameters -m 15 -q 30 -a CTGTCTCTTATACACATCTCCGAGCCCACGAGAC for ChIPmentation and -a GATCGGAAGAGCACACGTCTGAACTCCAGTCAC for ChIP-seq. Reads were mapped on mm10 using bowtie2 (v2.3.4.1) using default parameters. Only reads with a mapping quality above 30 were kept. A profile was obtained with macs2 (version 2.1.1.20160309 option –extsize 300). Bedgraphs were normalized to their number of million tags used in the profile and replicates were merged using the tool unionbedg (bedtools v2.27). An artifactual peak, which is due to a PCR contamination, was found in region chr2:74207282-74208158 in some datasets.
Genome_build: mm10
Supplementary_files_format_and_content: mean of normalized macs2 bedgraphs

source_name: Proximal forelimb mouse E12.5 Wild type H3K27me3 ChIPmentation


title: E12_DFL_delattP-Rel5_H3K27me3_ChIP-seq

source_name: Distal forelimb mouse E12.5 delHoxd(attP-Rel5)d9lac H3K27me3 ChIPmentation


title: d5_1mM_Input

description: d5 1mM_Glucose

source_name: d5 1mM_Glucose


title: Prev_ENV_F1

description: environmental sample


title: d9_1mM_Input

description: d9 1mM_Glucose

source_name: d9 1mM_Glucose


title: d5_1mM_H3K27ac

description: d5 1mM_Glucose

value: anti-H3K27ac (Diagenode, catalog number C15410196)


title: Pgra_ENV_F1

description: environmental sample

organism: Psychotria grandis


title: ATAC-seq_d9_1mM

description: d9 1mM_Glucose

data_processing:
Sequenced samples were trimmed with Trimmomatic (Bolger et al., 2014)
Mapped using Bowtie2 (Langmead and Salzberg, 2012)
Replicate mapped files merged with SAM tools (Li et al., 2009).
Coverage files were generated with deepTools (Ramírez et al., 2016). Open chromatin and differentially regulated chromatin was detected with MACS2 (Zhang et al., 2008) with a p value < 1x10-7 and a q value of less than 0.1 and a 2 fold enrichment threshold.
Genome_build: GRCm38
Supplementary_files_format_and_content: Coverage files in bigwig format

extract_protocol:
5x10^4 T cells treated as described were washed in PBS and then lysed in 10 mM Tris-HCl, pH 7.4,10 mM NaCl, 3 mM MgCl2 and 0.1% Igepal CA-630 (all Sigma). Nuclei were then spun down and then resuspend in 25 μl TD (2x reaction buffer), 2.5 μL TDE1 (Nextera Tn5 Transposase) and 22.5 μL nuclease-free water, incubated for 30 min at 37°C. DNA was purified with the Qiagen MinElute PCR Purification Kit (Thermo Fisher Scientific).
PCR amplification was performed with the NEBNext High-Fidelity 2x PCR Master Mix (New England Labs) using custom Nextera PCR Primers containing barcodes. Adaptors were removed with AMPure XP beads according to manufacturer’s protocol. Libraries were quantified with the Qubit and submitted for sequencing with a HISeq 3000 (Illumina) by the staff at the Deep-sequencing Facility at the Max-Planck-Institute for Immunobiology and Epigenetics.

treatment_protocol: After 3 days, cells were either kept in 25 mM glucose or switched to 1 mM glucose for 1 or 5 days (referred to as d5 and d9 respectively). 1 day before analysis, cells maintained in 1 mM glucose were treated with or without 5 mM sodium acetate (Sigma). Starting from day 2, and every day, cultured cells were harvested, washed, counted and plated at a concentration of 106 cells/ml in fresh medium with rhIL-2.


title: d9_Acetate_3

description: d9 1mM_Glucose + 5mM Acetate

data_processing:
Sequenced libraries were processed with the Galaxy platform and deepTools (Afgan et al., 2016; Ramírez et al., 2016)
STAR (Dobin et al., 2013), for trimming and mapping
featureCounts (Liao et al., 2014) to quantify mapped reads.
Raw mapped reads were processed in R (Lucent Technologies) with DESeq2 (Love et al., 2014), to determine differentially expressed genes and generate normalized read counts to visualize as heatmaps using Morpheus (Broad Institute).
Genome_build: GRCm38
Supplementary_files_format_and_content: raw read counts for every sample

extract_protocol:
RNA was extracted using the RNAsolv reagent (Omega) according to manufacturer instructions and quantified using Qubit 2.0 (Thermo Fisher Scientific) following the manufacturer’s instructions.
Libraries were prepared using the TruSeq stranded mRNA kit (Illumina) and sequenced in a HISeq 3000 (Illumina)


title: d5_Acetate_H3K27ac

description: d5 1mM_Glucose + 5mM Acetate


title: ATAC-seq_d5_Acetate

description: d5 1mM_Glucose + 5mM Acetate


title: d9_Acetate_2

description: d9 1mM_Glucose + 5mM Acetate


title: d9_Acetate_H3K27ac

description: d9 1mM_Glucose + 5mM Acetate


title: E12_DFL_Wt_H3K27me3_ChIP-seq

source_name: Distal forelimb mouse E12.5 Wild type H3K27me3 ChIPmentation


title: d9_1mM_3

description: d9 1mM_Glucose


title: ATAC-seq_d9_Acetate

description: d9 1mM_Glucose + 5mM Acetate


title: d9_Acetate_1

description: d9 1mM_Glucose + 5mM Acetate


title: ATAC-seq_d5_1mM

description: d5 1mM_Glucose


title: Pgra_ENV_F2

description: environmental sample


title: d9_1mM_2

description: d9 1mM_Glucose


title: d5_1mM_2

description: d5 1mM_Glucose


title: d5_1mM_1

description: d5 1mM_Glucose


title: d5_Acetate_1

description: d5 1mM_Glucose + 5mM Acetate


title: d5_Acetate_2

description: d5 1mM_Glucose + 5mM Acetate


title: PCGF2fl_TAM_Input_rep1

status: Public on Apr 25 2019

city: Oxford

first: Nadezda

last: Fursova

address: South Parks Rd

institute: University of Oxford

data_processing:
Paired-end reads were aligned using Bowtie 2 (with “--very-fast”, “--no-mixed” and “--no-discordant” options) against the concatenated mm10 and dm6 rRNA genomic sequence (GenBank: BK000964.3 and M21017.1), to filter out reads mapping to rRNA fragments. All unmapped reads from the first step, were aligned against the genome sequence of concatenated mm10 and dm6 genomes using the STAR RNA-seq aligner (Dobin et al. 2012). To improve mapping of intronic sequences for nascent transcripts abundant in nuclear RNA-seq, reads which failed to map using STAR were aligned against the mm10+dm6 concatenated genome using Bowtie 2 (with “--sensitive-local”, “--no-mixed” and “ --no-discordant” options). For gDNA-seq, paired-end reads were aligned to the genome sequence of concatenated mm10+dm6 genomes using Bowtie 2 with the “--no-mixed” and “--no-discordant” options specified. Reads that were mapped more than once were discarded. PCR duplicates were removed using SAMTools.
To internally calibrate RNA-seq, we spiked-in a fixed number of Drosophila SG4 to each experimental sample. For data visualisation, mm10 reads were randomly subsampled using factors that reflected the total number of dm6 reads in each sample. To correct for any variation in spike-in cell mixing in different biological replicates, the ratio of dm6/mm10 total read counts was calculated from corresponding genomic DNA-seq samples and later used to adjust downsampling factors.
Biological triplicate read counts were determined using custom scripts utilising SAMTools for a custom-built, non-redundant mm10 gene set. Briefly, mm10 refGene genes were filtered to remove very short genes with poor sequence mappability and highly similar transcripts which resulted in the final set of 20,633 genes. Raw mm10 reads prior to spike-in normalisation were used for read counts quantitation for differential expression analysis.
The final set of 20,633 genes were used for differential analysis using a custom R script adapting DESeq2 (Love et al., 2014) for spike-in calibrated RNA-seq data. To incorporate spike-in calibration into this analysis, read counts for the spike-in genome at a control set of intervals were supplied to calculate DESeq2 size factors which were then used for DESeq2 normalisation of raw mm10 read counts. A set of unique dm6 refGene genes was used for spike-in normalisation of cnRNA-seq. Prior to quantitation, spike-in reads were pre-normalised to accurately reflect the actual spike-in ratio derived from a corresponding Input or genomic DNA-seq sample. For a change to be called significant, we applied a threshold of p-adj < 0.05 and fold change > 1.5.
Genome_build: mm10
Supplementary_files_format_and_content: Stranded bigWig files generated with genomeCoverageBed from BEDTools representing genome coverage for merged biological replicates which have been pre-normalised using spike-in downsampling factors
Supplementary_files_format_and_content: DESeq2 results table with normalised read counts, estimated log2 fold changes and statistical parameters of differential expression gene analysis
Supplementary_files_format_and_content: Tables with RefSeq IDs and MGI symbols for selected groups of genes described in the paper (related to Figures S5 and S6)

organism:
Drosophila melanogaster
Mus musculus

source_name: Mouse embryonic stem cells with Drosophila spike-in, tamoxifen-treated (72hr TAM), gDNA-seq

extract_protocol:
1 x 10^7 mouse ESCs were mixed with 4 x 10^6 Drosophila SG4 cells in PBS. Nuclei were isolated in 1 ml HS Lysis buffer (50 mM KCl, 10 mM MgSO4.7H20, 5 mM HEPES, 0.05 % NP40 (IGEPAL CA630)), 1 mM PMSF, 3 mM DTT) for 1 min at room temperature. They were then recovered by centrifugation at 1000 × g for 5 min at 4°C, followed by a total of three washes with ice-cold RSB buffer (10 mM NaCl, 10 mM Tris (pH 8.0), 3 mM MgCl2). Nuclei integrity was assessed using 0.4% Trypan Blue staining (ThermoScientific). Next, nuclei were resuspended in 1 ml of TRIzol reagent (ThermoScientific) and RNA was extracted according to the manufacturer’s protocol, followed by treatment with the TURBO DNA-free Kit (ThermoScientific). Quality of RNA was assessed using 2100 Bioanalyzer RNA 6000 Pico kit (Agilent) and high-quality RNA samples were depleted of rRNA using the NEBNext rRNA Depletion kit (NEB). To quantitate the consistency of spike-in cell mixing for each individual sample, a small aliquot of nuclei was used to isolate genomic DNA using phenol-chloroform extraction. This was followed by sonication of DNA for 13-15 min using a BioRuptor Pico sonicator (Diagenode), shearing genomic DNA to an average size of less than 1 kb.
RNA-seq libraries were prepared using the NEBNext Ultra Directional RNA-seq kit (NEB) following manufacturer’s guidelines. Libraries from sonicated genomic DNA were constructed using NEBNext Ultra DNA Library Prep Kit for Illumina and indexing was done using NEBNext Multiplex Oligos, following manufacturer’s guidelines. Both RNA-seq and gDNA-seq libraries were sequenced as 80 bp paired-end reads on the Illumina NextSeq 500 platform in biological triplicate.

treatment_protocol: To induce conditional removal of distinct PRC1 complexes or their combinations in Pcgf4-/-; Pcgf2fl/fl, Pcgf1fl/fl, Pcgf3/5fl/fl, Pcgf1/3/5fl/fl, Pcgf1/3/5/2fl/fl, Pcgf1/3/5/6fl/fl and Ring1a-/-; Ring1bfl/fl ESC lines, cells were treated with 800 nM 4-hydroxytamoxifen (TAM) for 72 hours.

tag: replicate

tag: treatment agent

value: tamoxifen (OHT)

tag: treatment time point

value: 72 hr

tag: clone

tag: spike-in reference organism

value: Drosophila melanogaster

tag: spike-in cell line


title: d9_1mM_H3K27ac

description: d9 1mM_Glucose

tag: strain

tag: chip antibody


title: PCGF2fl_TAM_Input_rep2

tag: cell line


title: PCGF2fl_TAM_NucRNA_rep3

source_name: Mouse embryonic stem cells with Drosophila spike-in, tamoxifen-treated (72hr TAM), NucRNA-seq


title: PCGF2fl_UNT_Input_rep1

source_name: Mouse embryonic stem cells with Drosophila spike-in, untreated control, gDNA-seq

value: 0 hr


title: PCGF2fl_UNT_NucRNA_rep3

source_name: Mouse embryonic stem cells with Drosophila spike-in, untreated control, NucRNA-seq


title: PCGF2fl_UNT_Input_rep3


title: PCGF2fl_TAM_NucRNA_rep2


title: PCGF2fl_UNT_NucRNA_rep1


title: PCGF2fl_UNT_Input_rep2


title: PCGF2fl_TAM_NucRNA_rep1


title: PCGF1/3/5/6fl_UNT_Input_rep2


title: PCGF1/3/5/6fl_TAM_Input_rep2


title: PCGF1/3/5/6fl_UNT_Input_rep3


title: PCGF1/3/5/6fl_TAM_NucRNA_rep1


title: PCGF1/3/5/6fl_TAM_NucRNA_rep3


title: PCGF1/3/5/6fl_TAM_Input_rep3


title: PCGF1/3/5/6fl_UNT_NucRNA_rep3


title: PCGF1/3/5/6fl_TAM_Input_rep1


title: d9_1mM_1

description: d9 1mM_Glucose

tag: cell type

tag: genotype


title: PCGF2fl_TAM_Input_rep3


title: PCGF1/3/5/6fl_UNT_NucRNA_rep2


title: PCGF2fl_UNT_NucRNA_rep2


title: PCGF1/3/5/6fl_UNT_Input_rep1


title: PCGF1/3/5/2fl_TAM_Input_rep3


title: PCGF1/3/5/2fl_TAM_Input_rep1


title: PCGF1/3/5/6fl_UNT_NucRNA_rep1


title: PCGF1/3/5/2fl_UNT_Input_rep3


title: PCGF1/3/5/2fl_UNT_Input_rep2


title: PCGF1/3/5/6fl_TAM_NucRNA_rep2


title: PCGF1/3/5/2fl_TAM_NucRNA_rep2


title: PCGF1/3/5/2fl_TAM_NucRNA_rep3


title: PCGF1/3/5/2fl_TAM_NucRNA_rep1


title: A1

status: Public on May 07 2019

city: Fuzhou

first: Fei

last: He

address: No.1 Xuefubei Road

institute: Fujian Medical University

hyb_protocol: Total RNAs were hybridized using mRNA + lncRNA Human Gene Expression Microarray V4.0 (4×180K format, CapitalBio Corp, Beijing, China)

scan_protocol: Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505B) using one color scan setting for 1x44k array slides (Scan Area 61x21.6 mm, Scan resolution 10um, Dye channel is set to Green and Green PMT is set to 100%).

data_processing: The scanned images were analyzed with Feature Extraction Software 12.0 (Agilent) using default parameters (protocol GE1-v1_91 and Grid: 012391_D_20060331) to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.

source_name: lung adenocarcinoma tissues

label_protocol: The purity and concentration of RNA was determined by Nano Drop ND-1000 spectrophotometer according to OD260/280 reading.

extract_protocol: Tumor samples were microdissected to ensure the purity of the tumor. From the same patient, adjacent normal tissue samples were collected from resected, located approximately 3.5-5 cm away from the tumor site.

treatment_protocol: Samples were fresh frozen and were shipped on ice for subsequent RNA extraction

tag: tissue

value: lung adenocarcinoma


title: PCGF1/3/5/2fl_UNT_NucRNA_rep2


title: PCGF1/3/5fl_TAM_Input_rep3


title: PCGF1/3/5fl_TAM_Input_rep2


title: PCGF1/3/5fl_TAM_Input_rep1


title: PCGF1/3/5fl_UNT_Input_rep3


title: PCGF1/3/5fl_UNT_Input_rep2


title: PCGF1/3/5fl_UNT_Input_rep1


title: PCGF1/3/5fl_TAM_NucRNA_rep3


title: PCGF1/3/5fl_TAM_NucRNA_rep1


title: PCGF1/3/5fl_UNT_NucRNA_rep2


title: PCGF1/3/5fl_UNT_NucRNA_rep3


title: PCGF1/3/5fl_UNT_NucRNA_rep1


title: PCGF1/3/5/2fl_TAM_Input_rep2


title: PCGF3/5fl_TAM_Input_rep3


title: PCGF3/5fl_TAM_Input_rep2


title: PCGF1/3/5/2fl_UNT_Input_rep1


title: PCGF3/5fl_UNT_Input_rep2


title: PCGF3/5fl_TAM_Input_rep1


title: PCGF3/5fl_UNT_Input_rep3


title: PCGF1/3/5/2fl_UNT_NucRNA_rep3


title: PCGF3/5fl_UNT_Input_rep1


title: PCGF3/5fl_TAM_NucRNA_rep3


title: PCGF1/3/5/2fl_UNT_NucRNA_rep1


title: PCGF3/5fl_TAM_NucRNA_rep2


title: PCGF3/5fl_UNT_NucRNA_rep1


title: PCGF3/5fl_UNT_NucRNA_rep2


title: PCGF3/5fl_TAM_NucRNA_rep1


title: PCGF1fl_TAM_Input_rep3


title: PCGF1/3/5fl_TAM_NucRNA_rep2


title: PCGF1fl_TAM_Input_rep2


title: PCGF1fl_UNT_Input_rep2


title: PCGF1fl_TAM_NucRNA_rep3


title: PCGF1fl_UNT_Input_rep1


title: PCGF1fl_TAM_NucRNA_rep1


title: PCGF3/5fl_UNT_NucRNA_rep3


title: RING1Bfl_UNT_NucRNA_rep1


title: PCGF1fl_TAM_Input_rep1


title: M2_18h_3

status: Public on Mar 06 2019

description: mRNA

data_processing:
Sequenced libraries were processed with the Galaxy platform and deepTools (Afgan et al., 2016; Ramírez et al., 2016).
STAR (Dobin et al., 2013) for trimming and mapping.
featureCounts (Liao et al., 2014) to quantify mapped reads.
Raw mapped reads were processed in R (Lucent Technologies) with DESeq2 (Love et al., 2014) to determine differentially expressed genes and generate normalized read counts to visualize as heatmaps using Morpheus (Broad Institute).
Genome_build: GRCm38
Supplementary_files_format_and_content: *.txt: Tab-delimited text files include raw read counts for every sample.

source_name: Bone marrow-derived macrophage

growth_protocol: Mature bone marrow-derived macrophages (BMMs) were obtained at day 7 of culture of bone marrow cells in 20 ng/mL CSF-1 (Peprotech) in RPMI medium containing 10% fetal bovine serum, 4 mM L-glutamine, 100 U/mL penicillin/streptomycin (complete RPMI) (all Gibco).

extract_protocol:
Total RNA was extracted with the RNAqueous-Micro Total RNA Isolation Kit (Thermo Fisher Scientific) and quantified using Qubit 2.0 (Thermo Fisher Scientific) following the manufacturer’s instructions.
Libraries were prepared using the TruSeq stranded mRNA kit (Illumina) and sequenced in a HiSeq 3000 (Illumina).

treatment_protocol: BMMs were stimulated in complete RPMI supplemented with CSF-1 for indicated times with a combination of 20 or 100 ng/mL LPS (SIGMA), 50 ng/mL IFNg or 20 ng/mL IL-4 (all Peprotech) under 5% CO2, atmospheric oxygen, at 37°C in a humidified incubator.

tag: strain/background

tag: genotype/variation

value: Wild type

value: Bone marrow-derived macrophage

tag: treatment

value: IL-4 stimulation

tag: time point


title: PCGF1fl_UNT_Input_rep3


title: PCGF1fl_UNT_NucRNA_rep1


title: RING1Bfl_TAM_Input_rep3


title: RING1Bfl_TAM_NucRNA_rep2


title: RING1Bfl_UNT_Input_rep2


title: PCGF1fl_UNT_NucRNA_rep2


title: RING1Bfl_UNT_NucRNA_rep3


title: M2_18h_2

description: mRNA


title: RING1Bfl_TAM_Input_rep2


title: RING1Bfl_TAM_Input_rep1


title: RING1Bfl_UNT_Input_rep3


title: RING1Bfl_TAM_NucRNA_rep1


title: M2_18h_1

description: mRNA


title: RING1Bfl_TAM_NucRNA_rep3


title: RING1Bfl_UNT_Input_rep1


title: RING1Bfl_UNT_NucRNA_rep2


title: M2_1h_3

description: mRNA


title: M2_1h_2

description: mRNA


title: PCGF1fl_TAM_NucRNA_rep2


title: M1_18h_3

description: mRNA

value: LPS and IFNg stimulation


title: M1_18h_2

description: mRNA


title: PCGF1fl_UNT_NucRNA_rep3


title: M2_1h_1

description: mRNA


title: WT4_1_1

status: Public on Mar 26 2019

city: Nagoya

first: Chieko

last: Sugita

state: Aichi

address: Chikusa-ku Furo-cho

department: Center for Gene Research

country: Japan

institute: Nagoya University

description: Gene expression of WT protonema cells

hyb_protocol: 600 ng of Cy3-labelled cRNA (specific activity >16.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 25 ul containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 25 ul of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent custom Oligo Microarrays (G2509F) for 17 hours at 65°C in a rotating Agilent hybridization oven.  After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent).

scan_protocol: Slides were scanned immediately after washing on the Agilent Sure Scan using one color scan setting for 8x15k array slides (Scan Area 61x21.6 mm, Scan resolution 5um high sensitivity, Dye channel is set to Green and Green PMT is set to 100%).

data_processing: The scanned images were analyzed with Feature Extraction Software 11.5 (Agilent) using default parameters (protocol GE1-1105_Oct12 and Grid: 084494_D_F_20161121) to obtain background subtracted and spatially detrended Processed Signal intensities.  Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.

organism: Physcomitrium patens

source_name: 4 day old WT protonemata

label_protocol: Cyanine-3 (Cy3) labeled cRNA was prepared from 150 ng RNA using the One-Color Low Input Quick-Amp WT Labeling Kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA).  Dye incorporation and cRNA yield were checked with the NanoDrop 2000 Spectrophotometer.

growth_protocol: protonemal tissue was grown on cellophane-overlaid BCD media supplemented with 50mM ammonium tartarate  and 0.5% glucose under continuous light (30 micromole photons m-2 sec-1)at 25ºC

extract_protocol: Total cellular RNA was extracted with Isogen II (Nippon gene), purified with phenol/chloroform/isoamyl alcohol, and treated with DNase I (TaKaRa).

value: wild type

value: protonemata


title: WT6_2_1

description: Gene expression of WT protonema cells


title: M1_1h_3

first: Immunometabolism

last: Department

address: 1650 Orleans Street

institute: Johns Hopkins University

description: mRNA


title: M1_18h_1

description: mRNA


title: M1_1h_2

description: mRNA


title: M1_1h_1

description: mRNA


title: M0_18h_3

description: mRNA

value: No stimulus control 18h


title: M0_18h_2

description: mRNA


title: M0_1h_3

description: mRNA

value: No stimulus control 1h


title: LPS_18h_1

description: mRNA

value: LPS stimulation


title: LPS_18h_3

description: mRNA


title: LPS_1h_1

description: mRNA


title: M0_1h_2

description: mRNA


title: LPS_1h_3

description: mRNA