title: IgG ChIPseq_Ca_stimulated_rep1

data_processing:
Basecalls were performed using Real-Time Analysis (RTA) version 1.13.48.
ChIP-Seq reads were aligned to the current mouse reference sequence (NCBI build 38, mm10) using bowtie2 version 2.0.2 and only uniquely mapped reads were used for downstream analysis.
ChIP-Seq replicates with correlation values ≥0.9 as determined by DiffBind version 2.4.8 were merged and further analysed as one sample.
Peaks were called using MACS version 1.4 with a stringent p-value of 10-10.
Genome_build: mm10
Supplementary_files_format_and_content: Processed data files were created with MACS 1.4 and represent ChIP-seq peaks, including the MACS-specific score per identified peak region.

growth_protocol: Progenitor cells were enriched from BM single cell suspensions of PU.1 WT mice via density-gradient (Lymphocyte Separation Medium, Lonza) centrifugation (400g, 15 min, RT). Progenitors were prestimulated in RPMI (PAA) supplemented with 10 % FCS, 100 μg/ml Penicillin/Streptomycin, 2 mM L-glutamine and 10 ng/ml murine IL-3 (PeproTech), 20 ng/ml murine Il-6 (PeproTech) and 25 ng/ml murine SCF (PeproTech). Progenitor cells were immortalized by spin infection (4000 rpm, 1 h, RT) with a retrovirus expressing Hoxb8 fused to the estrogen receptor (HoxER) and by maintaining the cells in Opti-MEM® (Life Technologies) supplemented with 10 % FCS, 100 μg/ml Penicillin/Streptomycin, 10 ng/ml SCF, 30 µM 2-Mercaptoethanol and 2 µM β-Estradiol (Sigma). Differentiation into neutrophils was induced by withdrawal of Estradiol and addition of murine G-CSF (PeproTech) for 4 days.

extract_protocol:
2x107 HoxER-PU.1 WT neutrophils were harvested in a 50 ml Falcon tube. Cells were crosslinked with 1 % formaldehyde for 10 min at RT while shaking (seesaw rocker SSL4, Stuart). After crosslinking 5 ml 1 M HEPES (pH 7.5) was added and reaction was quenched by addition of 0.125 M glycine (5 min, RT, shaking). Cells were pelleted (1300 rpm, 8 min, 4°C) and washed two times with ice-cold PBS. Cell lysis was performed by swelling the pellet in ice-cold lysis buffer (10 mM Tris, pH 8.0, 10 mM NaCl, 0.2 % NP-40, 1:500 proteinase inhibitor cocktail (PIC, Sigma)) for 10 min on ice. Nuclei were pelleted by centrifugation (1800 rpm, 5 min, 4°C), and were dissolved in Non-SDS sonication buffer (10 mM Tris, pH 8.0, 1 mM EDTA, 0.5 M EGTA, 1:500 PIC). After incubation for 10 min on ice, chromatin was transferred to TPX hard plastic reaction tubes (1.5 ml or 15 ml, Diagenode) and was sonicated for 30 minutes sonication time on high power (30 sec on, 30 sec off, 0.5 sec pulse rate) using a Bioruptor (Diagenode) and a minichiller (Huber) for cooling. After sonication, chromatin was centrifuged (13200 rpm, 15 min, 4°C) to pellet cell debris and an aliquot (10% Input) was removed and was stored at -20°C for later usage. The chromatin was diluted 10x with ChIP dilution buffer (0.01 % SDS, 1.1 % Triton-X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl, pH 8.1, 167 mM NaCl) in a 2 ml reaction tube. To preclear the chromatin, 40 μl Protein A/G Agarose beads (Santa Cruz) were added, and the mixture was incubated for 30 min at 4°C under constant rotation (Rotator SB3, Stuart). Beads were pelleted (1000 rpm, 1min, 4°C) and the supernatant was transferred to a new reaction tube. 10 µg PU.1 antibody (Santa Cruz, sc-352X) or normal rabbit IgG (Upstate, 12-370) were added to the chromatin, and the mixtures were incubated overnight at 4°C under rotation. Protein A/G agarose beads (Santa Cruz) were blocked for one hour at RT in 1ml PBS containing 1.5% fish skin gelatin (Sigma). After washing, beads were resuspended in the initial volume of ChIP dilution buffer. For precipitation, 200 µl protein A/G agarose beads were added to the chromatin/antibody mix, and the solution was incubated for one hour at 4°C under rotation. The beads were pelleted (1000 rpm, 1 min, 4°C) and were washed two times in 1 ml ChIP low salt immune complex wash buffer (0.1 % SDS, 1 % Triton-X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1, 150 mM NaCl) while rotation for 5 min at 4°. Then, beads were washed two times in 1 ml ChIP high salt immune complex wash buffer (0.1 % SDS, 1 % Triton-X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.1, 500 mM NaCl) and two times in 1 ml ChIP LiCl immune complex wash buffer (0.25 M LiCl, 1 % NP-40, 1 % DOC, 1 mM EDTA, 10 mM Tris, pH 8.1). Afterwards, pelleted beads were washed two times in 1 ml TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, pH 8.0). DNA elution was performed by addition of 250 μl ChIP elution buffer (1 % SDS, 0.1 M NaHCO3) and incubation for 15 min at RT under rotation. The beads were pelleted (1000 rpm, 1 min, 4°C) and the supernatant was transferred to a new 1.5 ml Safe-Lock tube. The beads were conducted to a second elution step, and the two eluates were combined. Protein-DNA crosslinks of samples and inputs (10 %) were reversed by addition of 20 μl 5 M NaCl (overnight, 65°C). Next day, 2 μl (20 mg/ml) proteinase K (Invitrogen), 10 µl 0.5M EDTA and 20 µl Tris-HCl pH 6.5 were added and incubated 1h at 45°C. DNA was isolated by addition of one volume phenol/chloroform/isoamyl alcohol (Roth, 25:24:1) and subsequently centrifugation of the mixture (13200 rpm, 15 minutes, 4°C). The aqueous phase containing the DNA was transferred to a new tube, and 1 µl glycogen (20 mg/ml, Sigma) was added. The precipitated DNA was resuspended in 60 µl DNase and RNase free water (Gibco) and was stored at -20°C for further applications.
Libraries were prepared using the TruSeq ChIP Sample Preparation Kit (Set A, Illumina # IP-202-1012) following the instruction of Illumina. PU.1 ChIP-seq libraries were prepared using the TruSeq ChIP Sample Preparation Kit (Set A, Illumina) following the instruction of the manufacturer. Briefly, 5- 50 ng ChIP DNA was used as a start material. As a first step, overhangs resulting from the fragmentation were converted into blunt ends utilising the End Repair Mix. Next, the 3’ ends were adenylated, and indexing adaptors were ligated to the ends of the DNA fragments. For pooling three libraries in 1 lane, Index Adaptors AR002, AR007 and AR019 or AR005, AR006 and AR015 were used. Subsequently, the ligation products were gel-purified, and fragments were size-selected (250-300 bp). Library DNA was amplified by PCR, and the run was reduced to 12 cycles. DNA fragments were purified via bead selection, and ChIP-seq libraries were qualified on an Agilent Technologies 2100 Bioanalyzer by using the High Sensitivity DNA chip. The Core Facility Genomic Muenster performed library quantification by qPCR (Kapa Library Quant Kit) and single-end sequencing on an Illumina HighScanSQ.

treatment_protocol: HoxER-PU.1 WT neutrophils were left unstimulated or were stimulated for 1 hour with Candida albicans heat-inactivated (15 min at 80°C) cell material in an multiplicity of infection of 1.

value: normal rabbit IgG (Upstate, 12-370)


title: mRNAseq_WT_unstimulated_rep2

status: Public on Mar 19 2019

type: SRA

city: Münster

first: Frank

last: Rosenbauer

address: Robert-Koch-Str. 43

department: Institute of Molecular Tumor Biology

country: Germany

institute: University Hospital Münster

library_source: transcriptomic

data_processing:
Basecalls were performed using Real-Time Analysis (RTA) version 1.13.48.
mRNA-Seq reads were aligned to the current mouse reference sequence (NCBI build 38, mm10) using STAR version 2.3.1.
Differential gene expression was calculated using CuffDiff version 2.2.0. For normalisation, the classic reads per kilobase per million (RPKM) method was used. The default value of max bundle frags was elevated to 10 Mio.
For further analyses, only those genes were taken into consideration where the RPKM value of one condition in the differential analysis was at least one.
Genome_build: mm10
Supplementary_files_format_and_content: tab-delimited text files include RPKM values for each sample

molecule: total RNA

organism: Mus musculus

source_name: HoxER-PU.1 WT neutrophils

growth_protocol: Progenitor cells were enriched from BM single cell suspensions of PU.1 WT or KO mice via density-gradient (Lymphocyte Separation Medium, Lonza) centrifugation (400g, 15 min, RT). Progenitors were prestimulated in RPMI (PAA) supplemented with 10 % FCS, 100 μg/ml Penicillin/Streptomycin, 2 mM L-glutamine and 10 ng/ml murine IL-3 (PeproTech), 20 ng/ml murine Il-6 (PeproTech) and 25 ng/ml murine SCF (PeproTech). Progenitor cells were immortalized by spin infection (4000 rpm, 1 h, RT) with a retrovirus expressing Hoxb8 fused to the estrogen receptor (HoxER) and by maintaining the cells in Opti-MEM® (Life Technologies) supplemented with 10 % FCS, 100 μg/ml Penicillin/Streptomycin, 10 ng/ml SCF, 30 µM 2-Mercaptoethanol and 2 µM β-Estradiol (Sigma). Differentiation into neutrophils was induced by withdrawal of Estradiol and addition of murine G-CSF (PeproTech) for 4 days.

extract_protocol:
1x107 HoxER-PU.1 WT and HoxER-PU.1 KO neutrophils were left unstimulated or were stimulated for 2 hours with Candida albicans heat-inactivated cell material. RNA was extracted with the RNeasy Mini Kit (Qiagen) and DNase I digestion was performed directly on the column by using the DNase I Kit (Qiagen). RNA concentration and quality were determined by using the Bioanalyzer (Agilent Technologies) with the Agilent RNA 6000 Nano Kit (Agilent Technologies).
Libraries were prepared using the NEBNExt® UltraTM Directional RNA Library Prep Kit for Illumina (New England Biolabs) with the NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs) according to the manufacturer’s instructions. Briefly, 300 ng of total RNA was enriched for mRNA using NEBNExt Oligo d(T)25 beads and was fragmented to 200 nucleotides fragments. Next, two rounds of cDNA synthesis were performed to obtain double-stranded cDNA, which was purified using Agencourt AMPure XP Beads (Beckman Coulter Genomics). After the End Prep was completed, the NEBNext Adaptor for Illumina was ligated to the cDNA fragments. After another round of purification using the Agencourt AMPure XP Beads, adaptor-ligated DNA (library) was enriched with a PCR step. For pooling six libraries in one lane, NEBNext Index Primer # 2,4,5,6,7 and 12 were used in the PCR. Libraries were purified with Agencourt AMPure XP Beads and quality of libraries was determined on an Agilent Technologies 2100 Bioanalyzer by using the Agilent High sensitivity DNA Kit (2.2.1.2). The Core Facility Genomics of the Medical Faculty Muenster quantified, normalised and sequenced the libraries for 50 cycles on a HighScanSQ (Illumina).

treatment_protocol: HoxER-PU.1 WT or HoxER-PU.1 KO neutrophils were left unstimulated or were stimulated for 2 hours with Candida albicans heat-inactivated (15 min at 80°C) cell material in an multiplicity of infection of 1.

tag: cell type

value: HoxER-PU.1 WT neutrophils derived from immortalized progenitors

tag: strain

value: MRP8-Cre-ires/GFP (PU.1 WT)


title: PU.1_ChIPseq_Ca_stimulated_rep1

library_source: genomic

molecule: genomic DNA

tag: chip antibody

value: PU.1 (Santa Cruz, sc-352X)


title: IgG ChIPseq_unstimulated_rep1


title: PU.1_ChIPseq_Ca_stimulated_rep2


title: PU.1_ChIPseq_unstimulated_rep2


title: ATACseq_Entinostat_treated_WT_rep2

data_processing:
ATAC-seq reads were trimmed for Nextera adapters sequences using Skewer version 0.2.2 with the following parameters: -m pe, -x CTGTCTCTTATA, -y CTGTCTCTTATA, -q 20.
ATAC-seq reads were aligned to the murine reference genome (NCBI build 38, mm10) using bowtie2 version 2.3.2.
ATAC-seq reads were filtered for primary, properly-paired alignments with a MAPQ ≥ 30 (view -f 3 -F 2308 -q 30) followed by duplicate removal with SAMtools version 1.4.
ATAC-seq replicates were merged, and analysed as one sample; peaks were determined by using MACS version 1.4 with a stringent p-value of 10-10.
Target regions were determined based on differential ATAC-seq peaks (unstimulated, stimulated WT); average count profiles for other samples were calculated for these target regions.
Genome_build: mm10
Supplementary_files_format_and_content: wig file
Enti_rep2_rmdup_cutSites.wig

extract_protocol:
5x104 HoxER-PU.1 WT and HoxER-PU.1 KO neutrophils were harvested after 2 hour stimulation with heat-inactivated C. albicans material (MOI=1) or treament for 2h with DMSO, 1h with TSA [100 nM] and 2h with Entinostat [5 µM]. All centrifugations steps were carried out at 500 g at 4°C in a fixed-angle centrifuge. Cells were washed once in 50 μl cold PBS and were lysed in 50 μl cold ATAC lysis buffer (10 mM NaCl, 10 mM Tris, pH 7.4, 3 mM MgCl2, 0.1 % Igepal CA 630) while centrifuging for 10 min. The supernatant was discarded, and the crude nuclei extract (pellet) was resuspended in 50 μl transposition mix composed of 2.5 μl Tn5 Transposase, 25 μl tagmentation buffer (Nextera Sample Prep Kit) and 22.5 μl H2O. The mixture was incubated for 30 min at 37°C in an Eppendorf Thermomixer at 300 rpm. The reaction was purified using the MinElute kit following instructions of Qiagen and eluted in 10 μl of prewarmed TE buffer.
The transposed DNA was amplified for 12 cycles using preactivated (1 min at 98°C before use) KAPA HiFi Hotstart Ready Mix and single-index primers following the Nextera Low Plex Pooling guidelines. To obtain PCR-amplifiable fragments, PCR was initiated with an elongation step to obtain amplifiable product since the transposase inserts adapters only to the 5' end of each fragment. PCR was run as followed: holding stage (5 min @70°C; 3 min @95°C) and cycling stage (20 sec @98°C, 30 sec @ 63°C, 15 sec @ 72°C). PCR amplified samples were purified using AMPure XP beads (Beckman Coulter) at a beads/sample ratio of 1.2x. Library quality control was performed with an Agilent High-Sensitivity DNA chip on a Bioanalyzer 2100. The Core Facility Genomic Muenster performed library quantification by qPCR (Kapa Library Quant Kit) and paired-end sequencing on an Illumina Nextseq 500.


title: PU.1_ChIPseq_unstimulated_rep1


title: ATACseq_DMSO_treated_WT_rep1

data_processing:
ATAC-seq reads were trimmed for Nextera adapters sequences using Skewer version 0.2.2 with the following parameters: -m pe, -x CTGTCTCTTATA, -y CTGTCTCTTATA, -q 20.
ATAC-seq reads were aligned to the murine reference genome (NCBI build 38, mm10) using bowtie2 version 2.3.2.
ATAC-seq reads were filtered for primary, properly-paired alignments with a MAPQ ≥ 30 (view -f 3 -F 2308 -q 30) followed by duplicate removal with SAMtools version 1.4.
ATAC-seq replicates were merged, and analysed as one sample; peaks were determined by using MACS version 1.4 with a stringent p-value of 10-10.
Target regions were determined based on differential ATAC-seq peaks (unstimulated, stimulated WT); average count profiles for other samples were calculated for these target regions.
Genome_build: mm10
Supplementary_files_format_and_content: wig file
DMSO_rep1_rmdup_cutSites.wig


title: ATACseq_DMSO_treated_WT_rep2

data_processing:
ATAC-seq reads were trimmed for Nextera adapters sequences using Skewer version 0.2.2 with the following parameters: -m pe, -x CTGTCTCTTATA, -y CTGTCTCTTATA, -q 20.
ATAC-seq reads were aligned to the murine reference genome (NCBI build 38, mm10) using bowtie2 version 2.3.2.
ATAC-seq reads were filtered for primary, properly-paired alignments with a MAPQ ≥ 30 (view -f 3 -F 2308 -q 30) followed by duplicate removal with SAMtools version 1.4.
ATAC-seq replicates were merged, and analysed as one sample; peaks were determined by using MACS version 1.4 with a stringent p-value of 10-10.
Target regions were determined based on differential ATAC-seq peaks (unstimulated, stimulated WT); average count profiles for other samples were calculated for these target regions.
Genome_build: mm10
Supplementary_files_format_and_content: wig file
DMSO_rep2_rmdup_cutSites.wig


title: ATACseq_Entinostat_treated_WT_rep1

data_processing:
ATAC-seq reads were trimmed for Nextera adapters sequences using Skewer version 0.2.2 with the following parameters: -m pe, -x CTGTCTCTTATA, -y CTGTCTCTTATA, -q 20.
ATAC-seq reads were aligned to the murine reference genome (NCBI build 38, mm10) using bowtie2 version 2.3.2.
ATAC-seq reads were filtered for primary, properly-paired alignments with a MAPQ ≥ 30 (view -f 3 -F 2308 -q 30) followed by duplicate removal with SAMtools version 1.4.
ATAC-seq replicates were merged, and analysed as one sample; peaks were determined by using MACS version 1.4 with a stringent p-value of 10-10.
Target regions were determined based on differential ATAC-seq peaks (unstimulated, stimulated WT); average count profiles for other samples were calculated for these target regions.
Genome_build: mm10
Supplementary_files_format_and_content: Enti_rep1_rmdup_cutSites.wig


title: ATACseq_TSA_treated_WT_rep1

data_processing:
ATAC-seq reads were trimmed for Nextera adapters sequences using Skewer version 0.2.2 with the following parameters: -m pe, -x CTGTCTCTTATA, -y CTGTCTCTTATA, -q 20.
ATAC-seq reads were aligned to the murine reference genome (NCBI build 38, mm10) using bowtie2 version 2.3.2.
ATAC-seq reads were filtered for primary, properly-paired alignments with a MAPQ ≥ 30 (view -f 3 -F 2308 -q 30) followed by duplicate removal with SAMtools version 1.4.
ATAC-seq replicates were merged, and analysed as one sample; peaks were determined by using MACS version 1.4 with a stringent p-value of 10-10.
Target regions were determined based on differential ATAC-seq peaks (unstimulated, stimulated WT); average count profiles for other samples were calculated for these target regions.
Genome_build: mm10
Supplementary_files_format_and_content: wig file
TSA_rmdup_cutSites.wig


title: JWT (Tg-ACE) rep1

status: Public on Jan 18 2019

city: West Hollywood

first: AGCT

last: Core

state: CA

address: 8687 Melrose Ave, PDC B230

department: BMS

country: USA

institute: Cedars Sinai Medical Center

data_processing:
Raw reads obtained from RNA-Seq were aligned to the transcriptome using STAR (version 2.5.0) (Dobin A et al., 2013) / RSEM (version 1.2.25) (Li B and Dewey CN, 2011) with default parameters, using a custom human GRCh38 (or mouse CRCm38) transcriptome reference downloaded from http://www.gencodegenes.org, containing all protein coding and long non-coding RNA genes based on  Mouse GENCODE M8 annotation. Expression counts for each gene (TPM: transcripts per million) in all samples were normalized by the sequencing depth.
Genome_build: mm10
Supplementary_files_format_and_content: csv

source_name: Macrophages

extract_protocol:
RNA extracted by PureLink RNA Mini Kit.
Library construction was performed using the Illumina TruSeq Stranded mRNA library preparation kit (Illumina, San Diego, CA). Briefly, total RNA samples were assessed for concentration using a Qubit fluorometer (ThermoFisher Scientific, Waltham, MA) and quality using the 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). Up to one µg of total RNA per sample was used for poly-A mRNA selection. cDNA was synthesized from enriched and fragmented RNA using reverse transcriptase (Invitrogen, Carlsbad, CA) and random primers. The cDNA was further converted into doublestranded DNA (dsDNA), and the resulting dsDNA was enriched with PCR for library preparation. The PCR-amplified library was purified using Agencourt AMPure XP beads (Beckman Coulter, Brea, CA). The concentration of the amplified library was measured with a Qubit fluorometer and an aliquot of the library was resolved on a Bioanalyzer. Sample libraries are multiplexed and sequenced on a NextSeq 500 platform (Illumina) using 75bp single-end sequencing. On average, about 20 million reads were generated from each sample.

treatment_protocol: After thioglycollate i.p. injection, peritoneal cells were collected at day 4 and macrophages were purified by percoll gradient centrifugation. Isolated cells were culured for two hour in complete DMEM media and adhered cells were collected for RNA isolation

tag: isolation

value: Macrophages isolated by percoll gradient

tag: genotype

value: JWT (Tg-ACE)


title: JWT (Tg-ACE) rep2


title: mRNAseq_WT_unstimulated_rep1


title: mRNAseq_WT_Ca_stimulated_rep1


title: WT rep1

value: WT


title: WT rep3


title: WT rep2


title: Patient 3 Total Liver Homogenate

status: Public on Oct 22 2018

city: Toronto

first: Jeff

last: Liu

state: Ontario

address: 101 College Street, RM 4-601

department: Princess Margaret Cancer Center

country: Canada

institute: University Health Network

data_processing:
Raw sequencing data (bcl files) were converted to fastq files with Illumina bcl2fastq, version 2.19.1
The CellRanger (10X Genomics) secondary analysis pipeline was used to generate a digital gene expression matrix
Cells with a very small library size (< 1500) and a very high (> 0.5) mitochondrial genome transcript ratio were removed.  Genes detected (UMI count > 0) in less than three cells were removed.
Normalization by scran R package
Clustering and Gene Expression Analysis by Seurat package
Genome_build: GRCh38
Supplementary_files_format_and_content: log2CPM gene expression values for filtered cells in CSV text format

organism: Homo sapiens

source_name: Liver

extract_protocol:
The caudate lobe was cannulated with two or three 1.2-2mm diameter irrigation cannulae with olive tips, which were inserted into exposed vessels in the cut surface if the liver lobe (cannulae manufacturer: Ernst Kratz GmbH cat no: 1464LL, 1465LL). The lobe was flushed with HBS+EGTA at 4°C (perfusion rate of 10mL/min/cannulae), which removed any residual non-liver resident cells. Single cell isolation from the resected caudate liver lobe was performed with a modified  “two-step” collagenase procedure6,97 as described in Fig. 1b. Eight to 12 million viable cells were recovered per gram of tissue as determined by Beckman Vi-Cell trypan blue exclusion. Viability for TLHs by trypan blue exclusion varied between 50-90% viable for the five livers profiled. Caudate lobes weighed on average 30.2 g +/-SD of 8.367g. TLHs were taken directly after dissociation for scRNA Seq.
10X Genomics Single Cell 3' v2 Reagent Kit

tag: tissue

value: Total Liver Homogenate


title: E85-WT-8 rep 1

status: Public on Jan 08 2019

data_processing:
Raw reads obtained from RNA-Seq were aligned to the transcriptome using STAR (version 2.5.0) (Dobin A et al., 2013) / RSEM (version 1.2.25) (Li B and Dewey CN, 2011) with default parameters, using a custom mouse GRCm38 transcriptome reference downloaded from http://www.gencodegenes.org, containing all protein coding and long non-coding RNA genes based Mouse GENCODE M8 annotation. Expression counts for each gene  in all samples were normalized by the sequencing depth.
Genome_build: custom mouse GRCm38 transcriptome reference downloaded from http://www.gencodegenes.org, containing all protein coding and long non-coding RNA genes based Mouse GENCODE M8 annotation
Supplementary_files_format_and_content: text files include raw counts for each Sample calculated by RSEM

source_name: T cells

growth_protocol: Naïve splenic CD4+ T cells were isolated by flow cytometry (CD4+ CD25- CD62L high, CD44 low).

extract_protocol:
Total RNA was isolated from T cells using RNeasy kits (Qiagen, Valencia, CA) according to the manufacturer’s protocol
Library construction was performed using the Illumina TruSeq Stranded mRNA library preparation kit. Briefly, Total RNA samples were assessed for concentration using the Nanodrop 8000 (Thermo Scientific) and quality using the 2100 Bioanalyzer (Agilent). One microgram of total RNA per sample was used for poly-A mRNA selection using streptavidin-coated magnetic beads. cDNA was synthesized from enriched and fragmented RNA using reverse transcriptase (Super-Script II, Invitrogen) and random primers. The cDNA was further converted into double-stranded DNA, and the resulting dsDNA was enriched with PCR for library preparation. The PCR-amplified library was purified using Agencourt AMPure XP beads (Beckman Coulter). The concentration of the amplified library was measured with a NanoDrop spectrophotometer and an aliquot of the library is resolved on an Agilent 2100 Bioanalyzer. Sample libraries are multiplexed and sequenced on a NextSeq 500 platform (Illumina) using 75bp single-end sequencing. On average, about 20 million reads were generated from each sample.

treatment_protocol: Cells were treated with anti-CD3, anti-CD28, TGFb, IL-4, TL1A for 72 h.

tag: treatment

value: Th9+TLA1 condition


title: E87-WT-8 rep 2


title: E87-Batf3-8 rep 2


title: E85-Batf3-8 rep 1


title: shPU.1_kd_72h_CD14

status: Public on Oct 15 2018

data_processing:
Library strategy: 4C-seq
Base calling was performed with Illumina's CASAVA 1.7.
4C-seq reads were aligned against the human reference genome (hg19 /GRCh38.p9) using the Burrows-Wheeler Alignment Tool (BWA, v. 0.6.2;  max. 2 mismatches, default settings otherwise)
Aligned reads were mapped to a virtual restriction fragment library based on the chosen restriction enzyme sequences with the R/Bioconductor package Basic4Cseq.
Fragment data was RPM-normalized and filtered: Only uniquely aligned reads on non-blind restriction fragments were kept for analysis.
Genome_build: hg19
Supplementary_files_format_and_content: *_near-cis.bed: bed files were created with the R/Bioconductor package Basic4Cseq and include running median RPM values per valid fragment in the chosen near-cis region for the experiment's viewpoint.

source_name: acute monocytic leukemia - genetically modified

growth_protocol:
Inducible shRNA knockdown: 293TCeB cells were used for retroviral supernatant production after transfection with the shRNA-construct pRSMX_PG_along with the helper plasmids pHit/60 and pHIT/EA6x3. (PMID: 16572121, 11910072 and     12776180) Hereby, two tetracycline repressor (TETR) binding sites (Tet operators) were inserted into the histone H1 promoter for the inducible expression of shRNAs (TETON system). GFP+ selection of construct-positive cells was monitored by FACS analysis. Addition of 20 µg/ml doxycycline (Dox) activated transcription of the shRNA sequences
Primary human monocytes: Blood cells were collected from healthy donors in compliance with the Helsinki Declaration. Peripheral monocytes were separated by leukapheresis followed by density gradient centrifugation with Ficoll/Hypaque purification and subsequent countercurrent centrifugal elutriation as previously described (PMID:     8864140) Monocytes were >85% pure as determined by morphology and expression of CD14 (data not shown).
Human AML samples: Fresh AML blasts were collected at the University Hospital Muenster from bone marrow aspirates with high blast infiltration. Prior to freezing the cell suspensions, density gradient centrifugation was done using Ficoll-Paque to isolate mononuclear cells. Immediately before processing, cells were thawed and verified to have at least 80% viability. Use of the patient material was approved by the Ethics Committee of University Hospital Muenster (approval no. 2007-390-f-S)
THP-1 cell differentiation: The human monocytic AML cell line THP-1 was cultured in RPMI1640 (Thermo Fisher Scientific) medium supplemented with 10% fetal bovine serum (Superiour, Biochrom). Differentiation into macrophages was induced by treatment with phorbol 12-myristate 13-acetate (PMA, 10 nM final, Sigma-Aldrich) and Cholecalciferol (Vitamin D3, VD3, 100 nM final, Sigma-Aldrich). After incubation for the indicated time points, only adherent cells were harvested.

extract_protocol: 4C-seq was performed as previously described (PMID: 22609568). For chromatin digestion NlaIII (NEB) and Csp6I (Thermo Scientific) were used as first and second restriction endonucleases. Inverse PCR was performed to amplify specific viewpoints at the PU.1 promoter, the PU.1 URE or the TET2 gene as control Single-end sequencing was performed using the Illumina HiSeq2500 platform.

tag: 4c-seq viewpoint


title: LDB1_ChIPseq_rep2

data_processing:
LDB1 ChIP-seq data (80bp single-end reads, Nextseq500) were aligned to hg19 with BWA mem (arXiv:1303.3997).
Replicates were merged and further analysed as one sample.
Differential peaks were called with MACS2 (PMID: 22936215).
Genome_build: mm10
Supplementary_files_format_and_content: Processed data files represent MACS2 differential interactions for the ChIP-seq data on chromosome 11.

source_name: differentiated macrophages - acute monocytic leukemia derived

growth_protocol: The human monocytic AML cell line THP-1 was cultured in RPMI1640 (Thermo Fisher Scientific) medium supplemented with 10% fetal bovine serum (Superiour, Biochrom). Differentiation into macrophages was induced by treatment with phorbol 12-myristate 13-acetate (PMA, 10 nM final, Sigma-Aldrich) and Cholecalciferol (Vitamin D3, VD3, 100 nM final, Sigma-Aldrich). After incubation for the indicated time points, only adherent cells were harvested.

extract_protocol: ChIP and library construction were conducted essentially as described by using an unspecific IgG control antibody (rabbit, Merck Millipore #12-370) or LDB1 (rabbit, Abcam #ab96799). Tags for sequencing were mapped to the human reference genome (GRCh37.67/hg19) using BWA mem Aligner (arXiv:1303.3997) (LDB1 ChIPseq) and Bowtie software (all other).(PMID: 21154709) Downstream analysis of uniquely mapped tags, including quality control, peak calling, and motif analysis, was performed as described using MACS2 (PMID: 22936215) (LDB1 ChIP-seq) and Hypergeometric Optimization of Motif EnRichment (HOMER) software tools (all others). (PMID: 20513432) Motif enrichment in LDB1 binding peaks was calculated with findMotifsGenome.pl from the Homer suite.


title: LDB1_ChIPseq_rep1

value: differentiated macrophages - acute monocytic leukemia derived


title: IgG_ChIPseq

value: IgG


title: SC4_72h_URE_1

value: acute monocytic leukemia - genetically modified

value: PU.1 URE


title: SC4_72h_CD14_1


title: shPU.1_kd_72h_URE


title: JWT (Tg-ACE) rep3


title: PU.1-neutrophils_WT_rep1

type: RNA

hyb_protocol: 6 µg of fragmented and labeled aRNA were hybridized to Affymetrix Mouse Genome 430 PM peg plate arrays. For hybridization, washing and staining an Affymetrix GeneTitan system was used.

scan_protocol: GeneChip arrays were scanned using an Affymetrix GeneTitan system controlled by the Affymetrix GeneChip Command Console software (AGCC).

data_processing: Data was imported to R and RMA was normalized with the R package “affy”. Differential gene expression was calculated with the R package “limma” with a minimal fold change of 2 and a p-value of 0.05.

label: biotin

source_name: Neutrophils flow-sorted from BM of PU.1 WT mice.

label_protocol: The Affymetrix GeneChip 3’ IVT Express Protocol was used for the preparation of biotinylated amplified RNA (aRNA) from 300ng of total RNA.

extract_protocol: BM cells were obtained from adult mice by thoroughly flushing femurs and tibiae with PBS (PAA) supplemented with 1 % FSC (Biochrom). Single cell suspensions were generated by mashing the organ through a cell strainer of 40 µm diameter. Suspension of cells were incubated for 5 min with ice-cold ACK buffer (0.15 M NH4Cl, 10 M KHCO3, 0.1 mM EDTA, pH 7.3) to remove erythrocytes. Cells were first incubated for 10 min in PBS supplemented with 1% FCS and purified CD16/32 antibody to block Fc binding. Labeling with Gr-1 (Biolegend) was conducted for 10 min on ice in the dark. Discrimination of dead cells was performed by propidium iodide (PI, 0.1 mg/ml, Sigma Aldrich) shortly before measurement. Cells were flow-sorted with an FACS Aria III (BD) cytometer equipped with FACSDiva software. The RNAeasy Kit (Qiagen) was used for extraction of RNA according to the supplier’s instructions. DNAse I digestion was performed as part of the protocol to remove residual DNA from the RNA preparation. RNA was stored at -80 °C.

value: Neutrophils


title: PU.1-neutrophils_KO_rep2

source_name: Neutrophils flow-sorted from BM of PU.1 KO mice.

value: MRP8-Cre-ires/GFP:PU.1f/f (PU.1 KO)


title: LDB1_kd_a_12h_TET2

data_processing:
Library strategy: 4C-seq
Base calling was performed with Illumina's CASAVA 1.7.
4C-seq reads were aligned against the human reference genome (hg19 /GRCh38.p9) using the Burrows-Wheeler Alignment Tool (BWA, v. 0.6.2; max. 2 mismatches, default settings otherwise).
Aligned reads were mapped to a virtual restriction fragment library based on the chosen restriction enzyme sequences with the R/Bioconductor package Basic4Cseq.
Fragment data was RPM-normalized and filtered: Only uniquely aligned reads on non-blind restriction fragments were kept for analysis.
Genome_build: hg19 (GRCh37.67)
Supplementary_files_format_and_content: *_near-cis.bed: bed files were created with the R/Bioconductor package Basic4Cseq and include running median RPM values per valid fragment in the chosen near-cis region for the experiment's viewpoint.

growth_protocol:
Inducible shRNA knockdown: 293TCeB cells were used for retroviral supernatant production after transfection with the shRNA-construct pRSMX_PG_along with the helper plasmids pHit/60 and pHIT/EA6x3 (PMID: 16572121, 11910072 and 12776180). Hereby, two tetracycline repressor (TETR) binding sites (Tet operators) were inserted into the histone H1 promoter for the inducible expression of shRNAs (TETON system). GFP+ selection of construct-positive cells was monitored by FACS analysis. Addition of 20 µg/ml doxycycline (Dox) activated transcription of the shRNA sequences.
Primary human monocytes: Blood cells were collected from healthy donors in compliance with the Helsinki Declaration. Peripheral monocytes were separated by leukapheresis followed by density gradient centrifugation with Ficoll/Hypaque purification and subsequent countercurrent centrifugal elutriation as previously described (PMID: 8864140). Monocytes were >85% pure as determined by morphology and expression of CD14 (data not shown).
Human AML samples: Fresh AML blasts were collected at the University Hospital Muenster from bone marrow aspirates with high blast infiltration. Prior to freezing the cell suspensions, density gradient centrifugation was done using Ficoll-Paque to isolate mononuclear cells. Immediately before processing, cells were thawed and verified to have at least 80% viability. Use of the patient material was approved by the Ethics Committee of University Hospital Muenster (approval no. 2007-390-f-S).
THP-1 cell differentiation: The human monocytic AML cell line THP-1 was cultured in RPMI1640 (Thermo Fisher Scientific) medium supplemented with 10% fetal bovine serum (Superiour, Biochrom). Differentiation into macrophages was induced by treatment with phorbol 12-myristate 13-acetate (PMA, 10 nM final, Sigma-Aldrich) and Cholecalciferol (Vitamin D3, VD3, 100 nM final, Sigma-Aldrich). After incubation for the indicated time points, only adherent cells were harvested.

extract_protocol: 4C-seq was performed as previously described (PMID: 22609568). For chromatin digestion, NlaIII (NEB) and Csp6I (Thermo Scientific) were used as first and second restriction endonucleases. Inverse PCR was performed to amplify specific viewpoints at the PU.1 promoter, the PU.1 URE or the TET2 gene as control. Single-end sequencing was performed using the Illumina HiSeq2500 platform.


title: PU.1-neutrophils_KO_rep1


title: PU.1-neutrophils_WT_rep2


title: LDB1_kd_SC4_TET2


title: PU.1-neutrophils_KO_rep3


title: THP-1_PMAVD3_2_URE


title: THP1_2_URE

source_name: acute monocytic leukemia

value: acute monocytic leukemia


title: THP-1_PMAVD3_1_URE


title: THP1_1_URE


title: Patient5_URE


title: Patient1_URE


title: Monocytes_URE

source_name: monocytes

value: monocytes


title: shLDB1_kd_a_12h_URE


title: shLDB1_kd_a_12h_PP

value: PU.1 Promoter


title: SC4_12h_URE_2


title: SC4_72h_PP


title: shPU.1_kd_72h_URE 2


title: shPU.1_kd_12h_URE


title: shPU.1_kd_72h_URE 1


title: shPU.1_kd_12h_PP


title: SC4_12h_PP_2


title: SC4_72h_URE


title: shPU.1_kd_72h_PP


title: SC4_12h_URE_1


title: THP-1_PP


title: THP-1_PMAVD3_PP


title: SC4_12h_PP


title: Patient2_PP

source_name: acute myeloid leukemia

value: acute myeloid leukemia


title: Patient5_PP


title: Patient3_PP


title: Patient1_PP


title: CTR21d100-3

status: Public on Sep 29 2018

data_processing:
Raw reads obtained from RNA-Seq were aligned to the transcriptome using STAR (version 2.5.0) / RSEM (version 1.2.25) with default parameters, using a custom human GRCh38 transcriptome reference downloaded from http://www.gencodegenes.org , containing all protein coding and long non-coding RNA genes based on human GENCODE version 23 annotation. Expression counts for each gene in all samples were normalized by a modified trimmed mean of the M-values normalization method.
Genome_build: GRCh38
Supplementary_files_format_and_content: Tab-delimited text file includes read counts for each Sample.

source_name: shi et al differentiation protocol for iPSCs- day 100

growth_protocol: Human fibroblast lines were obtained from three HD patients with an expanded CAG HTT allele of 180 (CS97iHD180n), 109 (CS09iHD109n), or 77 CAG repeats (CS77iHD77n) and from three non-HD “controls” with 33 (CS83iCTR33n), 21 (CS00iCTR21n), or 18 (CS25iCTR18n) repeat CAG alleles. Reprogramming was conducted by non-integrating methods, as previously described (Mattis et al., 2015, Consortium, 2017). iPSCs were differentiated towards a cortical fate as described in Shi et al, with some modifications as described here. iPSCs were grown on Matrigel (BD Bioscience) in mTeSR (Stemcell Technologies 5850) until 70% confluency. They were then passaged with an EZ tool (Life Technologies 23181-010) and plated in a 2:1 ratio with rock inhibitor (10 µM Y27632, Tocris 1254) in mTeSR on matrigel. When the cells reached 100% confluency they were switched to cortical NIM (1:1 DMEM:F12+glutamax : Neurobasal, 2.5 µg/ml Insulin, 0.5% NEAA, 0.5% L-Glutamine, 1% anti-biotic anti-mycotic, 0.5% N2, 1% B27, 10 µM SB43142 (Tocris 1614), 2 µM LDN193189) with daily media changes. They were passaged at day 12 with TrypLE and replated at a 1:2 ratio. The next day they were refed with cortical NMM (1:1 DMEM:F12+glutamax : Neurobasal, 2.5 µg/ml Insulin, 0.5% NEAA, 0.5% L-Glutamine, 1% anti-biotic anti-mycotic, 0.5% N2, 1% B27) with 20ng/ml FGF2 for the next four days, after which FGF2 was removed from the media. Cells were passaged using TrypLE and expanded 1:2 as necessary after this point until day 32. At day 32-35 cells were single-cell dissociated using TrypLE (5 min at 37 °C), washed in DMEM and resuspended in NMM to plate onto PLO-treated and Matrigel-coated coverslips at 100,000 cells per coverslip. Cells were grown in NMM until time points described in the text. Half-media changes were performed every 2-3 days as needed.

extract_protocol:
Cells were manually scraped off of coverslips at time points 0, 60, 80, 100 and 130 of differentiation in PBS, spun down at 1000 RPM for 5 min and pellets were snap-frozen in liquid nitrogen. Frozen pellets were resuspended in 1 ml Trizol (). 200 ml of choloroform was added, then samples were shaken and incubated at room temperature for 3 min prior to spinning at 13,000 RPM for 15 min. The clear supernatant was moved to a new tube and 318 ml of 100% ethanol was added. The supernatant was then transferred to an RNeasy column (Qiagen) and spun 30 sec at 13,000 RPM. Column was washed once with 500 ml buffer RW1 and spun for 30 sec at 13,000 RPM. 70 ml of buffer RDD and 10ul of RNase-free DNAse (Qiagen) were added to column and incubated for 30 min. 500 ml of buffer RW1 was then added and column was spun at 13,000 RPM for 30 sec. Column was then washed again with 500 ml of RPE and spun at 13,000 RPM for 30 sec. The collection tube was changed and spun at 13,000 RPM for 1 min to remove residual buffer. 50 ml of RNase-free water was added and incubated at room temperature for 3 minutes before being spun into a clean collection tube at 13,000 RPM for 5 min. RNA was quantified using a nanodrop then 500 ng was run on a 1% gel to ensure quality. At least 1,200 ng of RNA was submitted for sequencing.
Library preparation and sequencing library construction were performed using the Illumina TruSeq Stranded mRNA library preparation kit. Briefly, total RNA samples were assessed for concentration using the Nanodrop 8000 (Thermo Scientific) and quality using the 2100 Bioanalyzer (Agilent). One microgram of total RNA per sample was used for poly-A mRNA selection using streptavidin-coated magnetic beads. cDNA was synthesized from enriched and fragmented RNA using reverse transcriptase (Super-Script II, Invitrogen) and random primers. The cDNA was further converted into double-stranded DNA, and the resulting dsDNA was enriched with PCR for library preparation. The PCR-amplified library was purified using Agencourt AMPure XP beads (Beckman Coulter). The concentration of the amplified library was measured with a NanoDrop spectrophotometer and an aliquot of the library is resolved on an Agilent 2100 Bioanalyzer. Sample libraries are multiplexed and sequenced on a NextSeq 500 platform (Illumina) using 75bp single-end sequencing. On average, about 20 million reads were generated from each sample.

tag: disease status

value: Control

tag: number cag repeats

tag: individual identifier

value: iPSC-derived cortical neurons

tag: differentiation time

value: 100 days


title: CTR18d100-2


title: CTR21d100-2


title: HD109d100-3

value: Huntington's disease


title: HD77d100-2


title: HD109d100-2


title: HD77d100-3


title: CTR18d80-2

source_name: shi et al differentiation protocol for iPSCs- day 80

value: 80 days


title: CTR21d80-3


title: CTR33d80-2


title: CTR21d80-2


title: Patient4_PP


title: HD109d80-3


title: HD109d80-2


title: HD77d80-3


title: HD77d80-2


title: HD77d60-2

source_name: shi et al differentiation protocol for iPSCs- day 60

value: 60 days


title: HD109d130

data_processing:
Raw reads obtained from RNA-Seq were aligned to the transcriptome using STAR (version 2.5.0) / RSEM (version 1.2.25) with default parameters, using a custom human GRCh38 transcriptome reference downloaded from http://www.gencodegenes.org , containing all protein coding and long non-coding RNA genes based on human GENCODE version 23 annotation. Expression counts for each gene in all samples were normalized by a modified trimmed mean of the M-values normalization method.
Genome_build: GRCh38
Supplementary_files_format_and_content: All.Count.txt: Tab-delimited text file includes read counts for each Sample.

source_name: Shi et al. differentiation protocol for iPSCs- day 130

growth_protocol: Human fibroblast lines were obtained from three HD patients with an expanded CAG HTT allele of 180 (CS97iHD180n), 109 (CS09iHD109n), or 77 CAG repeats (CS77iHD77n) and from three non-HD “controls” with 33 (CS83iCTR33n), 21 (CS00iCTR21n), or 18 (CS25iCTR18n) repeat CAG alleles. Reprogramming was conducted by non-integrating methods, as previously described (Mattis et al., 2015, Consortium, 2017). iPSCs were differentiated towards a cortical fate as described in Shi et al., with some modifications as described here. iPSCs were grown on Matrigel (BD Bioscience) in mTeSR (Stemcell Technologies 5850) until 70% confluency. They were then passaged with an EZ tool (Life Technologies 23181-010) and plated in a 2:1 ratio with rock inhibitor (10 µM Y27632, Tocris 1254) in mTeSR on matrigel. When the cells reached 100% confluency, they were switched to cortical NIM (1:1 DMEM:F12+glutamax : Neurobasal, 2.5 µg/ml Insulin, 0.5% NEAA, 0.5% L-Glutamine, 1% anti-biotic anti-mycotic, 0.5% N2, 1% B27, 10 µM SB43142 (Tocris 1614), 2 µM LDN193189) with daily media changes. They were passaged at day 12 with TrypLE and replated at a 1:2 ratio. The next day they were refed with cortical NMM (1:1 DMEM:F12+glutamax : Neurobasal, 2.5 µg/ml Insulin, 0.5% NEAA, 0.5% L-Glutamine, 1% anti-biotic anti-mycotic, 0.5% N2, 1% B27) with 20ng/ml FGF2 for the next four days, after which FGF2 was removed from the media. Cells were passaged using TrypLE and expanded 1:2 as necessary after this point until day 32. At day 32-35, cells were single-cell dissociated using TrypLE (5 min at 37 °C), washed in DMEM and resuspended in NMM to plate onto PLO-treated and Matrigel-coated coverslips at 100,000 cells per coverslip. Cells were grown in NMM until time points described in the text. Half-media changes were performed every 2-3 days as needed.

extract_protocol:
Cells were manually scraped off of coverslips at time points 0, 60, 80, 100 and 130 of differentiation in PBS, spun down at 1000 RPM for 5 min and pellets were snap-frozen in liquid nitrogen. Frozen pellets were resuspended in 1 ml Trizol. 200 ml of chloroform was added, then samples were shaken and incubated at room temperature for 3 min prior to spinning at 13,000 RPM for 15 min. The clear supernatant was moved to a new tube and 318 ml of 100% ethanol was added. The supernatant was then transferred to an RNeasy column (Qiagen) and spun 30 sec at 13,000 RPM. Column was washed once with 500 ml buffer RW1 and spun for 30 sec at 13,000 RPM. 70 ml of buffer RDD and 10ul of RNase-free DNase (Qiagen) were added to column and incubated for 30 min. 500 ml of buffer RW1 was then added and column was spun at 13,000 RPM for 30 sec. Column was then washed again with 500 ml of RPE and spun at 13,000 RPM for 30 sec. The collection tube was changed and spun at 13,000 RPM for 1 min to remove residual buffer. 50 ml of RNase-free water was added and incubated at room temperature for 3 minutes before being spun into a clean collection tube at 13,000 RPM for 5 min. RNA was quantified using a nanodrop, then 500 ng was run on a 1% gel to ensure quality. At least 1,200 ng of RNA was submitted for sequencing.
Library preparation and sequencing library construction were performed using the Illumina TruSeq Stranded mRNA library preparation kit. Briefly, total RNA samples were assessed for concentration using the Nanodrop 8000 (Thermo Scientific) and quality using the 2100 Bioanalyzer (Agilent). One microgram of total RNA per sample was used for poly-A mRNA selection using streptavidin-coated magnetic beads. cDNA was synthesized from enriched and fragmented RNA using reverse transcriptase (Super-Script II, Invitrogen) and random primers. The cDNA was further converted into double-stranded DNA, and the resulting dsDNA was enriched with PCR for library preparation. The PCR-amplified library was purified using Agencourt AMPure XP beads (Beckman Coulter). The concentration of the amplified library was measured with a NanoDrop spectrophotometer and an aliquot of the library is resolved on an Agilent 2100 Bioanalyzer. Sample libraries are multiplexed and sequenced on a NextSeq 500 platform (Illumina) using 75bp single-end sequencing. On average, about 20 million reads were generated from each sample.

value: 130 days


title: HD109d130-2

source_name: shi et al differentiation protocol for iPSCs- day 130


title: CTR18d130


title: Monocytes_PP


title: HD77d130


title: HD109d60-2


title: CTR33d100

source_name: Shi et al. differentiation protocol for iPSCs- day 100


title: HD109d80

source_name: Shi et al. differentiation protocol for iPSCs- day 80


title: CTR18d100


title: CTR21d100


title: HD77d100


title: HD77d80


title: HD109d60

source_name: Shi et al. differentiation protocol for iPSCs- day 60


title: HD77d60


title: CTR33d80


title: CTR18d80


title: CTR21d80


title: CTR33d60


title: CTR33d130


title: HD109d100


title: CTR18d60


title: HD109ipsc

source_name: Induced pluripotent stem cell colonies

value: Induced pluripotent stem cells (iPSCs)

value: 0 days


title: CTR33ipsc


title: HD77ipsc


title: CTR21d60


title: CTR21ipsc


title: CTR18ipsc


title: Treg10- 09-SpleenTregIL10neg

status: Public on Nov 13 2017

description: IL-10– splenic Treg cells

data_processing:
The raw reads were were aligned by Tophat v2.1.0 to the mouse GRCm38assembly with GENCODE M9 annotations downloaded fromhttp://www.gencodegenes.org/. RPKM (Reads Per Kilobase of transcript perMillion mapped reads) values were calculated for lncRNA and protein codinggenes with Cufflinks 2.2.1 software. RPKM levels below 1.0 were set to a “floor”value of 1.0.
Genome_build: GRCm38 assembly with GENCODE M9 annotations
Supplementary_files_format_and_content: xlsx files include RPKM values for each Sample calculated by Cufflinks

source_name: lung innate lymphoid type-2 cells

extract_protocol:
RNA was extracted and purified from lung (ILC2) or spleen (Treg) using the RNeasy Plus Mini Kit (Qiagen) per manufacturers instructions
Libraries of ILC2PBS and ILC2IL-33 samples were constructed using SMARTer Ultra Low Input RNA v3 kit (Clontech) as previously described. Briefly, the protocols for enzymatic fragmentation and ligation of the Ion Xpress Plus Fragment Library Kit (Life Technologies) were adjusted for our low-input total RNA of samples. Then resulting cDNA libraries were amplified onto Ion Sphere Particles with an Ion PI template OT2 200 Kit v3 (Life Technologies) and sequenced on an Ion Proton (Life Technologies) to generate an average depth of 20 million single-end 200bp reads, with less than 6.7% of the reads from ribosomal RNA and over 94% of the reads mapping to the mouse genome. Similarly, libraries of ILC2_act, ILC2_10, Treg_10- and Treg_10+ samples were constructed using SMARTer Ultra Low Input RNA v3 kit (Clontech), indexed using Nextera indices (Illumina) and sequenced on a NextSeq 500 (Illumina) using 75bp single-end sequencing kit per the manufacturer’s instructions. On average, about 10 million reads were generated from each sample.

value: IL-10- splenic Treg cells


title: ILC2act 07-10Bit22IL10neg

description: Thy-1.1– ILC2 cells

value: Thy-1.1- ILC2 ells


title: ILC210 04-10Bit26IL10pos

description: Thy-1.1+ ILC2 cells

value: Thy-1.1+ ILC2 cells


title: ILC2act 03-10Bit26IL10neg

description: Thy-1.1– ILC2 cells


title: ILC210 02-10Bit22IL10pos

description: Thy-1.1+ ILC2 cells


title: Treg10+ 06-10BitspleenIL10pos

description: IL-10+ splenic Treg cells

value: IL-10+ splenic Treg cells


title: Treg10- 05-10BitspleenIL10neg

description: IL-10– splenic Treg cells


title: ILC2IL-33 WT_ILC2_active

description: cells from mice injected with IL-33

value: cells from mice injected with IL-33


title: PP: EpCAM+ CCR10+

status: Public on Sep 04 2018

description: Single cell RNA sequencing of cultured IPF primary epithelial cells

data_processing:
The demultiplexed raw reads were aligned to the transcriptome using STAR (version 2.5.1) with default parameters, using a custom mm10 transcriptome reference from GENCODE M9 annotation, containing all protein coding and long non-coding RNA genes. Expression counts for each gene in all samples were collapsed and normalized to unique molecular identifier (UMI) counts using Cell Ranger software version 1.3.0 (10X Genomics). The result is a large digital expression matrix with cell barcodes as rows and gene identities as columns.
Genome_build: mm10
Supplementary_files_format_and_content: csv files include gene expression for each cell with or without normalization.

source_name: Explanted lung tissue

growth_protocol: Epithelial colonies were derived using CRC medium: 50% senescent lung fibroblast conditioned medium + 50% fibroblast complete medium (DMEM + 15% FBS + antiboitics) and 10 µM of Y27632. After approximately 2 weeks, cells were passaged and cultured overnight in Pneumacult Ex Plus medium (STEMCELL technologies) containing 10 µM Y27632.

extract_protocol: Single-cell RNA-seq libraries were prepared per the Single Cell 3′ v2 Reagent Kits User Guide (10x Genomics, Pleasanton, California). Cellular suspensions were loaded on a Chromium Controller instrument (10X Genomics) to generate single-cell Gel Bead-In-EMulsions (GEMs). GEM-RT were performed in a Veriti 96-well thermal cycler (Thermo Fisher Scientific, Waltham, MA). After RT, GEMs were harvested and the cDNAs were amplified, cleaned up with SPRIselect Reagent Kit.  Indexed sequencing libraries were constructed using Chromium Single-Cell 3′ Library Kit for enzymatic fragmentation, end-repair, A-tailing, adapter ligation, ligation cleanup, sample index PCR, and PCR cleanup. The barcoded sequencing libraries were quantified by quantitative PCR using the KAPA Library Quantification Kit (KAPA Biosystems, Wilmington, MA). Sequencing libraries were loaded on a NextSeq500 (Illumina, San Diego, CA) with a custom sequencing setting (26bp for Read 1 and 98bp for Read 2), to obtain a sequencing depth of 100K~200K reads per cell.

value: lung airway epithelial cells

value: EpCAM+ CCR10+

tag: processing

value: in vitro cultured and expanded

tag: diagnosis

value: Idiopathic Pulmonary Fibrosis


title: ILC2PBS ILC2_no_active_2

description: cells from mice injected with PBS

value: cells from mice injected with PBS


title: ILC2PBS ILC2_no_active_1

description: cells from mice injected with PBS


title: d21_SMA-Tm+

status: Public on Mar 29 2018

source_name: SMA-TdTomato+ cells from bleomycin treated d21 SMA-GFP;Tbx4cre;RosaTdTomato mice

extract_protocol:
Fluorescence- activated cell sorting (FACS) experiments were performed using fresh lung preparations. Mouse lung homogenates for single-cell flow cytometry were prepared as previously described (PMID 27400124). Briefly, fresh mouse lungs were perfused with 10 ml PBS, elastase (4 U/ml; Worthington Biochemical Corporation) were injected through the trachea to inflate the lung and dissociate epithelial cells. After that, samples were cut into approximately 1-mm3 pieces and digested with DNase I (100 U/ml; Sigma). Single cell homogenates were collected after passing through cell strainers and centrifugation. Flow cytometry was used to sort Epcam-, CD31-, and CD45- MCs. Primary antibodies to CD31, and CD45, and secondary antibody anti-streptavidin were all from eBioscience (San Diego, CA). Mouse anti-EpCAM (G8.8, catalog 118215) were from BioLegend (San Diego, CA). 7-AAD was from BD Biosciences (San Diego, CA). Singlet discrimination was sequentially performed using plots for forward scatter (FSC-A versus FSC-H) and side scatter (SSC-W versus SSC-H). Dead cells were excluded by scatter characteristics and viability stains. All FACS experiments were performed on an Aria III sorter (BD Immunocytometry Systems, San Jose, CA) at the Cedars-Sinai Medical Center Shared FACS Facility and FACS data were analyzed using FlowJo software (TreeStar, Ashland, OR).
Single-cell RNA-seq libraries were prepared per the Single Cell 3′ v2 Reagent Kits User Guide (10x Genomics, Pleasanton, California). Cellular suspensions were loaded on a Chromium Controller instrument (10X Genomics) to generate single-cell Gel Bead-In-EMulsions (GEMs). GEM-RT were performed in a Veriti 96-well thermal cycler (Thermo Fisher Scientific, Waltham, MA). After RT, GEMs were harvested and the cDNAs were amplified, cleaned up with SPRIselect Reagent Kit.  Indexed sequencing libraries were constructed using Chromium Single-Cell 3′ Library Kit for enzymatic fragmentation, end-repair, A-tailing, adapter ligation, ligation cleanup, sample index PCR, and PCR cleanup. The barcoded sequencing libraries were quantified by quantitative PCR using the KAPA Library Quantification Kit (KAPA Biosystems, Wilmington, MA). Sequencing libraries were loaded on a NextSeq500 (Illumina, San Diego, CA) with a custom sequencing setting (26bp for Read 1 and 98bp for Read 2), to obtain a sequencing depth of 100K~200K reads per cell.
10X single cell RNA-seq

value: mouse lung


title: d21_SMA+Tm+

source_name: SMA+TdTomato+ cells from bleomycin treated d21 SMA-GFP;Tbx4cre;RosaTdTomato mice


title: d21_SMA-Tm-

source_name: SMA-TdTomato- cells from bleomycin treated d21 SMA-GFP;Tbx4cre;RosaTdTomato mice


title: d0_SMA-TM+

source_name: SMA-TdTomato+ cells from normal SMA-GFP;Tbx4cre;RosaTdTomato mice


title: d0_SMA+TM+

source_name: SMA+TdTomato+ cells from normal SMA-GFP;Tbx4cre;RosaTdTomato mice


title: PN: EpCAM+ CCR10-

value: EpCAM+ CCR10-


title: ILC2act 01-10Bit22IL10neg

description: Thy-1.1– ILC2 cells


title: d0_SMA-TM-

source_name: SMA-TdTomato- cells from normal SMA-GFP;Tbx4cre;RosaTdTomato mice


title: ILC2IL-33 Expand_ILC2

description: cells from mice injected with IL-33


title: Treg10+ 10-SpleenTregIL10pos

description: IL-10+ splenic Treg cells


title: ILC210 08-10Bit22IL10pos

description: Thy-1.1+ ILC2 cells


title: ILC2PBS nA_ILC2

description: cells from mice injected with PBS


title: C1-48h

status: Public on Jun 13 2017

data_processing:
Raw reads obtained from RNAseq were aligned to the transcriptome using STAR (version 2.2.1) / RSEM (version 1.2.20) with default parameters, using a custom human GRCh38 transcriptome reference downloaded from http://www.gencodegenes.org, containing all protein coding and long non-coding RNA genes based on GENCODE version 23 annotation. Expression counts for each gene in all samples were normalized by a modified trimmed mean of the M-values normalization method and the unsupervised principal component analysis (PCA) was performed with DESeq2 Bioconductor package version 1.10.1 in R version 3.2.2. Each gene was fitted into a negative binomial generalized linear model and Wald test was applied to assess the differential expressions between two sample groups by DESeq2. Benjamini and Hochberg procedure was applied to adjust for multiple hypothesis testing and differential expressed gene candidates were selected with a false discovery rate less than 0.05. For visualization of coordinated gene expression in samples, a two-way hierarchical clustering with Pearson correlation distance matrix was performed with samples and differential expressed gene candidates using the Bioconductor g-plots package (version 2.14.2) in R. The pathway enrichment analysis was performed on these candidates with DAVID (https://david.ncifcrf.gov/summary.jsp) and Ingenuity IPA.
Genome_build: custom human GRCh38 transcriptome reference downloaded from http://www.gencodegenes.org, containing all protein coding and long non-coding RNA genes based on GENCODE version 23 annotation.
Supplementary_files_format_and_content: text files include raw counts for each Sample calculated by RSEM

source_name: First trimester trophoblast cells

growth_protocol: The immortalized human first trimester trophoblast cell line HTR8/SVneo (a gift from Dr. C. Graham; Queen’s University, Kingston, Ontario, Canada), was maintained in RPMI 1640 with Hepes and L-Glutamine (Life Technologies, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin in a humidified environment (5% CO2 and 95% air) at 37oC. HTR8/SVneo cells were plated on 6-well plates at a concentration of 2.5 x 105 cells/ml and cultured until they reached ~70% confluency.

extract_protocol:
RNA was extracted after siRNA transfection as previously described using the RNeasy Minikits (Qiagen ) and RNA concentration was quantified using spectroscopy (Eppendorf BioPhotometer ). Total RNA was quantified using both the NanoDrop to assess sample contamination by proteins or carryover reagents from RNA isolation, and the Qubit fluorometer (Invitrogen ). Samples were then qualified using the Fragment Analyzer (Advanced Analytical Technologies, Ankeny, IA) that analyzes the integrity of the total RNA by measuring the ratio between the 18S and 28S ribosomal peaks.
RNAseq libraries maintaining strand-specificity were made using 1ug of material per sample with the Illumina TruSeq Stranded mRNA kit (Illumina ), according to the manufacturer’s instructions. Final libraries were again quantitated with the Qubit fluorometer and checked for size via the Fragment Analyzer. Libraries were diluted to 4nM and pooled in equal volumes for denaturation, hybridization, and sequencing on the Illumina NextSeq 500 (Illumina ) with single end 75bp sequencing chemistry. On average, about 20 million reads were generated from each sample.

treatment_protocol: HTR8/SVneo cells were plated in 6-well plates at a concentration of 2.5 x 105 cells/ml and cultured until ~70% confluent. They were transfected with 50nM of Silencer Select GATA3 siRNA (4392420; Thermo Fisher Scientific, Grand Island, NY) or nonsense scrambled siRNA (4390843; Thermo Fisher), using Lipofectamine 2000 Transfection Reagent (Life Tech) and Opti-MEM I Reduced Serum medium (Life Tech), according to the manufacturer’s instructions. Transfection and gene knockdown efficiency was assessed by RT-qPCR.

tag: sirna-induced mrna knockdown

value: scrambled control siRNA


title: G2-48h

value: GATA3 siRNA


title: G3-48h


title: G1-48h


title: p53_knock_out (d30_p53_7)

status: Public on Nov 22 2016

description:
d30_p53_7
processed data file: low_input_RNA_d30_fpkm.txt

data_processing:
We used the Basespace RNA-Seq Alignment app, which uses the TopHat (Bowtie2) aligner. We then used Cufflinks Assembly and DE to get the significantly differentially expressed genes between wt and p53 samples
Genome_build: mm10 from Basespace
Supplementary_files_format_and_content: [.txt] table includes fpkm values for each Sample calculated by tophat

growth_protocol: the mice were 8-12 weeks old

extract_protocol:
RNA was isolated using the RNeasy Micro Kit (Quiagen)
Total RNA was quantified using both the NanoDrop to assess sample contamination by proteins or carryover reagents from RNA isolation, and the Qubit fluorometer (Invitrogen). Samples were then qualified using the Fragment Analyzer (Advanced Analytical Technologies, Ankeny, IA) that analyzes the integrity of the total RNA by measuring the ratio between the 18S and 28S ribosomal peaks. Libraries were constructed using SMARTer Ultra Low Input RNA v3 kit (Clontech), indexed using Nextera indices (Illumina). Final libraries were again quantitated with the Qubit fluorometer and checked for size via the Fragment Analyzer. Libraries were diluted to 4nM and pooled in equal volumes for denaturation, hybridization, and sequencing on an Illumina NextSeq 500 (Illumina) with single end 75bp sequencing chemistry.

treatment_protocol: GFP+, EpCAM+,CD31/34/45-,7AAD- cells were sorted from either Scgb1a1-CreERTM; Rosa26-mT/mG; p53+/+ or Scgb1a1-CreERTM; Rosa26-mT/mG; p53flox/flox mice at 70 days after tamoxifen exposure

value: lung


title: p53_knock_out (d30_p53_8)

description:
d30_p53_8
processed data file: low_input_RNA_d30_fpkm.txt


title: C2-48h


title: wild_type (d30-wt-6_S2)

description:
d30-wt-6_S2
processed data file: low_input_RNA_d30_fpkm.txt

value: wild type


title: wild_type (d30_wt_5_S10)

description:
d30_wt_5_S10
processed data file: low_input_RNA_d30_fpkm.txt


title: C3-48h


title: p53_knock_out (d30_p53_10)

description:
d30_p53_10
processed data file: low_input_RNA_d30_fpkm.txt


title: WT-H7-1-C46_S34

data_processing:
Raw reads obtained from RNA-Seq were aligned to the transcriptome using Bowtie (version 1.1.1) / RSEM (version 1.2.20) with default parameters
Expression counts for each gene in all samples were normalized by the total number of reads mapped to the transcriptome in order to account for sequencing depth (transcripts per million; TPM) with the correction of size factors derived from ERCC spike-in.
We imposed additional quality control criteria to remove low quality cells: 1), raw sequencing reads <1M; 2) among all sequencing reads, percentage of total mapped read is <50%; 3) among mapped reads, percentage of exonic reads <50% and 4) percentage of ERCC spike-in reads>40% to eliminating majority of the dying or dead cell.
The expression counts were transformed into log scale. To identify the subpopulation among the samples in an unsupervised way, principle component analysis (PCA) was performed to select most varied genes across all single cells based on their eigenvectors with FactoMineR v1.31 in R/Bioconductor v3.2. The top 250 most varied genes from PCA was clustered with Euclidean distance matrix with ggplots2 v1.0.1 in R/Bioconductor.
Genome_build: a custom reference containing both 92 ERCC sequences and the mouse transcriptome GRCm38 reference downloaded from http://www.gencodegenes.org, containing all protein coding and long non-coding RNA genes based on Release M3 annotation.
Supplementary_files_format_and_content: tab-delimited text files include tpm values for each Sample calculated by RSEM

growth_protocol:
Five thousand flow sorted epithelial cells were cultured with one hundred thousand mouse lung fibroblasts and Matrigel (BD Biosciences) seeded onto Transwell filter inserts (BD Biosciences) as previously described (Chen et al., 2012). Briefly, the epithelial/fibroblast mixture was added to an equal volume of growth factor reduced Matrigel (BD Biosciences) and seeded to the apical surface of 24-well transwell filter inserts (BD Biosciences) placed in 24-well flat-bottom culture plates. The solution was allowed to polymerize for 30 min at 37°C, then basic medium was added to the basal compartment of the well. Cell cultures were maintained for 7-14 days at 37ºC in a humidified incubator (5% CO2). Colony-forming efficiency was calculated as the percentage of seeded cells that give rise to colonies, imaged on a Zeiss Axiovert40 fluorescent microscope and quantitated using FIJI.
Scgb1a1-CreERTM; Rosa26-mT/mG and Scgb1a1-CreERTM; Rosa26R-Confetti mice were previously described (Farin et al., 2015; PMID 25564721). These mice were crossed to p53flox (JAX stock number 008462), p53-/- (JAX stock number 002101), or Super p53 (kindly provided by David Kirsch) mice to generate experimental animals. Mice were injected with 3 x 200mg/kg, 1 x 250mg/kg, or 1 x 5mg/kg body weighttamoxifen in corn oil for high dose, RNA-Seq, and low dose experiments respectively. All mice were maintained and treatments were carried out according to IACUC approved protocols.

extract_protocol:
Cell capture, lysis, reverse transcription and cDNA amplification was performed on the C1 IFC for mRNA-seq on Fluidigm C1 Single-Cell Auto Prep System following the manufacturer’s protocol. Medium sized C1 mRNA-Seq chips were used to capture each cell-cycle fraction. The C1 Auto-prep system captured the dissociated single cells across 96 wells and performed cell lysis, cDNA synthesis with reverse transcription and PCR reaction using the SMARTer Ultra Low Input RNA Kit. Cells captured across the 96 wells are manually inspected as a quality control measure to remove empty well, doublets or debris containing wells. 92 ERCC spike-ins of known concentration were added during the lysis step to assess and quantify technical variation. cDNA from several representative cells were checked by High Sensitivity DNA chips using Fragment Analyzer (Advanced Analytical).
Libraries for each of the 96 captured cells were prepared using the Illumina Nextera XT DNA sample preparation kit with 96 dual barcoded indices. Single cell libraries were multiplexed and sequenced across 4 lanes of a NextSeq 500 platform (Illumina) using 75 single-end sequencing. On average, about 6-7 million reads would be generated from each single cell library.

treatment_protocol: BrdU was injected at a concentration of 50mg/kg body weight at 12 and 2 hours before sacrifice. Sorted epithelial cells were fixed and stained using the FITC BrdU Flow Kit (559619, BD Biosciences). Cells were analyzed on an LSRFortessa (BD Biosciences) flow cytometer. After 7 days in vitro, cultures were treated with 5U/mL dispase in Ham’s F12 and incubate at 37°C for 30 minutes. Halfway through incubation, cultures were disrupted by pipetting. Cells were collected and resuspended in 0.05% trypsin and incubated at 37°C for 30 minutes. Samples were vortexed several times during incubation. Cells were then filtered through a 70μm filter stained with 7AAD and sorted for GFP+7AAD-. One thousand sorted epithelial cells were cultured with one hundred thousand mouse lung fibroblasts in 50% Matrigel as described previously.


title: WT-H8-1-C47_S17


title: WT-H3-1-C45_S49


title: WT-H12-1-C94_S10


title: WT-H2-1-C44_S66


title: Ctrl_Teff_1

status: Public on Sep 14 2017

hyb_protocol: Standard Illumina hybridization protocol

scan_protocol: Standard Illumina scanning protocol

data_processing: For data preprocessing, Illumina Genome Studio software was used to export expression intensity values and the significance of intensity over background signal for each probe of each sample. Genes identified as expressed over background in any two samples were retained for further analysis.Data was normalized by quantile normalization and differential expression analysis was performed by using R package ‘limma’(v3.30.11). Differential expressed genes with p-value < 0.05 and have at least 1.5 fold relative average difference between Ctrl and CKO signals were used to draw heat map. Ingenuity Pathway Analysis (IPA) software was employed to perform comparison analysis of diseases and bio functions (-log10 p value cut-off 1.3).

source_name: pooled Spleen, mesenteric lymph nodes and large intestines lamina propria

label_protocol: Biotinylated cRNA were prepared with the Ambion MessageAmp kit for Illumina arrays

extract_protocol: Qiagen RNAeasy kits (Qiagen)

treatment_protocol: in vitro 4 hours stimulation with PMA and ionomycin

tag: age

value: 8-12 weeks

value: Teff

tag: genotype/variation

value: Ctrl


title: WT-H1-1-C43_S65


title: Blimp1_CKO_pTreg_1

tag: cell type

value: pTreg

value: Blimp1 CKO


title: Blimp1_CKO_pTreg_3

tag: strain


title: Blimp1_CKO_Teff_2


title: Blimp1_CKO_pTreg_2


title: Ctrl_pTreg_3


title: wild_type (d30_wt_1)

description:
d30_wt_1
processed data file: low_input_RNA_d30_fpkm.txt


title: Ctrl_Teff_3


title: Ctrl_pTreg_2


title: Blimp1_CKO_Teff_3


title: Ctrl_Teff_2


title: Ctrl_pTreg_1


title: Blimp1_CKO_Teff_1


title: WT-G9-1-C42_S8


title: WT-F6-1-C79_S33


title: WT-G7-1-C40_S25


title: WT-G3-1-C39_S40


title: WT-G8-1-C41_S7


title: WT-G12-1-C88_S73


title: WT-G2-1-C38_S57


title: WT-F9-1-C36_S71


title: WT-F3-1-C33_S31


title: WT-F7-1-C34_S16


title: WT-G1-1-C37_S56


title: WT-F8-1-debrisvalve-C35_S70


title: WT-E9-1-C30_S62


title: WT-E6-1-C73_S24


title: WT-F12-1-C82_S64


title: WT-F2-1-C32_S48


title: WT-F1-1-C31_S47


title: WT-F4-1-C81_S14


title: WT-F10-1-C84_S54


title: WT-E7-1-C28_S6


title: WT-E2-1-C26_S39


title: WT-E1-1-C25_S38


title: WT-E10-1-C78_S45


title: WT-E3-1-C27_S22


title: WT-F11-1-C83_S19


title: WT-E12-1-C76_S55


title: WT-D7-1-C24_S69


title: WT-D1-1-C21_S29


title: WT-D11-1-C71_S9


title: WT-D5-1-C68_S41


title: WT-D6-1-C69_S15


title: WT-C3-1-C13_S13


title: WT-C4-1-C61_S4


title: WT-E8-1-C29_S61


title: WT-D2-1-C20_S30


title: WT-C7-1-C18_S60


title: WT-C2-1-C14_S21


title: WT-C12-1-C66_S46


title: WT-C1-1-C15_S20


title: WT-D9-1-C22_S53


title: WT-B8-1-C11_S43


title: WT-D10-1-C70_S36


title: WT-C9-1-C16_S44


title: WT-B6-1-C57_S68


title: WT-B7-1-C12_S51


title: WT-B5-1-C56_S32


title: WT-C6-1-C63_S5


title: WT-E5-1-C74_S50


title: WT-B4-1-C55_S67


title: WT-B2-1-C08_S12


title: WT-B11-1-C59_S72


title: WT-C8-1-C17_S52


title: WT-A9-1-C04_S26


title: WT-B10-1-C58_S27


title: WT-A5-1-C50_S23


title: WT-A4-1-C49_S58


title: WT-B1-1-C09_S11


title: WT-A2-1-C02_S2


title: WT-A3-1-C01_S3


title: WT-A6-1-C51_S59


title: WT-A7-1-C06_S42


title: WT-A12-1-C54_S28


title: WT-B9-1-C10_S35


title: WT-A1-1-C03_S1


title: p53-KO-OffChip-Pos-Ctrl_S36


title: WT-A10-1-C52_S18


title: p53-KO-H7-1-debris-C46_S66


title: p53-KO-H8-1-C47_S67


title: p53-KO-OffChip-Neg-Ctrl_S45


title: p53-KO-H9-1-C48_S60


title: p53-KO-H2-1-C44_S38


title: p53-KO-H3-1-C45_S39


title: WT-B12-1-C60_S37


title: p53-KO-H1-1-C43_S46


title: p53-KO-G9-1-C42_S52


title: WT-A11-1-C53_S63


title: p53-KO-G8-1-C41_S59


title: p53-KO-F5-1-C80_S57


title: p53-KO-G2-1-C38_S29


title: p53-KO-F2-1-3valve-C32_S20


title: p53-KO-E9-1-C30_S34


title: p53-KO-G4-1-C87_S13


title: p53-KO-E8-1-C29_S42


title: p53-KO-F9-1-C36_S43


title: p53-KO-E5-1-C74_S49


title: p53-KO-G3-1-C39_S30


title: p53-KO-E3-1-C27_S12


title: p53-KO-E6-1-1valve-C73_S23


title: p53-KO-F3-1-C33_S21


title: p53-KO-E2-1-C26_S11


title: p53-KO-E12-1-C76_S27


title: p53-KO-E1-1-C25_S37


title: p53-KO-D8-1-C23_S33


title: p53-KO-D5-1-C68_S40


title: p53-KO-D9-1-C22_S25


title: p53-KO-D2-1-C20_S2


title: p53-KO-D12-1-C72_S18


title: p53-KO-D4-1-C67_S4


title: p53-KO-G7-1-C40_S58


title: p53-KO-D3-1-C19_S3


title: p53-KO-D11-1-C71_S53


title: p53-KO-D1-1-C21_S28


title: p53-KO-C9-1-C16_S16


title: p53-KO-F8-1-C35_S51


title: p53-KO-C7-1-C18_S41


title: p53-KO-C6-1-C63_S14


title: p53-KO-C2-1-C14_S62


title: p53-KO-C4-1-C61_S64


title: p53-KO-C3-1-C13_S63


title: p53-KO-C5-1-C62_S31


title: p53-KO-C12-1-C66_S9


title: p53-KO-F7-1-C34_S50


title: p53-KO-C8-1-C17_S24


title: p53-KO-C11-1-C65_S44


title: p53-KO-C10-1-C64_S17


title: p53-KO-B8-1-C11_S15


title: p53-KO-B7-1-C12_S32


title: p53-KO-B6-1-C57_S5


title: p53-KO-B5-1-C56


title: p53-KO-B4-1-C55_S56


title: p53-KO-D10-1-C70_S26


title: p53-KO-B4-1-C55


title: p53-KO-B11-1-C59_S35


title: p53-KO-B1-1-C09_S10


title: p53-KO-B3-1-C07_S55


title: p53-KO-A8-1-C05_S6


title: p53-KO-B5-1-C56_S22


title: p53-KO-B2-1-C08_S54


title: p53-KO-B12-1-C60_S69


title: p53-KO-B10-1-C58_S8


title: p53-KO-A6-1-C51_S65


title: p53-KO-A4-1-C49_S48


title: p53-KO-A3-1-C01_S47


title: p53-KO-A12-1-C54_S61


title: p53-KO-B9-1-C10_S7


title: p53-KO-C1-1-C15_S19


title: p53-KO-A1-1-C03_S1


title: p53-KO-A10-1-C52_S68


title: Dmel MSL1 female

status: Public on Apr 25 2016

city: New York

first: Friederike

last: Dündar

state: New York

address: 1300 York Ave

department: Physiology & Biophysics

institute: Weill Cornell Medicine

data_processing:
Basecalls performed using CASAVA version 1.8.2
Sequenced reads were aligned to the genome (version dm3 for D. melanogaster samples; version Dvir1.2 for D. virilis samples) using bowtie (v2.0.5) with standard parameters.
Reads with mapping quality < 5 were discarded.10 bp) using the bamCoverage module of the deepTools suite (https://deeptools.github.io; PMID: 247994360
Peak calling was done with MACS2 (v. 2.0.10).
Genome_build: dm3; Dvir1.2
Supplementary_files_format_and_content: Bigwig files were generated using the bamCoverage module of deepTools, normalizing to 1x coverage with the parameters -f 200 --binSize 10 --normalizeTo1x [158736537 for D.mel | 189205863 for D. vir]

organism: Drosophila melanogaster

source_name: salivary glands

growth_protocol: Fly strains were reared at room temperature with standard medium. D. virilis stock number (UCSD): 15010-1051.00

extract_protocol:
Chromatin was obtained from male 3rd instar larvae salivary glands as previously described (PMID: 16547175). In brief, collected salivary glands (10 per ChIP) were fixed, sonicated for 8 min (pulsed eight times for 30 sec, paused for 30 sec, high) in a Bioruptor (Cosmo Bio), and chromatin immunoprecipitated with the respective antibody.
Libraries were prepared according to Illumina's instructions

tag: developmental stage

value: third instar

tag: Sex

value: male

tag: chip antibody

value: polyclonal MSL1 antibody raised against the N-terminus (AA1-584)


title: wk3_scrambled sponge_-IL7_36h_rep1

status: Public on Jan 01 2016

city: Innsbruck

first: Sebastian

last: Herzog

address: Innrain 80-82

department: Institute of Developmental Immunology

country: Austria

institute: Medical University of Innsbruck, Biocenter

description:
gene expression upon IL-7 withdrawal (36h)
wk3_scrambled sponge_-IL7_36h_1

hyb_protocol: 1.65 µg of each Cy3-labeled cRNA was fragmented and prepared for one-color based hybridisation (Gene Expression Hybridisation Kit, Agilent Technologies). cRNA samples were hybridized for 17 hours at 65°C on a Sureprint G3 Mouse Gene Expression Microarray (Agilent Technologies, AMADID 028005). Afterwards, microarrays were washed with increasing stringency using Geene Expression Wash Buffers (Agilent Technologies) followed by drying with acetonitril.

scan_protocol: Fluorescent signal intensities were detected with Scan Control 8.4.1 Software (Agilent Technologies) on the Agilent DNA Microarray Scanner.

data_processing: Raw data were extracted from images using Feature Extraction 10.7.1.1 Software (Agilent Technologies). Data analysis was performed using the Subio software package (Subio, Japan).

source_name: wk3_scrambled sponge_-IL7_36h

label_protocol: 100 ng total RNA per sample were used for an RT-IVT reaction. Total RNA was reverse transcribed into cDNA and then converted into labelled cRNA by in-vitro transcription (Low Input Quick-Amp Labeling Kit One-Color; Agilent Technologies) incorporating Cyanine-3-CTP according to the manufacturer's instructions. RNA absorbance ratio, cRNA concentration and Cy3 dye concentration were checked with the NanoDrop ND-1000 UV-VIS spectrophotometer. Furthermore, the quality of labeled non-fragmented cRNA was analyzed with the 2100 Bioanalyzer (Agilent Technologies).

growth_protocol: wk3 cells were cultured in Iscove’s medium containing 7.5% FCS (Biochrom Superior), 100 U/ml penicillin, 100 U/ml streptomycin (PAN) and 50 µM 2-ME supplemented with excessive IL-7 obtained as a supernatant of IL-7-expressing J558L cells. Following infection with retroviral vectors, transduced cells were selected with puromycin at a concentration of 1 µg/ml for 3-4 days.

extract_protocol: Total RNA was prepared using the RNeasy RNA Purification kit (Qiagen) including on-column DNase digestion according to the manufacturer's protocol. RNA was quantified using a NanoDrop-1000 spectrophotometer (Peqlab) and integrity was monitored with the Agilent 2100 Bioanalyzer (Agilent Technologies).

treatment_protocol: wk3 cells selected for expression of a scrambled sponge as a control or a vector encoding a miR-15 sponge designed to target all miR-15 family members (miR-15a, miR-15b, miR-16, miR-497, miR-195, miR-322) were cultured in excessive IL-7 or without IL-7 for 36 hours to induce pre-B cell differentiation.

tag: cell line

value: pre-B cell

tag: transduced with

value: control vector scrambled sponge

tag: cultured

value: without IL-7 for 36 h


title: Myc/Bcl2+ GMP-derrived myeloid leukemia (rep 3)

status: Public on Sep 01 2016

data_processing: Gene level normalized expression signals were calculated from Affymetrix CEL files using the RMA algorithm as implemented in the R/Bioconductor package affy.

source_name: myeloid leukemic splenocytes

growth_protocol: We used 4-8-week-old recipient mice that received 4.5–6 Gy total body irradiation. 2x104–1´106 malignant cells were intravenously injected. Leukocytosis with blast counts >10% in peripheral blood derived from donor cells indicated acute leukemia (20% bone marrow infiltration). If recipient animals showed physical signs of full-blown leukemia (hind limb paralysis, loss of body weight, palpable spleen or submandibular lymph nodes), they were analyzed.

extract_protocol: The RNAeasy Kit (Qiagen) was used for extraction of RNA according to the supplier’s instructions. DNAse I digestion was performed as part of the protocol to remove residual DNA from the RNA preparation. RNA was stored at -80 °C.

tag: genotype

value: wildtype


title: Myc/Bcl2+ DN2-derrived myeloid leukemia (rep 2)


title: wk3_miR-15 sponge_-IL7_36h_rep2

description:
gene expression upon IL-7 withdrawal (36h)
wk3_miR-15 sponge_-IL7_36h_2

source_name: wk3_miR-15 sponge_-IL7_36h

value: miR-15 sponge (designed to target all miR-15 family members)


title: Myc/Bcl2+ GMP-derrived myeloid leukemia (rep 2)


title: wk3_miR-15 sponge_+IL7_rep1

description:
gene expression in the presence of IL-7
wk3_miR-15 sponge_+IL7_1

source_name: wk3_miR-15 sponge_+IL7

value: in excessive IL-7


title: DN2 (rep 2)

source_name: healthy thymic DN2 cells


title: wk3_scrambled sponge_+IL7_rep2

description:
gene expression in the presence of IL-7
wk3_scrambled sponge_+IL7_2

source_name: wk3_scrambled sponge_+IL7


title: Myc/Bcl2+ DN2-derrived myeloid leukemia (rep 5)


title: GMP (rep 2)

source_name: healthy bone marrow GMP cells


title: Myc/Bcl2+ DN2-derrived myeloid leukemia (rep 3)


title: Myc/Bcl2+ GMP-derrived myeloid leukemia (rep 4)


title: GMP (rep 1)


title: wk3_scrambled sponge_-IL7_36h_rep2

description:
gene expression upon IL-7 withdrawal (36h)
wk3_scrambled sponge_-IL7_36h_2


title: wk3_miR-15 sponge_-IL7_36h_rep1

description:
gene expression upon IL-7 withdrawal (36h)
wk3_miR-15 sponge_-IL7_36h_1


title: Myc/Bcl2+ DN2-derrived myeloid leukemia (rep 1)


title: wk3_scrambled sponge_+IL7_rep1

description:
gene expression in the presence of IL-7
wk3_scrambled sponge_+IL7_1


title: Myc/Bcl2+ GMP-derrived myeloid leukemia (rep 5)


title: Myc/Bcl2+ LSK-derrived myeloid leukemia (rep 3)


title: Myc/Bcl2+ LSK-derrived myeloid leukemia (rep 2)


title: Pam + TSA 4hours stimulation rep1

status: Public on Jan 01 2011

city: Geneva

first: FRANCOIS

last: Patrice

state: Ge 4

address: Rue Gabrielle-Perret-Gentil, 4

department: Service of Infectious Diseases

country: Switzerland

institute: Genomic Research Laboratory

description:
Mus musculus BMDM cRNA11 vs cRNA pool UMRR after 4 hours cell growth in presence of Pam + TSA replicate 1/2
cRNA23

hyb_protocol: A mixture of sample cRNA and pool RNA was diluted in 50 µl Agilent hybridization buffer, and hybridized at a temperature of 60°C for 17 hours in a dedicated hybridization oven

scan_protocol: 100% PMT using Agilent scanner

data_processing: Local background-subtracted signals were corrected for unequal dye incorporation or unequal load of labelled product. The algorithm consisted of a rank consistency filter and a curve fit using the default LOWESS (locally weighted linear regression) method

source_name: BMDM Pam + TSA 4hours stimulation rep1

label_protocol: First cDNA Synthesis from Total RNA then fluorescent cRNA Syntesis: in vitro transcription and incorporation of cyanine 5-CTP

extract_protocol: Fixation followed by RNeasy purification

tag: gender

value: female

value: BALB/c

value: BM-derived macrophage

value: 12 weeks

tag: agent

value: Pam + TSA

tag: time

source_name: Universal mouse RNA mixture (UMRR)

tag: reference pool

value: Universal mouse RNA mixture


title: 1676 control_+IL-7_rep2

status: Public on Jan 01 2015

description: gene expression in the presence of IL-7

source_name: mouse pre-B cell line, control vector, +IL7, replicate 2

growth_protocol: 1676 cells were cultured in Iscove’s medium containing 7.5% FCS (Biochrom Superior), 100 U/ml penicillin, 100 U/ml streptomycin (PAN) and 50 µM 2-ME supplemented with excessive IL-7 obtained as a supernatant of IL-7-expressing J558L cells. Following infection with retroviral vectors, transduced cells were selected with puromycin at a concentration of 1 µg/ml for 3-4 days.

treatment_protocol: 1676 cells selected for expression of a control vector or a vector encoding hsa-miR-125b were cultured in excessive IL-7 or without IL-7 for 24 hours to induce pre-B cell differentiation.


title: Myc/Bcl2+ GMP-derrived myeloid leukemia (rep 1)


title: Myc/Bcl2+ LSK-derrived myeloid leukemia (rep 1)


title: 1676 control_+IL-7_rep1

description: gene expression in the presence of IL-7

source_name: mouse pre-B cell line, control vector, +IL7, replicate 1


title: DN2 (rep 1)


title: Myc/Bcl2+ DN2-derrived myeloid leukemia (rep 4)


title: wk3_miR-15 sponge_+IL7_rep2

description:
gene expression in the presence of IL-7
wk3_miR-15 sponge_+IL7_2