title: HiC Neutrophils replicate 1

status: Public on Feb 06 2017

city: La Jolla

first: Yina

last: Zhu

state: CA

address: 9500 Gilman Drive

department: Biological Sciences

institute: university of california,san diego

description: genomic interactions

library_source: genomic

data_processing:
The paired-end Hi-C reads were truncated at GATCGATC site, aligned separately to the mm9 genome using BOWTIE2, only keeping read pairs if both ends mapped to unique locations. The two reads were merged into pairs using HOMER (http://biowhat.ucsd.edu/homer/). Read alignment filtering and other Hi-C processing including normalization was performed using HOMER. Reads passing these filters are included in the Hi-C summary files. Principle component analysis of normalized Hi-C interaction frequency matrices was performed using HOMER and R. Hi-C statistics was processed using HiCUP(http://www.bioinformatics.babraham.ac.uk/projects/hicup/).
For ChIP-Seq, MeDIP-Seq and GRO-Seq samples (single end reads), reads were aligned to the mm9 genome using BOWTIE2, keeping only reads that mapped unique locations. Aligned read files were analyzed with HOMER to find peaks, generate bedGraph, calculate RPKM from the gene bodies of RefFlat genes, and perform other analyses in the study.
RNA-Seq reads were aligned to the mm9 reference genome using Tophat2. RPKMs for each RefFlat gene were calculated from aligned reads using Cufflink. For a gene to be considered expressed the cutoff of FPKM is 0.5.
Genome_build: mm9
Supplementary_files_format_and_content: Processed files include BED files (ChIP-Seq peak or region positions, Hi-C PC1 domain positions), FKPM CSV files (RNA-Seq expression data), RPKM CSV files (GRO-Seq expression data), BEDGRAPH (ChIP-Seq, duplicates-combined GRO-Seq and MeDIP-Seq tracks, principle component values along the genome for combined Hi-C data), and TXT files, including Hi-C summary files (filtered read pairs from combined Hi-C data processed using HOMER; filtered read pairs from each Hi-C replicates processed using HiCUP), columns: Read Name, chromosome for read 1, positions for read 1 (5' end of read, one-indexed), strand of read 1, chromosome for read 2, positions for read 2 (5' end of read, one-indexed), strand of read 2), as well as Hi-C background files from HOMER.

molecule: genomic DNA

source_name: in-vitro differentiated neutrophils

growth_protocol:
GM-CSF-dependent ECOMG cells were grown in RPMI 1640 with 10% FBS and penicillin-streptomycin-glutamine, by the addition of 1:100 conditioned media (about 10 ng/mL GM-CSF) isolated from a B16 melanoma cell line stably transfected with a murine GM-CSF construct. b-estradiol where applicable, was added to the medium at a final concentration of 1 mM from a 10,000 × stock in 100% ethanol. Differentiation of ECOMG cells into granulocytes was induced by withdrawal β-estradiol for 5 days.
C57BL/6J mice were housed in specific pathogen-free conditions in accordance with the Institutional Animal Care and Use Committee of University of California, San Diego. Bone marrow derived neutrophils were separated by EasySepTM Mouse Neutrophil Enrichment Kit (Stem Cell Technologies, 19762). Bone marrow cells were harvested from femur, tibia and crista iliac. Bone marrow cells were incubated with a cocktail of biotinylated lineage specific antibodies (EasySepTM Mouse Neutrophil Enrichment Cocktail), conjugated with EasySepTM Biotin Selection Cocktail, labeled with EasySepTM D Magnetic Particles, followed by depletion of biotin-labeled cells with EasySep™ Magnet.

extract_protocol:
The in-situ Hi-C experiments were performed similarly as described (Rao SSP, et al. 2014. A 3D map of the human genome at kilobase resolution reveals principles of chromatin looping. Cell 159: 1665-1680).
The GRO-Seq experiments were performed as described (Core LJ, Waterfall JJ, Lis JT. 2008. Nascent RNA sequencing reveals widespread pausing and divergent initiation at human promoters. Science 322: 1845-8).
The ChIP-Seq experiments were performed similarly as described (Lin YC, et al. 2012. Global changes in the nuclear positioning of genes and intra- and interdomain genomic interactions that orchestrate B cell fate. Nat Immunol 13: 1196-1204).
The MeDIP-Seq experiments were performed similarly as described (Pomraning KR, Smith KM, Freitag M. 2009. Genome-wide high throughput analysis of DNA methylation in eukaryotes. Methods 47: 142-150).
For RNA-Seq: total RNA was isolated using RNeasy Mini kit (Qiagen). RNA was treated with TURBO DNase (Life Technologies). mRNA was purified from total RNA using Dynabeads mRNA purification kit (Life Technologies).cDNA was generated with the First-strand synthesis kit (Life Technologies) using a combination of random hexamers and oligo(dT) in presence of actinomycin D. Second-strand synthesis was performed with dUTP instead of dTTP. The ds-cDNA was sonicated to 200-400 bp using the S220 Focused-ultrasonicator (Covaris).
Hi-C libraries were prepared with the NEBNext primer set and were selected by size by 6% PAGE and sequenced for 50 cycles on Illumina HiSeq 2500.
GRO-Seq libraries were prepared with custom GRO-Seq PCR primers and were selected by size by 8% PAGE and sequenced for 50 cycles on Illumina HiSeq 2000.
ChIP-Seq and MeDIP-Seq libraries were prepared with the NEBNext primer set and were selected by size by 8% PAGE and sequenced for 50 cycles on Illumina HiSeq 2000 or 2500.
RNA-Seq ibraries were prepared with the TruSeq primer set and were selected by size by 8% PAGE and sequenced for 50 cycles on Illumina HiSeq 2000.

tag: cell type

value: Ecomg derived neutrophils

tag: genotyping

value: human E2A_ER_Pbx1b


title: HiC Progenitors replicate 1

type: SRA

country: USA

description: genomic interactions

organism: Mus musculus

source_name: undifferentiated myeloid progenitor cells

value: Ecomg myeloid progenitor cell line

tag: strain


title: HiC Progenitors replicate 6

description: genomic interactions


title: HiC Neutrophils replicate 7 PCR replicate 1

description: genomic interactions


title: HiC Progenitors replicate 7

description: genomic interactions


title: RHC3555

status: Public on Mar 20 2017

city: Singapore

first: Huipeng

last: Li

address: 60 Biopolis St, #02-01

country: Singapore

institute: Genome Institute of Singapore

description: processed data file = All_CRC_tumor_cells_FPKM.csv

library_source: transcriptomic

data_processing:
Raw FASTQ files were processed with Tophat-2.1.0 for alignment. Tophat parameters were tuned for single cell data. Namely, "--read-edit-dist" was set to 3, "--read-realign-edit-dist" was set to 0. hg19 was used as the genome reference. The "max-multihits" parameter was set to 1 so that only uniquely mapped reads were exported to the aligned BAM file.
Gene abundances were quantified as fragments per kilobase of transcript per million mapped reads (FPKM) using Cuffdiff-2.2.1, with "--frag-bias-correct" parameter enabled, "--library-norm-method" set to be "classic-fpkm". To remove the systematic scaling effect of short RNAs on the FPKM values of the whole transcriptome, we masked out rRNAs, tRNAs, snRNA and snoRNAs by introducing a mask file for the "--mask-file" option of Cuffdiff. Raw count values were also exported by Cuffdiff for the QC analysis.
Genome_build: hg19
Supplementary_files_format_and_content: comma separated files include raw FPKM values of all cells that passed QC

molecule: polyA RNA

organism: Homo sapiens

source_name: CRC01_Colorectal Tumor

growth_protocol: Cell lines: H1 human ES cell line was grown in mTESR medium (STEMCELL). GM12878 were grown in suspension in RPMI 1640 supplemented with 2mM L-glutamine and 15% fetal bovine serum. A549, HCT116, K562, IMR90 and H1437 cell lines were cultivated in DMEM medium supplemented with 10% fetal bovine serum and 2mM L-glutamine. Cell lines were maintained at 37 degrees, in 5% CO2 and 95% air controlled humid chamber.

extract_protocol:
Dissociated single cells were stained for viability assessment using Calcein AM and Ethidium-Homodimer-1 (Live/Dead Kit, Lifetech) before being loaded onto RNAseq IFC-C1 chips (5-10 and 10-17µm size chips). RNAseq assay was done according to the manufacturer's instructions (Fluidigm). Briefly, dissociated single cells optimal buoyancy was achieved by mixing them with Cell Suspension Buffer and Cell Wash Buffer before loading onto the IFC chips. Once the cell capture was achieved, all the IFC were imaged using  Nikon microscope. Based on bright field and Calcein AM imaging, only single cells were selected for subsequent library preparation and sequencing analysis. RNAseq protocol was conducted according to the manufacturer indications, onto C1 machines using SMARTer® Kit Designed for the C1TM System (Clontech). Briefly, after cell lysis, mRNA was isolated and converted to cDNA using modified oligo dT primers. PCR based pre-amplification of the cDNA was achieved using the Advantage 2 PCR kit (Clontech) for 21 cycles (C1 protocol). cDNAs were subsequently harvested for each single cell, individually quantified using Quant-ITTM PicoGreen® dsDNA Assay Kit (Lifetech) and checked for quality using High Sensitivity DNA Chips on a Bioanalyzer instrument (Agilent Technologies).  RNAseq assays were identically conducted from cell lines: A549 (Human Lung carcinoma cell line, ATCC), GM12878 (human B-Lymphoide cancer cell line, Coriell Institute), H1437 (Human Lung adenocarcinoma cancer cell line, ATCC), IMR90 (human fetal lung fibroblast cell line, ATCC), H1 (human embryonic cell line), K562 (human immortalised myelogenous leukemia cell line, ATCC). For GM12878 and H1 ESC cell lines, two independent experiments were conducted.
Library prep were prepared only with selected single cells showing correct cDNA concentration and quality. We used Nextera XT DNA Sample Preparation Kit (illumina) in combination with Nextera XT DNA Sample Preparation Index Kit (96 indices) (Illumina) to complete individual library preparation, according to manufacturer's instruction. We subsequently pooled up to 96 libraries for clean up using Agencourt AMPure® XP (Agencourt Bioscience Corp). Pooled and multiplexed libraries were processed on HiSeq HighOutput v3000 for 101-paired-end sequencing (Illumina).

treatment_protocol: Tumor tissues were obtained from patients diagnosed with colorectal cancer, after written informed consent was obtained, from the National Cancer Center of Singapore, Singapore General Hospital. Patients (n=11) diagnosed with stage 2-4 Colorectal Carcinoma (CRC) were selected for this study. Primary tumors were surgically removed and kept in sterile and cold RPMI medium until processing. Normal Mucosa (NM) was sampled far from neoplastic tissue. Fresh resected tissues were processed as soon as possible after surgery. Briefly, samples were minced on a plate and enzymatically digested with Collagenase IV (Gibco) and DNAse I (Worthington) for 30 min at 37 degrees celcius, with agitation. After digestion, samples were sieved on a 100µm cell strainer, washed with PBS/BSA 1%/EDTA 2mM and centrifuged for 8 min at 500g. The washing  step was repeated using a 40µm cell strainer disposal. Pelleted cells were then resuspended in PBS/EDTA 2mM and assessed for viability and size in a Countess instrument (Lifetech). Cells were counted and resuspended in Cell suspension buffer (Fluidigm) and loaded onto IFC-C1 chips as per manufacturer recommendations.

tag: patient id

tag: sample type

value: Primary CRC patient tissue sample

tag: tissue type

value: Colorectal Tumor

tag: primary tissue sample id

tag: patient gender

value: Female

tag: tnm

tag: tumor grade

tag: stage ajcc

value: IIC


title: HiC Neutrophils replicate 2

description: genomic interactions


title: HiC Neutrophils replicate 7 PCR replicate 2

description: genomic interactions


title: WT_polyA-mRNA_rep2

status: Public on Oct 27 2016

city: Colorado Springs

first: Andrew

middle: David

last: Klocko

state: Colorado

address: 278 Centennial Hall, 1420 Austin Bluffs Pkwy

department: Chemistry and Biochemistry

institute: University of Colorado Colorado Springs

data_processing:
Paired-end reads were demultiplexed, trimmed to 50bp (if needed)
Hi-C contact maps were produced according to Imakaev et al. (Imakaev et al. 2012). Briefly, an attempt was made to uniquely map the first 15 base pairs (bp) of each read to the Neurospora genome. If a unique match was not found, the read was extended 5 bp and another attempt was made. This process continued until a unique match was found or the end of the read reached (50 bp).
Read pairs were then assessed using a number of quality control filters, typically removing reads that covered unligated products (Dangling Ends), products that were too close to the restriction site, Self-circles, and extremely large or small fragments.
Read pairs passing all filters (typically 10% of total read pairs) were used to build uncorrected contact maps (arrays) that were then corrected to account for the unequal representation of genomic loci in HiC datasets. Corrected maps were used in all subsequent analyses
To calculate the ratio of observed contacts to expected contacts, the median contact frequency was calculated at each genomic distance (up to a distance of ~3.5 Mb at which point contact frequency had reached background levels) and used as the expected contact frequency at each respective distance.
To directly compare datasets, a fixed number of reads (prior to filtering) were sampled with the program seqtk, such that the sampled number of raw reads from each dataset produced approximately equal numbers of reads that passed the filtering steps.
To make ChIPseq enrichment files across 10kb bins to use for Circos plots, .bcf files were produced using samtools' mpileup function from ChIPseq .bam files, the .bcf files were "fixed" to replace empty positions with zeros, and processed with a custom script to report the ChIP-seq enrichment over 10kb bins (the resulting seven files were catenated into one file).
All python scripts (".py" files) used for Hi-C data processing are available as supplemental information in the manuscript Galazka et al, 2016, Genome Research, except for the modified scripts, plotIntraTelomeres.py and plotInterTelomeres.py, which were used to calculate intra and inter telomeric enrichment in our Hi-C datasets. In addition, the python script K9-K27Quant.py counts the number of Hi-C links originating from a H3K9me3-marked 10kb bin and end in a bin marked by H3K27me2/3, while the python script K9-K27only_Quant.py counts the number of Hi-C links originating from a H3K9me3-marked 10kb bin and end in a bin marked ONLY by H3K27me2/3 (and not H3K9me3). The program count_links_K27marked.py counts the number of Hi-C links in each 10Kb bin marked by H3K27me2/3. The program hicEigenvectorAnalysis.py derives the number of principle components for the whole genome specified using non-iteratively corrected Hi-C .hdf5 datasets. The program hicEigenvector_singleLG.py extracts the Eigenvector of a single Linkage Group from the whole genome data derived from the "hicEigenvectorAnalysis.py" script. The program hicPearsonCorrelationHeatmap.py calculates the Pearson Correlation between all genomic regions and displays them as a heatmap for a specific Linkage Group. These scripts are provided here.
To determine differences in the genome organization caused by mutants, mutants were compared to the WT Hi-C data (NMF39 [GSM1825702] within the GEO dataset GSE71024). See GSM2186732 in this GSE82222 dataset.
To make ChIPseq enrichment files for display in Integrative Genomics Viewer (IGV, Robinson et al, 2011, Nat. Biotechnol.), .bam files (and the corresponding .bai index files) were counted with the "Count" command of igvtools, using the mean window function and 200bp windows. Output file was a .tdf file, displayable on IGV.
For Poly-A mRNA seq, high quality, adapter-less reads were identified (Stacks); Kmer filtering reduced sequencing errors. Reads were mapped (TopHat) to the modified Neurospora genome assembly 12, and sorted (SAMTools), and directionality-preserved read numbers were calculated at each gene ID (HTSeq), adding one read count to prevent abnormal fold changes. Differential gene expression analysis (DESeq) examined replicate differences and pair-wise analyses between WT and mutants. Output .bed files of genes with log2 ≥ 2 or log2 ≤ -2 expression and adjusted p-values ≤ 0.05 were modified as .txt files for Circos plot display.
Genome_build: Neurospora crassa assembly 12 Fixed (files: neurospora_crassa_or74a_12_genome_FIXED.fasta, and neurospora_crassa_or74a_12_transcripts_FIXED.gtf)
Supplementary_files_format_and_content: for Hi-C files: "...array.txt" files are the generated tab-delininated files with raw counts (on a log2 scale) for each bin (the entire genome is divided into 40kb, 50kb, or 10kb segments, and a bin indicates the intersection of two of these segments in an array format) across the genome. "...observed_expected.txt" is a file where the above array file containing observed interactions in our HiC dataset was normalized by the "expected" interaction if interactions were locally strong and stochastically decreased as genomic distance increased.
Supplementary_files_format_and_content: For ChIP-seq files: The tdf files were generated using the "count" function in igvtools (Integrative Genomics Viewer; Broad Institute, Robinson et al, 2011), using a window size of 200bp (.tdf files are binary files that shows enrichment peaks for each ChIP sample and have been processed for faster display of the data in IGV). The bcf files were produced from bam files (mapped to the Neurospora crassa assembly 12 Fixed by samtools), and report enrichment values.
Supplementary_files_format_and_content: For RNA-seq files: the .txt files were generated by the program HTseq (using sequencing reads aligned to the Neurospora crassa assembly 12 Fixed genome with the program samtools). These HTseq.txt files report read number counts per gene for each replicate sample (two replicates total) from our four strains (WT N3752, delta set-7 N4718 and N4730, delta npf N4721, delta set-7;delta dim-5 N5916). The three Dataset.xls files were produced using the replicate HTseq.txt files in the program DESeq to determine genes that were differentially expressed > four fold and were significant (p < 0.05) in each mutant strain when compared to the WT N3752 reference expression datasets.

organism: Neurospora crassa

source_name: germinated conidia

growth_protocol: 2.5x10^8 conidia were grown in cultures of 50mL 1xVogels, 1.5% Sucrose medium for 3-4 hours at 30oC with shaking at 200rpm (At this time, >70% of the conidia for all strains had germination tubes >4x their original size, as roughly measured by light microscopy)

extract_protocol:
[HiC] To determine the concentration of genomic DNA (gDNA) per aliquot of spheroplasts, 1 aliquot per replicate was resuspended in 200 μL decrosslinking buffer (50 mM Tris-HCl [pH 8], 5 mM Na Ethylenediaminetetraacetic acid (Na-EDTA), 0.5% (w/v) sodium dodecyl sulfate (SDS)) plus 100 mg proteinase K and incubated at 65 °C overnight. The next day, the volume was brought to 500 uL with TE buffer (10 mM Tris-HCl [pH 8], 1 mM Na-EDTA) plus 40 mg RNase A and aliquots were incubated at 37°C for 30 minutes. gDNA was then extracted twice with a 1:1 mixture of buffered phenol (pH 8) and chloroform, and once with chloroform. gDNA was precipitated by adding 1/10 volume of Na acetate (pH 5.2) and 1 volume of isopropanol. DNA pellets were washed 3x with 70% (v/v) ethanol, dried at RT for 10 minutes and resuspended in 50 μL TE. The DNA concentration was determined from the absorbance at 260 nm. Spheroplasts corresponding to 3.5 μg of gDNA were resuspended in 270 μL of HindIII buffer and 30 μL of 10% (w/v) SDS was added to a final concentration of 0.1% (w/v). Spheroplasts were then lightly decrosslinked by incubation at 65°C for 10 minutes and then placed immediately on ice. 33 μL of 10% (v/v) Triton X-100 and 8.3 μL of 10x HindIII buffer was added and gDNA digested with 400 U (20 μL of 20 U/μL from NEB) HindIII for 16 hours at 37 °C while nutating. The next day, samples were placed on ice and split into two aliquots. To construct HiC libraries, one aliquot of HindIII digested chromatin was labeled with biotin-14-dCTP in a 210 μL reaction consisting of 30 μM dATP, 30 μM dTTP, 30 μM dGTP, 30 μM biotin-14-dCTP and 25 U of Klenow (large fragment). This reaction was incubated at 37 °C for 45 minutes with nutating. Klenow (large fragment) and HindIII was then inactivated by the addition of SDS to 1.5% (w/v) and incubation at 65°C for 30 minutes. This chromatin was then used in a 3.5 mL ligation reaction consisting of 50 mM Tris-HCl [pH 7.5 @ 25 °C], 10 mM MgCl2, 0.9% (v/v) Triton X-100, 10 mM Dithiothrireitol (DTT), 100 μg/mL BSA, 1 mM adenosine triphosphate (ATP) and 1675 U T4 DNA ligase (4.2 μL of 400,000 U/mL T4 DNA ligase from NEB). This reaction was incubated at 16°C for 4 hours. At the end of the 4 hour 3C and HiC ligation reactions, 250 mg of proteinase K was added and all reactions were allowed to decrosslink at 65°C overnight. The next day, another 250 mg of proteinase K was added and samples were decrosslinked at 65°C for another 2 hours. Samples were cooled to ~25°C on ice, and extracted with 5 mL of phenol (pH 8) by vortexing for 2 minutes. After centrifugation (3500 RPM, 10 minutes), the aqueous phase was removed and extracted with 5 mL of phenol (pH 8):chloroform (1:1) by vortexing for 2 min. After centrifugation (3500 RPM, 10 minutes), the aqueous phase was removed and brought to a volume of 5 mL with TE. DNA was precipitated by the addition of 0.5 mL 3 M Na Acetate [pH 5.2], 50 mg glycogen, 12.5 mL 95% ethanol and incubation at -80°C for 1 hour. DNA was pelleted by centrifugation (20,000 g, 20 minutes, 4°C), the supernatant decanted and DNA resuspended in 450 μL TE by vortexing. DNA was again extracted with 1 volume of phenol (pH 8):chloroform (1:1) and then chloroform as before. DNA was then ethanol precipitated as before and resuspended in 25 μL of TE/10 buffer (10 mM Tris-HCl [pH 8 @ 25°C], 0.1 mM Na-EDTA). To remove biotin-dCTP from unligated ends, the HiC libraries were digested for 2 hours at 12°C in a 50 μL reaction consisting of 5 μL of NEB buffer 2, 100 μg/mL BSA, 100 μM dATP, 100 μM dGTP and 5 U of T4 DNA polymerase. These reactions were quenched by adding Na-EDTA to 10 mM and DNA extracted with 1 volume of phenol (pH 8):chloroform and then with 1 volume of chloroform. DNA was ethanol precipitated, resuspeneded in 500 μL of TE and sheared by sonication (duty cycle 80, power 1.2, 10 s, 5x with 1 minute rest on ice between pulses). Sheared, biotinylated DNA was captured by adding 25 μL of streptavidin beads (Invitrogen) that had been equilibrated in BW buffer (5 mM Tris-HCl [pH 8 @ 25°C], 0.5 mM Na-EDTA, 1 M NaCl, 0.05% (v/v) Tween-20) and incubating for 30 minutes with shaking. Beads were washed 2x with BW buffer, 2x with TE/10 buffer and resuspended in 25 μL TE/10 buffer.
[HiC] To prepare HiC libraries for Illumina sequencing, either Illumina TruSeq kits was used according to the manufacturers’ protocol, or essentially a “homemade version” of that protocol was used. In the homemade version, DNA ends were repaired in reactions consisting of 25 μL of HiC libraries on streptavidin beads, 5 μL of T4 DNA ligase buffer, 2 μL 10 mM (each) dNTPs, 0.5 μL end repair enzyme mix (0.5 U T4 DNA polymerase, 0.25 U Klenow Fragment, 2.25 U T4 DNA Polynucleotide Kinase) in a final volume of 50 μL and incubated at 20°C for 30 minutes. Beads were washes 2x with BW buffer and 2x with TE/10, resuspended in 16.5 μl TE/10. A-tailing was performed by adding 2 ul 10x NEB Buffer #2, 1 ul 4 mM dATP, 0.5 ul Klenow 3’ to 5’ exo minus (5 U/ul) and incubating at 37 C for 30 minutes, mixing every 10 minutes. Beads were again washed with BW and TE/10 buffers as above and resuspended in 20 ul TE/10. Indexed adaptors were ligated by adding 25 ul 2x ligase buffer (132 mM Tris-HCl [pH 7.6], 20 mM MgCl2, 2 mM DTT, 2 mM ATP, 15% (w/v) PEG 6000), 2.5 ul T4 DNA ligase (400 U/ul) and 2.5 ul Illumina TruSeq DNA adapters diluted 1:10 and incubating at RT for 20 minutes, mixing every 10 minutes. Ligation reactions were quenched with 5 ul of 0.5 M Na-EDTA [pH 8] and beads washed 3x with BW buffer and 2x with TE/10. Beads were then resuspended in 50 ul TE/10 and HiC libraries amplified in 50 ul PCR reactions using the Phusion polymerase and the following cycle: 45 s at 98°C; 18 cycles of 15 s at 98°C, 30 s at 63°C, 30 s at 72°C; 1 minute at 72°C. The PCR product was separated from the beads, cleaned with Ampure XP beads, and resuspended in 25 ul TE/10. Library quality was assessed by gel electrophoresis before deeps sequencing.
[ChIP] Cultures were grown in Vogels medium and 1.5% sucrose (standard minimal medium), shaking for 16 hours at 32oC. Mycelia were washed in PBS prior to crosslinking. Mycelia were cross-linked for 10 min in 0.5% formaldehyde for histone modification ChIP.  Tissue added to ChIP lysis buffer (50mM Hepes pH 7.5, 90mM NaCl, 1mM EDTA, 1% triton + proteinase inhibitors) and was disrupted by sonication for thirty pulses before chromatin was sheared using a Bioruptor (Diagenode) for 15 min with a cycle of 30 sec on followed by 30 sec off, at high power. Lysates were cleared by centrifugation, 1/20th Input was saved, and histone modification antibody was added (typically 1uL) and samples were rotated overnight at 4oC. The next day, equilibrated Protein A or Protein G slurry (agarose or magnetic beads) was added to bind the antibody, incubated for 3 hours at 4oC, and washed twice in cold ChIP Lysis buffer, once with ChIP Lysis buffer + 0.5M NaCl, once with LiCl was buffer (10mM Tris pH 8.0, 250mM LiCl, 1mM EDTA, 0.5% NP40), and DNA/protein was eluted with TES buffer (50mM Tris pH 8.0, 10mM EDTA, 1% SDS) and incubated at 65oC.  Samples (IP and input) were decrosslinked at 65oC overnight.  The next day, samples were proteinase K treated for 2 hours at 50oC, and DNA was purified using the QIAquick PCR purification kit  and eluted in 30 μl of water.  Approximately 10 ng of DNA was used to generate ChIP-Seq libraries.  Each library was prepared using the Illumina Tru-Seq kits A and B (Illumina, IP-202-1012 and IP-202-1024) according to the manufacturer’s instructions.  “Invisible” fragments between 250-400 bp were excised and purified using the MinElute gel extraction kit (Qiagen, 28606).  Final libraries were PCR-amplified using one cycle at 98 °C for 30 sec, 8 cycles at 98 °C for 10 sec, 60 °C for 30 sec and 72 °C for 30 sec and a final extension at 72 °C for 5 min.
[RNA] Cultures were grown in Vogels m

treatment_protocol: [HiC] 2.6 mL of methanol-free formaldehyde (20%) was added to a final concentration of 1%. After a 10 minute room temperature (RT) incubation with shaking at 100 RPM, crosslinking reactions were quenched by adding 3.4 mL of 2 M Tris-HCl [pH 8 @ 25 °C] to a final concentration of 125 mM and incubating at room temperature (RT) for another 10 minutes with shaking at 100 RPM. Conidia were harvested by centrifugation (3000 RPM, 5 minutes) and resuspended gently in 40 mL spheroplasting buffer (1 M sorbitol, 100 mM KPO4  [pH 7.5]) plus 30 mM β-mercaptoethanol, pelleted again (3000 RPM, 5 minutes) and resuspended in 40 mL spheroplasting buffer. 200 mg of VinoTaste Pro (Novozymes) was added and strains were spheroplasted for 60 minutes at 30°C with shaking at 100 RPM. Spheroplasts were harvested by centrifugation (2000 RPM, 5 minutes) and washed 3x with 20 mL HindIII buffer (50 mM NaCl, 10 mM Tris-HCl [pH 7.9 @ 25°C], 10 mM MgCl2, 100 μg/mL bovine serum albumin (BSA)). Washed pellets were resuspended in 6 mL HindIII buffer, split into 1 mL aliquots in 1.7 mL eppendorf tubes. Spheroplasts were pelleted (1500 g, 5 minutes) and the supernatant was aspirated away. Aliquots were frozen in liquid nitrogen and stored at -80°C.

tag: genotype/variation

value: wild type

tag: tissue

value: mycelia


title: WT_polyA-mRNA_rep1


title: NIH 3T3 MEF genomic DNA

status: Public on Jan 08 2016

city: Seattle

first: Jie

last: Yao

state: WA

address: 615 Westlake Ave N

institute: Allen Institute

data_processing:
Basecalls performed using CASAVA version 1.8.
Reads were aligned to mm9 by Bowtie2 using the default setting.
Only one copy of uniquely mapped reads was retained.
Genome_build: mm9
Supplementary_files_format_and_content: A bedGraph file presents uniquely mapped reads.

source_name: NIH 3T3 mouse embryonic fibroblasts (MEF)

growth_protocol: Cells were grown on 10 cm plates to a confluency of 80% in appropriate growth media.

extract_protocol:
Genomic DNA was isolated using Dneasy blood and tissue kit (Qiagen).
Genomic DNA was fragmented by NEBNext® dsDNA Fragmentase, then subject to end repair, addition of "A" bases, ligation of Illumina adaptors, size selection of libary and PCR enrichment of adaptor-modified DNA fragments according to the protocol provided by Yale Center for Genome Analysis (http://ycga.yale.edu/sequencing/Illumina/protocols.aspx)

treatment_protocol: No specific treatment was performed for this study.

tag: cell line

value: NIH 3T3

value: mouse embryonic fibroblast cell line

tag: culture media

value: DMEM high glucose + 10% newborn calf serum, 100 unit/ml penicillin and 100ug/ml streptomycin


title: HiC Progenitors replicate 2

description: genomic interactions


title: HiC Neutrophils replicate 3

description: genomic interactions


title: HiC Neutrophils replicate 5

description: genomic interactions


title: HiC Progenitors replicate 5

description: genomic interactions


title: HiC Neutrophils replicate 6

description: genomic interactions


title: HiC Progenitors replicate 4

description: genomic interactions


title: HiC Progenitors replicate 3

description: genomic interactions


title: All_samples_bulk_RNA-seq_TPM

status: Public on Aug 02 2016

city: Stanford

first: Rahul

last: Sinha

address: 265 Campus Drive

department: ISCBRM

institute: Stanford University School of Medicine

description: These files are matrices containing TPM values, where each row is a gene and each column a sample.

data_processing:
Sequenced reads were trimmed for adaptor sequences using skewer 0.1.127 with parameters -x AGATCGGAAGAGCACACGTCTGAACTCCAGTCACNNNNNNATCTCGTATGCCGTCTTCTGCTTG -y AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTAGATCTCGGTGGTCGCCGTATCATT -t 16 -q 21 -l 21 -n -u -f sanger for bulk-population RNA-seq and parameters -x CTGTCTCTTATACACATCTCCGAGCCCACGAGACNNNNNNNNATCTCGTATGCCGTCTTCTGCTTG -y CTGTCTCTTATACACATCTGACGCTGCCGACGANNNNNNNNGTGTAGATCTCGGTGGTCGCCGTATCATT -t 16 -q 21 -l 21 -n -u -f sanger for single-cell RNA-seq.
Trimmed reads were then mapped to hg38 using STAR 2.4 with parameters  --outSAMunmapped Within --outFilterType BySJout --outSAMattributes NH HI AS NM MD  --outFilterMultimapNmax 20 --outFilterMismatchNmax 999 --outFilterMismatchNoverReadLmax 0.04   --alignIntronMin 20   --alignIntronMax 1000000   --alignMatesGapMax 1000000  --alignSJoverhangMin 8   --alignSJDBoverhangMin 1 --sjdbScore 1 --twopassMode Basic --twopass1readsN -1 --outWigStrand Stranded
Normalized expression values (TPM) and transcript counts were then calculated using RSEM 1.2.21 with parameters --bam --estimate-rspd --seed 12345 --paired-end --forward-prob 0
Genome_build: hg38

molecule: total RNA

source_name: H7 ESC

extract_protocol:
Total RNA or chromatin was extracted as described below
Bulk RNA-seq: Total RNA from flow-sorted or differentiated cell populations was isolated using Trizol (ThermoFisher) as per manufacturer’s recommendation and was facilitated by addition of linear polyacrylamide (Sigma) as a carrier during RNA precipitation. Purified total RNA was treated with 4 units of RQ1 RNase-free DNase (Promega) at 37 dgrees Celsius for 1 hour to remove trace amounts of genomic DNA. The DNase-treated total RNA was cleaned-up using RNeasy micro kit (QIAGEN). Subsequently, the integrity of extracted RNA was assayed by on-chip electrophoresis (Agilent Bioanalyzer) and only samples with a high RNA integrity (RIN) value were used for subsequent cDNA preparation. Purified total RNA (10-50 ng) was reverse-transcribed into cDNA and amplified using the Ovation  RNA-seq System V2 (NuGEN). Amplified cDNA was sheared using the Covaris S2 (Covaris) using the following settings: total volume 120 l, duty cycle 10%, intensity 5, cycle/burst 100, total time 2 min. The sheared cDNA was cleaned up using Agencourt Ampure XP (Beckman Coulter) to obtain cDNA fragments >= 400 base pairs (bp). 500 ng of sheared and size-selected cDNA were used as input for library preparation using NEBNext Ultra DNA Library Prep Kit for Illumina (New England BioLabs) as per manufacturer’s recommendations. Resulting libraries (fragment distribution: 300-700 bp; peak 500-550 bp) were pooled (multiplexing) and sequenced using HiSeq 4000 or NextSeq 500 (Illumina) at the Stanford Functional Genomics Facility to obtain 2x150 base pair paired-end reads. For each RNA-seq library, the effectiveness of adapter ligation and the effective library concentration was determined by qPCR and bioanalyzer (Agilent) prior to pooling and loading them onto the sequencers. The reads obtained were trimmed for base call quality (PHRED score >= 21) and the presence of adapter sequences at the 3' end using Skewer (version: 0.1.127; Jiang et al, BMC Bioinformatics, 2014). Single-cell RNA-seq: Cells were briefly washed (DMEM/F12), dissociated (TrypLE Express), strained (100 micron filter), pelleted and resuspended in DMEM/F12 for counting. Before single-cell capture, two quality control steps were implemented. Firstly, cell size was estimated in order to determine whether cells should be loaded onto C1 capture arrays of either 10-17 micron or 17-25 micron size. Arrays were chosen for each lineage by estimating the median cell size of each given population on a flow cytometer on the basis of the FSC-W signal and choosing an array with an appropriate pore size to accommodate such cells. Secondly, to ensure the high viability of in vitro-differentiated cells prior to commencing single-cell RNA-seq, for each population a separate aliquot of cells was stained with 1.1 micromolar DAPI and analyzed by flow cytometry; for all cell populations that were used for single-cell RNA-seq, 98% of cells were viable (i.e., DAPI negative). For single-cell capture, cells were diluted to a concentration of 1000 cells/microliter, diluted 3:2 in C1 Cell Suspension Reagent, and loaded onto a Fluidigm C1 single-cell capture array chip for automated capture on a Fluidigm C1 Machine (Stanford Stem Cell Institute single-cell Core). After loading, the efficiency of single-cell capture was verified using an automated microscope that imaged each catured cell on the chip. The subsequent cell lysis, cDNA synthesis, and aplification was carried on the array chip inside the microfuidic chamber in an automated fashion using C1 machine using the SMARTer Ultra Low RNA Kit (Clontech, 634833) reagents as per the manufacturers' instructions (Fluidigm, PN 100-7168 Rev. A2). The amplified cDNA from individual cells was harvested into a 96 well and diluted using the harvesting reagent (Fluidigm). The concentration and integrity of amplified cDNA were assessed using a Fragment Analyzer (Advanced Analytical) in a 96 well plate format. Amplified cDNAs form only thos wells that (1) were not degraded, and (2) originated from wells that were microscopically verified to contain a single cell, were carried forward for subsequent library construction. Using a single-channel liquid handeling robot, Mosquito (TTP Labtech), amplified cDNAs from single-cells from all the lineages were diluted to a concentration range of 0.05-0.16 ng/microliter using C1 Harvest Reagent (Fluidigm) as a diluent and consolidated into 384 well plates. The diluted single-cell cDNAs were tagmented and converted to sequencing libraries in the 384 well plates using the Nextera XT DNA Sample Prep Kit (Illumina, FC-131-1096) in an automated fashion using another 16-channel Moquito robot (TTP Labtech) and 384 distinct illumina compatiblr molecular barcodes. The resulting sequencing libraies from a single such 384 plate were then pooled and cleaned up using Agencourt AMPure XP beads (Beckman Coulter). The pooled libraries were then analyzed for quality and concentration using bioanalyzer (Agilent) and qPCR and loaded on a single lane of NextSeq 500 or two lanes of Hiseq 4000 to obtain 1-2 million 2x150 reads per cell. The reads obtained were trimmed for base call quality (PHRED score >= 21) and the presence of adapter sequences using Skewer (version: 0.1.127; Jiang et al, BMC Bioinformatics, 2014). ATAC-seq: For each replicate, 50,000 cells were lysed in lysis buffer containing 0.01% IGEPAL CA-630 (Sigma, I8896) to obtain nuclei, which were directly used in the Tn5 transposition reaction (reagents from Nextera DNA Sample Preparation Kit; Illumina, FC-121-1030). Immediately following transposition, DNA fragments were purified (MinElute Kit, Qiagen) and PCR amplified for a total of 12-13 cycles using previously-designed primer sequences that include illumina compatible adapters and barcodes. The resulting ATAC-seq libraries were purified (MinElute Kit, Qiagen), pooled and final library-pool concentrations were assessed (Bioanalyzer) prior to next-generation sequencing. The quality of ATAC-seq libraries was confirmed by a shallow sequencing run using a MiSeq v3 (Stanford Functional Genomics Facility, 2x75bp reads) before deep sequencing was performed on a NextSeq 500 (2x75bp reads).

treatment_protocol: H7 hESC  Design: H7 human embryonic stem cells (WiCell) were cultured feeder-free in mTeSR1 (StemCell Technologies; supplemented with 1% penicillin/streptomycin) on a Geltrex basement membrane (Gibco), with mTeSR1 media changed daily.  Anterior Primitive Streak (APS)  Design: Partially confluent cultures of mTeSR1-maintained H7 human embryonic stem cells (WiCell) were dissociated into very fine clumps using Accutase (Gibco) and sparsely passaged ~1:12-1:20 onto new Geltrex-coated cell culture plates in mTeSR1 supplemented with 1 μM thiazovivin (Tocris; a ROCK inhibitor to prevent cell death after dissociation) overnight. The following morning, H7 human embryonic stem cells were briefly washed (in DMEM/F12) before treatment for 24 hours with 30 ng/mL Activin A + 4 μM CHIR99021 + 20 ng/mL FGF2 + 100 nM PIK90 in CDM2 basal media to induce differentiation into anterior primitive streak.  Mid Primitive Streak (MPS)  Design: Partially confluent cultures of mTeSR1-maintained H7 human embryonic stem cells (WiCell) were dissociated into very fine clumps using Accutase (Gibco) and sparsely passaged ~1:12-1:20 onto new Geltrex-coated cell culture plates in mTeSR1 supplemented with 1 μM thiazovivin (Tocris; a ROCK inhibitor to prevent cell death after dissociation) overnight. The following morning, H7 human embryonic stem cells were briefly washed (in DMEM/F12) before treatment for 24 hours with 30 mg/mL Activin A + 40 ng/mL BMP4 + 6 μM CHIR99021 + 20 ng/mL FGF2 + 100 nM PIK90 in CDM2 basal media to induce differentiation into mid primitive streak.  Paraxial Mesoderm (DLL1+)  Design: H7-derived day 1 anterior primitive streak was briefly washed (DMEM/F12) and differentiated towards day 2 paraxial mesoderm for 24 hours (1 μM A-83-01 + 3 μM CHIR99021 + 250 nM LDN-193189 + 20 ng/mL FGF2) in CDM2 basal medium. Subsequently, day 2 paraxial mesoderm-containing cultures were dissociated into single cells (TrypLE Express) and stained with an anti-DLL1 PE antibody (Biolegend) and DAPI (Biolegend). Then, viable DLL1+ paraxial mesoderm cells were purified using a fluorescence-activated cell sorter (FACSAria II, BD Biosciences).     Non-Paraxial Mesoderm (DLL1-)  Design: H7-derived day 1 anterior primitive streak was briefly washed (DMEM/F12) and differentiated towards day 2 paraxial mesoderm for 24 hours (1 μM A-83-01 + 3 μM CHIR99021 + 250 nM LDN-193189 + 20 ng/mL FGF2) in CDM2 basal medium. Subsequently, day 2 paraxial mesoderm-containing cultures were dissociated into single cells (TrypLE Express) and stained with an anti-DLL1 PE antibody (Biolegend) and DAPI (Biolegend). Then, viable DLL1- non-paraxial mesoderm cells were purified using a fluorescence-activated cell sorter (FACSAria II, BD Biosciences).  Early Somites  Design: H7-derived day 2 paraxial mesoderm was briefly washed (DMEM/F12) and further differentiated into day 3 early somites for 24 hours (1 μM A-83-01 + 250 nM LDN-193189 + 1 μM C59 + 500 nM PD0325901; sometimes with the use of 1 μM XAV939 in lieu of C59) in CDM2 basal medium.  Sclerotome  Design: H7-derived day 3 early somites were briefly washed (DMEM/F12) and further differentiated into day 5-6 ventral sclerotome for 48-72 hours (5 nM 21K + 1 μM C59) in CDM2 basal media.  Dermomyotome  Design: H7-derived day 3 early somites were briefly washed (DMEM/F12) and further differentiated into day 5 dermomyotome (3 μM CHIR99021 + 150 nM Vismodegib) for 48 hours, optionally with the addition of 50 ng/mL BMP4.  Central Dermomyotome  Design: H7-derived day 3 early somites were briefly washed (DMEM/F12), treated with 3 μM CHIR99021 + 150 nM Vismodegib + 50 ng/mL BMP4 for 24 hours, and then washed again (DMEM/F12) and subsequently treated with CHIR99021 (3 μM) + 21K (5 nM) for 24 hours to yield day 5 central dermomyotome.  Lateral Mesoderm  Design: H7-derived day 1 mid primitive streak was briefly washed (DMEM/F12) and differentiated towards day 2 lateral mesoderm for 24 hours (1 μM A-83-01 + 30 ng/mL BMP4 + 1 μM C59) in CDM2 basal medium.  Cardiac Mesoderm (GARP+)  Design: H7-derived day 2 lateral mesoderm was differentiated into day 4 cardiac mesoderm by briefly washing (DMEM/F12), treatment with 1 μM A8301 + 30 ng/mL BMP4 + 20 ng/mL FGF2 for 24 hrs followed by brief washing (DMEM/F12) and then treatment with 25 ng/mL Activin + 30 ng/mL BMP4 + 1 μM C59 for the next 24 hrs. Subsequently, day 4 cardiac mesoderm-containing cultures were dissociated into single cells (TrypLE Express) and stained with an anti-GARP APC antibody (Biolegend) and DAPI (Biolegend). Then, viable GARP+ cardiac mesoderm cells were purified using a fluorescence-activated cell sorter (FACSAria II, BD Biosciences)  D2.25 Somitomeres (single-cell RNA-seq)  Design: H7-derived day 2 paraxial mesoderm was briefly washed (DMEM/F12) and further differentiated into day 2.25 somitomeres for 6 hours (1 μM A-83-01 + 250 nM LDN-193189 + 1 μM XAV939 + 500 nM PD0325901) in CDM2 basal medium.

tag: source

value: H7 ESC

value: In vitro cultured H7 human embryonic stem cells (WiCell) and H7-derived downstream early mesoderm progenitors


title: H8083 Red1 ChIP in pREC8-SCC1 biological replicate 1

status: Public on Aug 06 2015

city: New York

first: Andreas

last: Hochwagen

state: NY

address: 1009 Silver Center 100 Washington Square East

institute: New York University

data_processing:
Sequencing reads were mapped to the S288C (sacCer2) genome using Bowtie unless specified otherwise, only perfect matches across all 51bp were considered during mapping. Multiple alignments were not taken into account, which means each read only mapped to one location in the genome. Reads were also mapped to the SK1 genome with similar results. Reads were extended towards 3’ ends to a final length of 200bp in MACS (http://liulab.dfci.harvard.edu/MACS/) (Zhang et al., 2008).
To identify axis protein enriched regions (peaks), the observed sequence read tag density along the chromosome was normalized against the measured background (INPUT DNA). Median coverage of all base positions in the ChIP sample was calculated. The coverage at each position was then divided by the calculated median. The same division was also performed for the input sample, followed by subtraction of the input from the ChIP sample at each position. Peak identification was performed using MACS (http://liulab.dfci.harvard.edu/MACS/) (Zhang et al., 2008) using a P-value of 1e-15. Peak summits were defined as the local maximum of read coverage within Red1 peaks.
Genome_build: SacCer2
Supplementary_files_format_and_content: Wiggle file: ChIP enrichment at each base position.

organism: Saccharomyces cerevisiae

growth_protocol: To induce synchronous meiosis, strains were inoculated at OD600 = 0.3 in BYTA medium for 16.5 hours at 30°C. Then cultures were washed twice with water and resuspended into SPO medium at OD600 = 1.9 at 30°C (details are described in (Blitzblau et al., 2012)).

extract_protocol:
25 ml of the meiotic culture was harvested and fixed for 30 min in 1% formaldehyde. The formaldehyde was quenched by addition of 125 mM glycine. Samples were processed as described in (Blitzblau and Hochwagen, 2013).
Library preparation was performed using Illumina TruSeq DNA Sample Prep Kits v1 but adaptors were used at 1:20 dilution. Amplified ChIP DNA between 300 and 500bp was gel purified on 1.8% agarose and sequentially quantified using the Qubit assay HS kit as well as an Agilent 2100 Bioanalyzer. Fragments were sequenced on an Illumina HiSeq 2500 instrument using a 51-bp single-end sequencing protocol.

tag: meiotic timepoint

value: 3 hour

tag: genotype

value: pREC8-SCC1::3HA, rec8 delta

tag: antibody

value: Anti-Red1 (S. Roeder)


title: 1KO plus DNMT1

status: Public on Sep 15 2016

city: Jefferson

first: Zhiguang

state: AR

address: 3900 NCTR Road

department: National Center for Toxicological Research

institute: Food and Drug Administration

data_processing:
Illumina Casava1.7 software used for basecalling.
For detailed mapping, mouse reference genome (UCSC mm9) was applied to two types of nucleotide replacement by changing C to T (C2T genome) or G to A (G2A genome) and indexes were made using bowtie-build. Reads also underwent nucleotide replacement by changing cytosine in read1 of pair-end sequencing to thymine and guanine in read2 to adenine. Mapping was done using bowtie (version 0.12.7) by allowing three mismatches in the leading 40 bases with up to 140 of phred score at mismatched positions. Twenty multiple alignments were allowed at most for each read, i.e. using k=20 when running bowtie program.
Monoclones were chosen by selecting only one pair of alignments with the maximum phred score from the pairs that have the same mapping positions for both ends.
The methylation statuses of cytosine were determined by considering read end types, DNA strand that the reads align against, nucleotide on reference genome, and nucleotide on reads.
Genome_build: mm9
Supplementary_files_format_and_content: Space-delimited text files include methylation scores for each C/G base. We use the following formula: S = rC / (rC + rT)  where rC is the number of reads that C still remains as C after bisulfite treatment, rT is the number of reads that C becomes T after bisulfite treatment.

source_name: ES cells

growth_protocol: Mouse ES Cells were maintained without feeder cells on 0.1% gelatin coated Petri dish in DMEM medium supplemented with 15% FBS (ES cell grade), 2mM glutamine, 10uM mercapto-ethanol, 100U/ml LIF, Penicillin/Streptomycin mixture 100ug/ml, 1X non-essential amino acid.

extract_protocol:
DNA was havest using DNeasy Blood & Tissue Kit(Qiagen, Germany)
One to five microgram of genomic DNA was fragmentized to 200~500bp by Covaris S2 sonicator (Covaris, USA). End repairing was then performed following manufacturer’s instruction (End-It DNA end repair kit, Epicentre, USA).  After Ampure XP (Sigma USA) purification, adenine was added to 3’ end with 3ml DNA Taq polymerase (M0267S, NEB) and 1mM dATP in 50ml reaction solution incubated at 70°C for 30 mins. After Ampure XP purification, 1ml of Illumina Trueseq adaptors were ligated with 4ml T4 DNA ligase (M0202L, NEB) in 40ml reaction solution and incubated at 16°C overnight. Fragments at 300-600 bp from adaptor-ligated DNA were collected from 2% agarose gel, and then bisulfite-treated using Imprint DNA modification Kit (MOD50-1 KIT, Sigma USA) as manufacturer instructed.  PCR enrichment was performed to amplify the libraries, which were then collected from 2% agarose gel electrophoresis at size 300-600bp.

treatment_protocol:
This cell,i.e.,"1KO plus DNMT1",was derived by transfecting a full functional DNMT1 construct into DNMT1/DNMT3a/DNMT3b triple knockout cells (TKO) kindly provided by Dr Masaki Okano (Tsumura A, Hayakawa T, Kumaki Y, Takebayashi S, Sakaue M, Matsuoka C, et al. Maintenance of self-renewal ability of mouse embryonic stem cells in the absence of DNA methyltransferases Dnmt1, Dnmt3a and Dnmt3b. Genes Cells. 2006;11:805-14.).
Since the DNMT1 gene is restored, this cell is now only DNMT3a and DNMT3b deficient.

tag: strain

tag: cell type

value: ES cell

tag: treatment

value: a DNMT1 construct was transfected into DNMT1/DNMT3a/DNMT3b triple knockout cells


title: YapS87A rep3

city: Milwaukee

first: Brian

last: Link

state: WI

address: 8701 Watertown Plank Rd

institute: Medical College of Wisconsin

data_processing:
RNA quality was determined using an Agilent BioAnalyzer.  50-bp single read sequencing was performed in triplicate for each genotype using an Illumina HiSeq2000 at VANTAGE (Vanderbilt University, Nashville, TN). Sequencing results were analyzed by VANGARD (Vanderbilt University, Nashville, TN). RNA-seq reads were mapped to D. rerio cDNA sequences from EnsEMBL release 66.
30M reads per sample
Genome_build: Danio rerio EnsEMBL release 66.
Supplementary_files_format_and_content: tab delimited txt include fkpm for each sample.

organism: Danio rerio

source_name: whole eye

extract_protocol:
Yap S87A and sibling control whole eyes were dissected at 36 hpf and immediately frozen on dry ice until ~60 pooled retinas were obtained for each genotype. RNA was purified as described in (Uribe et al., 2012) except that RNA was eluted in a 50μl final volume.
RNA quality was determined using an Agilent BioAnalyzer.  50-bp single read sequencing was performed in triplicate for each genotype using an Illumina HiSeq2000 at VANTAGE (Vanderbilt University, Nashville, TN). Sequencing results were analyzed by VANGARD (Vanderbilt University, Nashville, TN). RNA-seq reads were mapped to D. rerio cDNA sequences from EnsEMBL release 66.

tag: developmental stage

value: 36 hpf

value: whole eye


title: YapS87A rep2


title: WT rep3

value: vsx2:Gal4 sibling control


title: YapS87A rep1


title: HiC Neutrophils replicate 4

description: genomic interactions

value: Ecomg derived neutrophils

tag: genotyping

value: human E2A_ER_Pbx1b


title: WT rep2


title: WT rep1


title: H8083 Input biological replicate 2

source_name: Input of H8083 biological replicate 2


title: C2C12 myoblast genomic DNA

source_name: C2C12 myoblasts

value: mouse myoblast cell line

value: DMEM high glucose + 10% fetal bovine serum, 100 unit/ml penicillin and 100ug/ml streptomycin


title: H8083 Input biological replicate 1

source_name: Input of H8083 biological replicate 1


title: Input DNA

status: Public on Jun 12 2015

city: Le Kremlin-Bicêtre

first: Florian

last: Le Billan

address: 63, Gabriel Péri street

country: France

institute: INSERM

data_processing:
Illumina bcl2fastq 1.8.4 software used for basecalling.
ChIP-Seq data analysis was done using the Eoulsan software version 1.2.2. Before mapping, poly N read tails were trimmed, reads ≤11 bases were removed, and reads with quality mean ≤12 were discarded. Reads were then aligned against the hg19 reference genome using Bowtie mapper (version 0.12.9) using the "-n 2 -l 28 -e 70 -k 2 --best" parameters. Only one alignment was kept in a given locus for each read and reads alignments matching on more than one locus were removed.
Peaks were called using macs 1.4.2 with the following setting: -gs hs -m 5,100, the other parameters remaining the default ones
Genome_build: hg19
Supplementary_files_format_and_content: bed format containg relative enrichments: vehicle vs input (C11.5 vs C11.1), aldosterone vs input (C11.6 vs C11.1), aldosterone vs vehicle (C11.6 vs C11.5) and H3K4me3 vs input (C11.8 vs C11.1).

source_name: HK-GFP-MR cells_input

growth_protocol: Human Kidney GFP-hMR cells (HK-GFP-MR) were routinely cultured at 37°C, in an humidified incubator gassed with 5% CO2, seeded on petri dishes (BP100) with 10 mL of DMEM (DMEM High Glucose medium with L-glutamine) containing 2.5% fetal bovine serum (FBS) (Biowest, Courtaboeuf, France), 100X Penicillin Streptomycin. For the HK GFP-MR cells, the medium was supplemented with 200 µg/mL geneticin.

extract_protocol:
Treated cells were fixed with 1% paraformaldehyde (Electron Microscopy Sciences, Saint-Germain-en-Laye, France) for 10 min at RT. Fixation was quenched with 0.125 M glycine for 8 min. Cells were washed twice with ice-cold PBS. Cell lysis and chromatin shearing were carried out using the #HighCell ChIP kit buffers, according to manufacturer’s recommendations, with the following modifications; chromatin shearing was performed in 500 µL of shearing buffer S1 + Protease Inhibitor mix, applying 2 runs of 10 cycles at high intensity (Bioruptor®, Diagenode). Each cycle was composed of 30 s with effective application of ultrasounds (ON) and 30 s without (OFF). Between each run, heated water had to be removed and replaced by ice-cold water and ice. Concentrations of sheared chromatin protein were measured, using the bicinchoninic acid assay (Interchim, Montluçon, France) on a spectrophotometer Victor3® (Perkin Elmer, Courtabœuf, France), in parallel to a BSA standard range (Interchim).  Protein A-coated magnetic beads (25 µl of the stock suspension) were washed three times in buffer C1 and re-suspended in C1 buffer supplemented with 1% PIC (100 µl final volume). Sheared chromatin (total amount of 1.3 mg protein) and 5 µg of one of the tested antibodies were then added to the beads and the final volume was adjusted to 500 µL with C1 buffer supplemented with 1% PIC and 0,1% BSA (final concentrations). Samples were incubated overnight at 4°C using a rotary incubator. Magnetic beads were then isolated and washed three times with C1 buffer and once with W1 buffer. DNA fragments from the immunoprecipitated chromatin were eluted with the DNA Isolating Buffer (DIB) supplemented with 1% Proteinase K, for 15 min at 55°C and finally for 15 min at 100°C.  Reference for Antibody:  Viengchareun S., et al, Osmotic stress regulates mineralocorticoid receptor expression in a novel aldosterone-sentitive cortical collecting duct cell line, Mol Endo, 2009, doi:  10.1210/me.2009-0095.  In brief: Rabbit polyclonal anti-MR antibody (39N) was generated using the human MR 1 18 peptide and purified by affinity chromatography (Double X/XP boosting antibody production program, Eurogentec, Seraing, Belgium).
Library preparation and Illumina sequencing were performed at the Ecole normale superieure Genomic Platform (Paris, France). Libraries were prepared using NEXTflex ChIP-Seq Kit (Bioo Scientific), using 1 ng of IP or Input DNA. Libraries were multiplexed by 6 on 1 flow cell lane. A 50 bp read sequencing was performed on a HiSeq 1500 device.

treatment_protocol: HK-GFP-MR cells, 7-8.10+6 by BP100, maintened in steroid-free medium for 24h, was stimulated by 100 nM of aldosterone or by the same volume of vehicle, for 1h.

value: Human Kidney GFP-hMR cells

tag: passage

tag: chip antibody


title: H8083 Red1 ChIP in pREC8-SCC1 biological replicate 2


title: Hs_CTCF_X_ChIP_(20141204_25)

first: Jorja

last: Henikoff

address: 1100 Fairview AV N, A1-162

department: Basic Sciences

institute: Fred Hutchinson Cancer Research Center

description:
High-resolution X-ChIP-seq for CTCF was performed in human K562 celss using a specific antibody against CTCF.
library strategy: Modified ChIP-seq (see PMID 22025700)

data_processing: 1. We used Bowtie2 (v2.2.3) with options "--no-unal --local --very-sensitive-local --no-discordant --no-mixed --contain --overlap --dovetail --phred33 -I 10 -X 700" to map paired-end 25bp reads to release hg19 of the human genomic sequence obtained from UCSC. 2. We extracted properly paired reads (Supplementary file .bed). 3. For each base pair in the genome, we counted the number of paired-end fragments aligned over it. 4. We normalized base pair counts by dividing by the total number of counts for all base pairs and then multiplying by the total number of base pairs in the genome (Supplementary file .wig)

source_name: Crosslinked MNase digested chromatin

extract_protocol: Human K562 cells were crosslinked using formaldehyde for 10 minutes at room temperature. Chromatin was fragmented using MNase and then sonicated to ensure complete extraction of chromatin and associated proteins. Chromatin was immunoprecipitated using an antibody against CTCF. DNA was extracted from the immunoprecipitated chromatin and subjected to Illumina (Solexa) sequencing.

value: K562 cells

value: CTCF (Millipore catalog #07-729)


title: Vehicle

source_name: HK-GFP-MR cells_vehicle-treated

value: Rabbit polyclonal 39N anti-MR antibody (custom-made)


title: RNA-seq-MHG

status: Public on Nov 22 2013

first: Kyle

middle: M.

last: Loh

department: Department of Developmental Biology

data_processing:
For RNA-seq, sequenced read were aligned using Tophat  and FPKM (fragments per kilobase of exon per million mapped reads) calculated using Cufflinks
Genome_build: hg19

source_name: hESC derived anterior foregut (MHG)

growth_protocol: Most hESC lines employed in this study were originally cultured on irradiated mouse embryonic fibroblast (MEF) feeder layers. To adapt hESC lines to feeder-free culture, MEF-grown hESCs were initially transferred onto Matrigel-coated plates and propagated in MEF-conditioned medium (CM). hESCs were continually passaged on Matrigel-coated plates in MEF-CM until they efficiently could plate on Matrigel (in lieu of a feeder layer) and until residual irradiated MEFs were eliminated after serial passaging to prevent feeder carryover into future defined culture conditions.All hESC and hiPSC lines were propagated feeder-free in mTeSR1. Differentiation was conducted feeder-free in fully-defined. Prefacing differentiation, confluent hPSC cultures were passaged as small clumps with collagenase IV (typically 1:3 split ratio) onto new plates coated with either human fibronectin or Matrigel. After 1-2 days of recovery in mTeSR1, hPSCs were washed with F12 (Gibco) to evacuate all mTeSR1 and then were treated for 24 hours with Activin A (100 ng/mL, R&D Systems), CHIR99021 (2 μM, Stemgent), and PI-103 (50 nM, Tocris) in CDM2 to specify APS. Afterwards, cells were washed (F12), then treated for 48 hours with Activin A (100 ng/mL), LDN-193189/DM3189 (250 nM, Stemgent), and optionally, also FGF2 (10 ng/mL, Invitrogen) in CDM2 to generate DE by day 3. Media was refreshed every 24 hours. Day 3 DE was patterned into AFG, PFG, or MHG by 4 days of continued differentiation in CDM2. DE was washed (F12), then differentiated as follows: AFG, A-83-01 (1 μM, Tocris) and DM3189 (250 nM); PFG, RA (2 μM, Sigma) and DM3189 (250 nM); MHG, BMP4 (10 ng/mL, R&D Systems), CHIR99021 (3 μM), and FGF2 (100 ng/mL), yielding day 7 anteroposterior domains.

extract_protocol:
For RNA-seq, RNA was harvested from adherent cells grown in individual wells of a 12-well plate by  the addition of 350 μL of RLT Buffer for several minutes. RNA could be indefinitely  frozen at -80 °C or could be directly extracted. RNA extraction was conducted with the  RNeasy Micro Kit (Qiagen) generally as per the manufacturer’s recommendations,  however with an intermediate 1 hour on-column DNase digestion to eliminate residual  genomic DNA, and RNA was finally eluted from the column in 30 μL H2O.
RNA-seq library construction was conducted with the TruSeq RNA Library Preparation Kit (Illumina) as per the manufacturer’s instructions.

value: hESC derived anterior foregut (MHG)


title: Aldosterone

source_name: HK-GFP-MR cells_aldosterone-treated


title: C2C12MB-Dam rep 1

status: Public on Oct 30 2013

data_processing: Basecalls performed using CASAVA version 1.8. Reads were aligned to mm9 by Bowtie2-2.0.0-beta5 using the following command: bowtie2 -phred3 -x mm9 -U <in.fastq> -S <out.sam>. Genome build: mm9. Supplementary_files_format_and_content: Alignment of the reads to mm9 in BED format.

growth_protocol: Lamin B1 cDNA was cloned into the pLGW-dam-V5 vectors. Lentivirus with Dam-V5 and Dam-V5-Lmnb1 vectors was generated by the Virapower Lentivirus Expression system (Invitrogen) and was stored at -80 °C before use. The lentivirus was diluted with the growth medium and incubated with the cells for two days before changing to the growth media.

extract_protocol:
Genomic DNA was isolated using Dneasy blood and tissue kit (Qiagen).
PCR-enriched methylated DNA fragments from the DamID assay were fragmented by NEBNext® dsDNA Fragmentase, then subject to end repair, addition of "A" bases, ligation of Illumina adaptors, size selection of libary and PCR enrichment of adaptor-modified DNA fragments according to the protocol provided by Yale Center for Genome Analysis (http://ycga.yale.edu/sequencing/Illumina/protocols.aspx)

tag: expressing protein

value: E. coli DNA Adenine Methyltransferase


title: C2C12MB-Dam rep 2


title: C2C12MB-LmnB1 rep 2

value: E. coli DNA Adenine Methyltransferase tethered to mouse Lamin B2


title: 3T3MEF-Dam rep 1

source_name: 3T3 fibroblasts


title: 3T3MEF-Dam rep 2


title: 3T3MEF-LmnB1 rep 2

value: E. coli DNA Adenine Methyltransferase tethered to mouse Lamin B1


title: C2C12MB-LmnB1 rep 1


title: 3T3MEF-LmnB1 rep 1


title: H3K4me3

value: anti-H3K4me3 (Diagenode, included in catalog numbers C01010050 AB-001-0010, C01010051 AB-001-0024)