title: nuclearRNAseq_control3

description:
Sample 39
RNAseq_normalizedRPM_CTRLRUN.txt
RNAseq_raw_read_counts_CTRL_RUN.txt
nuclear RNA

source_name: control 3


title: HA-Empty_SKM_rep2

status: Public on Dec 05 2016

type: SRA

city: Madison

first: Aseem

last: Ansari

state: WI

address: 440 Henry Mall

department: Biochemistry and The Genome Center

country: USA

institute: University of Wisconsin-Madison

description: Replicate 2 for Empty+SKM early cells (HA tag on ATFs); negative control

library_source: genomic

data_processing:
Illumina Casava 1.8.2 software used for basecalling
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence, then mapped to mm10 using Bowtie2 version 2.2.5.
Output sam files were converted to bam files, sorted, and indexed with Samtools 1.3.
Differential peak signals were determined by DiffBind 1.16.2 for H3K27ac and H3K9me3 histone marks.
Genome_build: mm10
Supplementary_files_format_and_content: tab-delimited text files include peaks called by MACS; comma separated files for pairwise comparisons

molecule: genomic DNA

organism: Mus musculus

source_name: mouse embryonic fibroblasts

growth_protocol: Transduced MEFs were grown in knock-out DMEM supplemented with 15% fetal bovine serum, 1% nonessential amino acids, 2 mM L-glutamine, 1 x 10^3 units/mL leukemia inhibitory factor, 1 mM sodium pyruvate, and 100 µM beta-mercaptoethanol on 0.1% gelatin. iPS cells were treated with 100 nM 4-hydroxytamoxifen to induce expression of the Oct4-MerCreMer; Tomato/GFP reporter. Cells were maintained in a humidified 37°C incubator with 5% CO2.

extract_protocol:
For histone marks, 6 x 10^6 cells were harvested, and for ATFs, 2.5 x 10^7 cells were harvested. Cells were fixed in 1.5% formaldehyde, then flash frozen. Cells were lysed with a series of 3 buffers: LB1, LB2, and LB3. LB1 consisted of 10 mM HEPES, 10 mM EDTA, 0.5 mM EGTA, and 0.25% Triton X-100. LB2 consisted of 200 mM NaCl, 10 mM HEPES, 1 mM EDTA, and 0.5 mM EGTA. LB3 consisted of 50 mM Tris-HCl, 10 mM EDTA, 0.5% Empigen BB, and 1% SDS. Samples were sonicated in a Misonix sonicator (S-4000) at 60% power, 10 sec on and 10 off, for a total of 32 min total pulse time. Samples were cleared by centrifugation 17,000 x g for 10 min. For preclearing, samples were incubated with magnetic Protein G beads (Life Technologies #10003D) for 1 hour at 4°C. After preclearing, 1% of sample was saved as input. Samples were incubated with the appropriate antibody, H3K27ac antibody (Abcam #ab4729), H3K9me3 antibody (Abcam #ab8898), or HA antibody (Abcam #ab9110), overnight at 4°C with IP Buffer (2 mM EDTA, 150 mM NaCl, 20 mM Tris-HCl, and 1% Triton X-100). Protein-DNA complexes on magnetic beads were washed in WB1 once, WB2, once, WB3 once, and TE Buffer twice. WB1 consisted of 2 mM EDTA, 20 mM Tris-HCl, 0.1% SDS, 1% Triton X-100, 150 mM NaCl). WB2 consisted of 2 mM EDTA, 20 mM Tris-HCl, 0.1% SDS, 1% Triton X-100, and 500 mM NaCl. WB3 consisted of 1 mM EDTA, 10 mM Tris-HCl, 250 mM LiCl, 1% deoxycholate, and 1% NP-40. DNA was eluted in 0.1 M NaHCO3, 0.2 M NaCl, and 1% SDS. Protein-DNA complexes were reverse crosslinked by incubation at 65°C for 6 hours. DNA was treated with RNase A and Proteinase K. Captured DNA was column purified (Epoch Life Sciences #1920-250).
Samples were prepared for sequencing with the TruSeq ChIP Sample Preparation Kit (Illumina #IP-202-1012) as per the manufacturer instructions and quantified with a Qubit fluorometer (Life Technologies #Q32866). Three, four or six TruSeq indexed ChIP samples were pooled per lane. All samples were loaded at a final concentration of 8 pM and sequenced as single reads on the Illumina HiSeq 2500 following the manufacturer's protocols.

tag: cell type

value: MEF-derived cells

tag: strain

tag: transduction

value: Empty, Sox2, Klf4, c-Myc

tag: chip antibody

value: HA (abcam #9110)


title: nuclearRNAseq_control2

city: Cambridge

first: Felix

last: Krueger

address: Granta Park

department: Bioinformatics

country: United Kingdom

institute: Altos Labs

description:
Sample 38
RNAseq_normalizedRPM_CTRLRUN.txt
RNAseq_raw_read_counts_CTRL_RUN.txt
nuclear RNA

library_source: transcriptomic

data_processing:
All reads were aligned using Bowtie against the rat genome (build 3.4) using settings  -n 1 -m 1 --strata --best (i.e. requiring at most one unique mapping per read, allowing one mismatch).  Results were converted to the BAM file format using SAMtools. Annotations of functional genomic features and their coordinates were obtained from Ensembl (Release 69). ChIP-seq reads were extended in silico to 150 bp, for them to correspond to the average length of the mononucleosomal DNA fragments observed upon 2100 Bioanalyzer measurements. Read counts of genomic features of interest were determined using the SeqMonk Mapped Sequence Data Analyser (Version 0.22.0; www.bioinformatics.babraham.ac.uk/projects/seqmonk/), and further processed using R version 2.15.1.
Analysis of gene expression was based on strand-specific read counts, with only reads mapping to the strand opposite of the annotated gene orientation being counted. To assess issues related to strand-specificity of the RNA-seq, we assessed the fraction of all reads per gene mapping to the opposite strand. Differential RNA expression was assessed based on differences between each treatment and control sample, paired per experiment, using a Chi-squared test. The average read number per million reads was determined, as well as the median of the resulting P value per gene from 4 independent experiments was calculated, and corrected for multiple testing. Differential expression in read counts from PCM1-positive and -negative samples was determined using EdgeR.
Except for cluster analyses, histone methylation was always assessed in autosomal 10 kb windows, with a 10 kb step size. Windows with no or poor mapping (resp. gaps in the genome build or highly repetitive sequences) were not considered. Such windows were identified as having read counts that were over 6 times the third interquartile range different from the median input read counts. Read counts for input samples were highly concordant (average Pearson’s r = 0.994) and failed to show significant differences between H3K9me2 and H3K27me3, or between the different treatments (q-value exceeding 0.5 for all). All input samples were therefore binned and treated as one dataset in subsequent analyses. To identify regions of estimated equal H3K9me2 or H3K27me3 levels, circular binary segmentation was applied on the combined control datasets using the DNAcopy package using default parameters. Circular binary segmentation was applied using the segment function, using the hybrid approach for the computation of the P-value of the maximal t-statistic, requiring a significance level lower than 0.01 to accept change-points, and a minimum of 5 probes per segment.
Differences in ChIP-seq read density between these segments between paired treated and control samples were assessed using a paired t-test on the 10kb window read counts. Counts were normalized in SeqMonk by adjusting them to have a matched distribution across all samples. Average histone methylation levels per segment were required to differ at least 20%, and to differ in at least 3 out of 4 paired treatment-control experiments in the same direction. P-values from student t-tests were corrected for multiple testing, and segments were only considered significantly different if q-values were below 0.1.
Genome_build: rn3.4
Supplementary_files_format_and_content: Quantitated ChIP-Seq data files are tab-delimited and contain the following 7 columns: position of chromosome segments as identified by CBS (1) Chromosome, (2) Start and (3) End; (4-5) log2 reads per million
Supplementary_files_format_and_content: Quantitated RNA-Seq data files for hypertrophic vs control are tab-delimited and contain the following 4 columns: (1) gene name (Ensembl); (2-3) log2 reads per million; (4) FDR-adjusted P-value
Supplementary_files_format_and_content: Quantitated RNA-Seq data files for PCM1+ vs - are tab-delimited and contain the following 4 columns: (1) gene name (Ensembl); (2) log2 fold change; (3) log2 counts per million; (4) FDR-adjusted P-value

molecule: total RNA

organism: Rattus norvegicus

source_name: control 2

extract_protocol:
End repair or second strand synthesis reactions were purified using Agencourt AMPure XP (Beckman Coulter) and eluted in 15 µL dA-tailing mix (NEBNext dA-Tailing Module; New England Biolabs). This mix was incubated at 37 °C for 30 min, purified using Agencourt AMPure XP (Beckman Coulter) and eluted in 14 µL of pre-annealed barcoded adapters at a 20× molar amount relative to the estimated amount of dA-tailed DNA. Next, 4 µL 5× Ligation Buffer and 2 µL Quick Ligase (NEBnext Quick Ligation Module; New England Biolabs) were added, and the reaction was incubated for 15 min at 37 °C.  These reactions were again purified using Agencourt AMPure XP (Beckman Coulter) and eluted in 50 µL water for ChIP sequencing, or 49 µL TE buffer and 1 µL USER enzyme mix (New England Biolabs) for nuclear RNA sequencing. The latter mix was incubated at 37 °C for 30 min and at 95 °C for 5 min. To amplify these libraries, 50 µL Phusion High-Fidelity PCR Master Mix with HF Buffer (Thermo Scientific, Loughborough, UK) was added to each sample, together with primer 1 and 2 oligonucleotides to a final concentration of 500 nM. Each reactions was distributed between 4 PCR tubes and PCR was performed in a thermal cycler using the following program: initial denaturation at 98°C for 30 seconds, amplification over 18 cycles of 98 °C, 65 °C and 72 °C for respectively 10, 30 and 30 seconds, and a final extention at 72 °C for 7 min. PCR reactions were purified using the QIAquick PCR purification kit, eluted in 25 µL, mixed with 10 µL loading buffer (200 mg/mL Ficoll 400, 15 mg/mL Orange G and 2× Tris/Borate/EDTA buffer) and loaded on a 1.7% TBE agarose gel, prestained with SYBR safe DNA gel stain (Life Technologies). DNA was separated by electrophoresis at 4V/cm for 2 hours at 4 °C, and 200 to 400 bp fragments were excised and purified using the QIAquick Gel Extraction Kit (QIAGEN) as per the manufacturer’s instructions. DNA fragment size was verified on a 2100 Bioanalyzer Instrument using a High Sensitivity DNA Analysis Kit (both from Agilent Technologies Inc., Santa Clara, USA), and DNA concentration was verified using the Quant-iT PicoGreen dsDNA Assay Kit (Life Technologies).
nuclei sorting protocol: To minimize handling times and to control potential inter-experimental artefacts, nuclei were isolated and sorted from 2 left ventricular wall samples per experiment, one hypertrophic sample and one corresponding control sample. Methods for nuclear isolation were based on a previously described method.To minimize handling times and to control potential inter-experimental artefacts, nuclei were isolated and sorted from 2 left ventricular wall samples per experiment, one hypertrophic sample and one corresponding control sample. Methods for nuclear isolation were based on a previously described method. All buffers were precooled, supplemented with dithiothreitol (1 µM), actinomycin D (1mg/L), cOmplete protease inhibitor (PI) cocktail (Roche; 0.5 µL/mL) and SUPERase.In (Ambion; 200 U/mL), and buffered with 10 mM Tris-HCl to pH 8.0. Frozen hearts where thawed in 10 mL lysis buffer (0.32 M sucrose, 5 mM CaCl2, 5 mM EDTA, 0.5 mM EGTA, 3 mM Mg(CH3COO)2) and homogenized using an IKA ultra-Turrax (20,000 RPM, 15 sec). After addition of 30 mL lysis buffer, the homogenate was dounced 10× on ice, filtered consecutively over mesh, a 100 µm and a 70 µm cell strainer and spun for 10 minutes (700×G; 4°C). The resulting pellet was dissolved in 10 mL centrifuge buffer (2.1 M sucrose, 3 mM Mg(CH3COO)2, and 5 mM EDTA), and superimposed upon 5 mL centrifuge buffer in an autoclaved Thinwall Ultra-Clear Tube (Beckman Coulter, High Wycombe, UK). Tubes were loaded in a precooled rotor (SW32.1Ti) and spun for 60 minutes (26,000×G; 4°C) in an OPTIMA MAX Ultracentrifuge (both from Beckman Coulter). The resulting pellet was dissolved in 1.5 mL sorting buffer (0.43 M sucrose, 70 mM KCl, 2 mM MgCl2 and 5 mM EDTA) and labelled for 1 hour on ice using 3 µL anti-PCM1 antibody (HPA023374) that was pre-incubated for 15 minutes with 2 µL Alexa Fluor 488 Goat Anti-Rabbit IgG (H+L) Antibody (Life Technologies). Nuclei were sorted on an Influx streaming air high-speed cell sorter (BD Biosciences, Oxford, UK), into Protein LoBind tubes (Eppendorf, Stevenage, UK) on a cooled tube holder. For comparison of unsorted and sorted nuclei, the input samples corresponded to a 350 µL aliquot of the original homogenized sample, and the nuclear samples corresponded to 400,000 nuclei not gated for PCM1 staining.  All buffers were precooled, supplemented with dithiothreitol (1 µM), actinomycin D (1mg/L), cOmplete protease inhibitor (PI) cocktail (Roche; 0.5 µL/mL) and SUPERaseIn (Ambion; 200 U/mL), and buffered with 10 mM Tris-HCl to pH 8.0. Frozen hearts where thawed in 10 mL lysis buffer (0.32 M sucrose, 5 mM CaCl2, 5 mM EDTA, 0.5 mM EGTA, 3 mM Mg(CH3COO)2) and homogenized using an IKA ultra-Turrax (20,000 RPM, 15 sec). After addition of 30 mL lysis buffer, the homogenate was dounced 10× on ice, filtered consecutively over mesh, a 100 µm and a 70 µm cell strainer and spun for 10 minutes (700×G; 4°C). The resulting pellet was dissolved in 10 mL centrifuge buffer (2.1 M sucrose, 3 mM Mg(CH3COO)2, and 5 mM EDTA), and superimposed upon 5 mL centrifuge buffer in an autoclaved Thinwall Ultra-Clear Tube (Beckman Coulter, High Wycombe, UK). Tubes were loaded in a precooled rotor (SW32.1Ti) and spun for 60 minutes (26,000×G; 4°C) in an OPTIMA MAX Ultracentrifuge (both from Beckman Coulter). The resulting pellet was dissolved in 1.5 mL sorting buffer (0.43 M sucrose, 70 mM KCl, 2 mM MgCl2 and 5 mM EDTA) and labelled for 1 hour on ice using 3 µL anti-PCM1 antibody (HPA023374) that was pre-incubated for 15 minutes with 2 µL Alexa Fluor 488 Goat Anti-Rabbit IgG (H+L) Antibody (Life Technologies). Nuclei were sorted on an Influx streaming air high-speed cell sorter (BD Biosciences, Oxford, UK) using a gating strategy as described.15 Post-sorting flow cytometric analyses were done using FlowJo (v9.6.3) and Bioconductor’s flowCore package.52 Nuclei were sorted into Protein LoBind tubes (Eppendorf, Stevenage, UK) on a cooled tube holder. For comparison of unsorted and sorted nuclei, the input samples corresponded to a 350 µL aliquot of the original homogenized sample, and the nuclear samples corresponded to 400,000 nuclei not gated for PCM1 staining.
DNA libraries were sequenced on an Illumina HiSeq 1000 (Illumina, Essex, UK) using either the default RTA software, or a custom program that consisted of annealing of Illumina Read 1 sequencing primer, 4 dark cycles, 46 light cycles, denaturation, annealing of Illumina Read 1 sequencing primer and 5 light cycles to read the indexing barcode. The latter modification was introduced because the low complexity of the first bases from the indexing barcode yielded incorrect cluster definitions and consequently lowered the number of high quality reads.

value: PCM1-positive nuclei from left ventricle of the heart

value: Sprague Dawley

tag: hypertrophy induction

value: Male Sprague Dawley rats of ~280 g not assigned to the exercise protocol were used as control.

tag: isolation of material from sorted nuclei

value: A solution of at least 100000 sorted nuclei was mixed with 3× its volume of TRIzol LS reagent (Life Technologies) and 2 µL glycogen (Life Technologies), and stored for at most one month at -80°C until further processing according to manufacturer's instructions. Genomic DNA was removed using the TURBO DNA-free kit (Life Technologies) as per the kit’s instructions. RNA was subsequently reverse transcribed using SuperScript III Reverse Transcriptase (Life Technologies) as follows:3 µg Random Primers (mostly hexamers; Life Technologies) were added to 10 µL RNA and 8 µL 5× First-Strand Buffer. This mixture was heated to 94 °C for 3 minutes for RNA fragmentation, and incubated on ice for at least 1 minute. Next 2 µL dNTP Mix (10 mM) (Life Technologies) were added, as well as 4 µl DTT (100 mM), 40U SUPERase.In, 400U SuperScript™ III Reverse Transcriptase and 8 µg Actinomycin D. Water was added to make up the volume to 40 µL, and the sample was incubated at 25 °C for 5 min 50 °C for 30 minutes and inactivated at 70 °C for 15 min. cDNA-RNA duplexes were purified using 72 µL Agencourt AMPure XP (Beckman Coulter) and eluted in 20 µL of the following second-strand synthesis reaction mix:0.33 mM dATP, dCTP and dGTP (Life Technologies), 0.33 mM dUTP (Sigma Aldrich), 0.25 U Ribonuclease H (Life Technologies) and 10 U DNA polymerase I (New England Biolabs). The reaction was allowed to proceed for 2.5 hours at 16°C.


title: Oocyte B6 [Oo_B6_18]

city: Paris Cedex 05

first: Nicolas

last: Servant

address: 26 rue d'ulm

country: France

institute: Institut Curie

data_processing:
Quality control: Reads characterized by at least one of the following criteria were discarded from the analysis i) >50% of low quality bases (Phred score <5) ii) >5% of N bases iii) >80% of AT rate iv) ≥30% of continuous A and/or T
Genome reconstruction: The VCF file (mgp.v5.merged.snps_all.dbSNP142.vcf) reporting all SNP sites from 36 mouse strains based on mm10 was downloaded from the Sanger database. Using SNPsplit tool (v0.3.0), these SNPs were filtered based on their quality values (FI value) and used to reconstruct the Cast genome from mm10 genome assembly.
Mapping: Single-end reads were then aligned to the mouse mm10 and Cast/EiJ genomes using the TopHat2 software (v2.1.0). Only random best alignments with less than 2 mismatches were reported for downstream analyses. The resulting mapping files for both parental genomes were then merged for each sample.
Allelic Ratio: The SAMtools mpileup utility (v1.1) was then used to extract base-pair information at each genomic position from the merged alignment file. At each SNP position, the number of paternal and maternal allele was counted. The allele-specific origin of the transcripts (or allelic ratio) has been measured by the total number of reads mapped on the paternal genome divided by the total number of reads for each gene: allelic ratio = paternal reads / (paternal+maternal) reads.
Estimation of gene expression levels: To estimate the transcript abundance, count tables of gene expression were generated using HTSeq v0.6.1 software. Read counts are then normalized based on the amplification size of each transcript (RPRT for Reads Per Retro-Transcribed length per million mapped reads)
Genome_build: mm10

source_name: oocyte

growth_protocol: natural matings, no embryo culture

extract_protocol:
PolyA+ mRNA extracted from each single cell was reverse transcribed from the 3’UTR and amplified following the Tang et al protocol. Care was taken to process only embryos and single blastomeres of the highest quality based on morphology, number of cells and on amplification yield. A total of 72 BC and 110 CB single blastomeres have been processed and passed quality controls.
Illumina manufacturer protocol
50 bp single end

treatment_protocol: single cell cDNA amplification (Tang et al, 2010)

tag: developmental stage

value: Oocyte

tag: genotype

value: Xist wt/wt

tag: Sex

value: Female


title: nuclearRNAseq_control1

description:
Sample 37
RNAseq_normalizedRPM_CTRLRUN.txt
RNAseq_raw_read_counts_CTRL_RUN.txt
nuclear RNA

source_name: control 1


title: HA-Empty_SKM_rep1

description: Replicate 1 for Empty+SKM early cells (HA tag on ATFs); negative control


title: ChIPseq_input_banded_aorta

description:
H3K9me2_normalized_RPM_in_CBS_bins_SHAM_AB.txt
H3K27me3_normalized_RPM_in_CBS_bins_CTRL_RUN.txt
nuclear RNA

source_name: banded aorta

value: Male Sprague Dawley rats of ~170 g were anesthetized and ventilated through endotracheal intubation with a mixture of 69% N2O, 29% O2 and 2% isoflurane. A silk suture 3-0 was tightened around the ascending aorta proximal to the brachiocephalic trunk. Buprenorphine was given as postoperative analgesia. After 6 weeks, only those rats were retained, that had non-failing hearts with detectable hypertrophy, defined as increased posterior wall thickness in diastole (>1.9 mm), increased left ventricular weight (>0.75 g) and preserved lung weight (<2.0 g).

value: A solution of at least 100000 freshly sorted nuclei for nucleosome extraction was topped up with MNase buffer (0.32 M sucrose, 4 mM MgCl2, 1 mM CaCl2 and 1 µL/mL cOmplete PI cocktail) to a final volume of 1.5 mL and spun for 10 minutes (1,000×G, 4°C). All but 100 µL supernatant was removed, and nuclei were gently suspended in an additional 1.4 mL MNase buffer and spun for 10 minutes (1,000×G, 4°C). All but 100 µL supernatant was again removed, and the pelleted nuclei were suspended to a concentration of 4,000 nuclei (as counted by flow cytometry) per µL in MNase buffer. Micrococcal nuclease (Affymetrix USB, Staufen, Germany) was subsequently added (20 U/mL) to the nuclei suspension. In order to digest the chromatin to a predominantly mononucleosomal resolution, the reaction was stopped after 8 minutes of incubation at 37°C by transfer on ice and by the addition of ½ volume of nucleosome extraction buffer (15 mM EDTA, 0.3% Triton X-100, 10 mM Na2HPO4, 3.5 mM KH2PO4, 1.5 M NaCl, 20 µg/mL and 3 µL/mL cOmplete PI cocktail at pH 7.4). Nucleosomes were subsequently extracted at 4°C overnight on a vertical rotor (30 RPM). The next day, nuclear debris was removed by centrifugation for 10 minutes (21,000×G, 4°C), after which 50 µL was used as input. These solutions were kept overnight on a vertical rotor at 4 °C (5 RPM). Proteinase K (2 mg) was subsequently added to each tube (including input fractions), and after incubation at 42°C for 30 minutes, DNA was extracted using the QIAquick PCR purification kit (QIAGEN, Manchester, UK) as follows:binding buffer PB with pH indicator was added directly to the suspension, pH adjusted using Na(CH3COO) and after an incubation of 10 minutes, the DNA binding, washing and elution in 50 µL water was done as per the kit’s instructions. Purified DNA was rendered blunt ended using the NEBnext End Repair Module (New England Biolabs) as per the manufacturer’s instructions.


title: nuclearRNAseq_exercise2

description:
Sample 34
RNAseq_normalizedRPM_CTRLRUN.txt
RNAseq_raw_read_counts_CTRL_RUN.txt
nuclear RNA

source_name: exercise 2

value: Male Sprague Dawley rats of ~280 g were assigned to interval training on a treadmill (Columbus Instruments, OH, US) with 25 degree inclination, with 5 days of acclimatization with running velocity for 6 m/min for 30, 45, 60, 75, 90 and 120 min. Thereafter interval training was performed 6 days a week with 12 x 8 min intervals with 2 min resting period (6 m/min) after a warm-up period at 10 m/min. Exercise training was performed over six weeks at a running speed of 15 m/min increasing with 2 m/min each week. Rats not able to fulfill the exercise protocol were withdrawn from the study.


title: nuclearRNAseq_exercise4

description:
Sample 36
RNAseq_normalizedRPM_CTRLRUN.txt
RNAseq_raw_read_counts_CTRL_RUN.txt
nuclear RNA

source_name: exercise 4


title: nuclearRNAseq_sham_operated2

description:
Sample 46
RNAseq_normalizedRPM_SHAM_AB.txt
RNAseq_raw_read_counts_SHAM_AB.txt
nuclear RNA

source_name: sham-operated 2

value: Male Sprague Dawley rats of ~170 g were anesthetized and ventilated through endotracheal intubation with a mixture of 69% N2O, 29% O2 and 2% isoflurane. Sham-operated animals underwent the same surgical procedure as those receiving aortic banding, except tightening the silk suture around the ascending aorta proximal to the brachiocephalic trunk. Buprenorphine was given as postoperative analgesia.


title: Sox17E57KOct4_coopseq (unbound)

city: St. Louis

first: Gary

middle: D.

last: Stormo

state: MO

address: 4515 McKinley Ave. Room 4208.

department: Genetics

institute: Washington University School of Medicine

description:
Contains unbound for downstream processing. Contains raw data from 2 replicates.
Processed data file: coopseq_Chang_allData.txt

data_processing:
Library strategy: Coop-seq
Extracting binding sites sequence from raw reads. Each read follows the format of GAGTCGTCTCGTCAGCAC-binding site-CCGTAGAGCACTCAGGTC.
Count read numbers for each variant, classified by dimer bound, Sox bound, POU bound, or unbound in coopseq and classfied by bound or unbound in specseq.
For specseq, calculate the bound/unbound ratio for each variant, then rank variants according to their relative affinities.
For coopseq, the cooperativity, omega, is calculated by (Dimer bound x unbound)/(Sox bound x POU bound).
Supplementary_files_format_and_content: Tab delimited text files.  See Processed_data_description.txt.

molecule: other

organism: synthetic construct

source_name: Synthesized DNA

extract_protocol:
All EMSA were done with Sox and Oct proteins in 1X NEB Cutsmart buffer (50mM Potassium Acetate, 20mM Tris-acetate, 10mM Magnesium Acetate, 100μg/ml BSA, pH 7.9@25°C) supplemented with 10% glycerol with 1mM of dsDNA and were incubated for 30 minutes on ice. Electrophoresis mobility assay (EMSA) were done using native 12% PAGE prepared as Tris/Glycine (25mM Tris pH 8.3; 192mM glycine) mini-gels. These gels were first pre-run using 1X Tris/Glycine buffer (25mM Tris pH 8.3; 192mM glycine) at 200V for 30 minutes, then samples were loaded for an additional run of 60 minutes at 200 V at 4°C. After EMSA, the gels were stained with ethidium bromide and visualized using Biorad gel imager. Each band detected in the EMSA were excised with a plastic tool and the DNA in the gel extracted by incubating for 30 mins at 50°C in 50ml acrylamide gel extraction buffer (500mM Ammonium acetate, 10mM Magnesium Acetate, 1mM EDTA, 0.1% SDS). 0.01 pmole of PCR Control DNA with the sequence of GAGTCGTCTCGTCAGCACCGGCGGCGGTTCCCGGAAAGACCGTAGAGCACTCAGGTC (primer binding sites underlined) were added to each extracted sample. Sample in the extraction buffer were purified with QIAquick Nucleotide Removal Kit (Qiagen) following the manufacturer’s instructions. Each fraction of DNA was barcoded and amplified using HotStart PCR Master Mix (Lambda Biotech). DNA was denatured at 94°C for 30 seconds, annealed at 55°C for 30 seconds and extend at 72°C for 45 seconds per round for 12 to 20 rounds with modified Indexed-Illumina primers (PE1-Genetics1/2, PE2.0). The PCR product was then purified again using QIAquick Nucleotide Removal Kit.
DNA library was constructed by mixing the following oligos GAGTCGTCTCGTCAGCACCGGCCATTGTNTGCTAATAGACCGTAGAGCACTCAGGTC, GAGTCGTCTCGTCAGCACCGGCATTGTNNTGCTAATAGACCGTAGAGCACTCAGGTC, GAGTCGTCTCGTCAGCACCGCATTGTNGNTGCTAATAGACCGTAGAGCACTCAGGTC, GAGTCGTCTCGTCAGCACCCATTGTNGGNTGCTAATAGACCGTAGAGCACTCAGGTC, GAGTCGTCTCGTCAGCACCATTGTNGGCNTGCTAATAGACCGTAGAGCACTCAGGTC, GAGTCGTCTCGTCAGCACCATTGTNGGCGNTGCTAATGACCGTAGAGCACTCAGGTC, GAGTCGTCTCGTCAGCACCATTGTNGGCGGNTGCTAATACCGTAGAGCACTCAGGTC, GAGTCGTCTCGTCAGCACCATTGTNGGCGGCNTGCTAATCCGTAGAGCACTCAGGTC, GAGTCGTCTCGTCAGCACCGGCATTGTTATGCTAATAGACCGTAGAGCACTCAGGTC, GAGTCGTCTCGTCAGCACCGCATTGTTNATGCTAATAGACCGTAGAGCACTCAGGTC, GAGTCGTCTCGTCAGCACCCATTGTTNNATGCTAATAGACCGTAGAGCACTCAGGTC, GAGTCGTCTCGTCAGCACCATTGTTNGNATGCTAATAGACCGTAGAGCACTCAGGTC, GAGTCGTCTCGTCAGCACCATTGTTNGGNATGCTAATGACCGTAGAGCACTCAGGTC, GAGTCGTCTCGTCAGCACCGGCATTGTTATTAGCATAGACCGTAGAGCACTCAGGTC, GAGTCGTCTCGTCAGCACCGCATTGTTNATTAGCATAGACCGTAGAGCACTCAGGTC, GAGTCGTCTCGTCAGCACCCATTGTTNNATTAGCATAGACCGTAGAGCACTCAGGTC, GAGTCGTCTCGTCAGCACCATTGTTNGNATTAGCATAGACCGTAGAGCACTCAGGTC, GAGTCGTCTCGTCAGCACCATTGTTNGGNATTAGCATGACCGTAGAGCACTCAGGTC, GAGTCGTCTCGTCAGCACCGGAACAATGATGCTAATAGACCGTAGAGCACTCAGGTC, GAGTCGTCTCGTCAGCACCGAACAATGNATGCTAATAGACCGTAGAGCACTCAGGTC, GAGTCGTCTCGTCAGCACCAACAATGNNATGCTAATAGACCGTAGAGCACTCAGGTC, GAGTCGTCTCGTCAGCACAACAATGNGNATGCTAATAGACCGTAGAGCACTCAGGTC, GAGTCGTCTCGTCAGCACAACAATGNGGNATGCTAATGACCGTAGAGCACTCAGGTC, GAGTCGTCTCGTCAGCACCGGAACAATGATTAGCATAGACCGTAGAGCACTCAGGTC, GAGTCGTCTCGTCAGCACCGAACAATGNATTAGCATAGACCGTAGAGCACTCAGGTC, GAGTCGTCTCGTCAGCACCAACAATGNNATTAGCATAGACCGTAGAGCACTCAGGTC, GAGTCGTCTCGTCAGCACAACAATGNGNATTAGCATAGACCGTAGAGCACTCAGGTC, and GAGTCGTCTCGTCAGCACAACAATGNGGNATTAGCATGACCGTAGAGCACTCAGGTC. DNA library was designed by flanking the degenerate sequences of interest with sequences GAGTCGTCTCGTCAGCAC and CCGTAGAGCACTCAGGTC for downstream processing. To make double-stranded DNA (dsDNA) libraries, 100 pmole single-strand degenerate template sequences were mixed with an equal amount of reverse complement primer (GACCTGAGTGCTCTACGG). In the presence of Taq Polymerase, brief 10-sec denaturing followed by 10 min of 55 degree annealing/extension is sufficient to make dsDNA libraries. Because any unextended single-stranded DNA (ssDNA) could contaminate the unbound band, the reaction mix was digested by 1 ml NEB Exo I exo- nuclease (New England Biolabs, Beverly, MA) for 30 min. All final dsDNA products were purified by QIAGEN (Valencia, CA) PCR purification columns.

tag: library

value: DNA target oligos

tag: transcription factor(s)


title: Sox18Oct4_coopseq (unbound)


title: H3K27me3_ChIPSeq_sham_operated4

description: H3K27me3_normalized_RPM_in_CBS_bins_SHAM_AB.txt

source_name: sham-operated 4

value: anti-H3K27me3, 07-449 (Millipore, Watford, UK)

value: A solution of at least 100000 freshly sorted nuclei for nucleosome extraction was topped up with MNase buffer (0.32 M sucrose, 4 mM MgCl2, 1 mM CaCl2 and 1 µL/mL cOmplete PI cocktail) to a final volume of 1.5 mL and spun for 10 minutes (1,000×G, 4°C). All but 100 µL supernatant was removed, and nuclei were gently suspended in an additional 1.4 mL MNase buffer and spun for 10 minutes (1,000×G, 4°C). All but 100 µL supernatant was again removed, and the pelleted nuclei were suspended to a concentration of 4,000 nuclei (as counted by flow cytometry) per µL in MNase buffer. Micrococcal nuclease (Affymetrix USB, Staufen, Germany) was subsequently added (20 U/mL) to the nuclei suspension. In order to digest the chromatin to a predominantly mononucleosomal resolution, the reaction was stopped after 8 minutes of incubation at 37°C by transfer on ice and by the addition of ½ volume of nucleosome extraction buffer (15 mM EDTA, 0.3% Triton X-100, 10 mM Na2HPO4, 3.5 mM KH2PO4, 1.5 M NaCl, 20 µg/mL and 3 µL/mL cOmplete PI cocktail at pH 7.4). Nucleosomes were subsequently extracted at 4°C overnight on a vertical rotor (30 RPM). The next day, nuclear debris was removed by centrifugation for 10 minutes (21,000×G, 4°C), after which 10 µL Protein G Dynabeads (Life Technologies) were added to the supernatant for preclearing the solution and removing the remainder of the antibodies that were used for nuclei labelling. After 1 hour at 4°C on a vertical rotor (30 RPM), the beads were removed by magnetic separation, and the solution split in 2 equal fractions. Per volume, 2.5 volumes of ChIP buffer (3 mM Na2HPO4, 1 mM KH2PO4, 50 mM NaCl, 5 mM EDTA, 2 µg/mL BSA and 1 µL/mL cOmplete PI cocktail at pH 7.4) were added, as well as per fraction either the mouse monoclonal anti-H3K9me2 (clone MABI 0307; Active Motif, La Hulpe, Belgium) or the rabbit polyclonal anti-H3K27me3 (07-449; Millipore, Watford, UK) antibody, each at 1 µg/mL. These solutions were kept overnight on a vertical rotor at 4 °C (5 RPM) to allow for antibody binding. On the next day, 20 µL Protein G Dynabeads were added to each solution, and after mixing on a vertical rotor at 4°C (5 RPM) for 2 hours, beads were magnetically separated and the cleared solution transferred to a new tube to be kept as input fraction. Beads were washed 8 times with RIPA buffer (50 mM Tris-HCl, 1 mM EDTA, 1% NP-40, 0.7% sodium deoxycholate, 0.5 M LiCl and 1 µL/mL cOmplete PI cocktail at pH 7.5) and once with TE buffer (50 mM Tris-HCl, 1 mM EDTA and 1 µL/mL cOmplete PI cocktail at pH 7.5), after which beads were resuspended in 200 µL TE and transferred to a new tube. Proteinase K (2 mg) was subsequently added to each tube (including input fractions), and after incubation at 42°C for 30 minutes, DNA was extracted using the QIAquick PCR purification kit (QIAGEN, Manchester, UK) as follows:binding buffer PB with pH indicator was added directly to the suspension, pH adjusted using Na(CH3COO) and after an incubation of 10 minutes (and magnetic separation of the beads from the ChIP samples), the DNA binding, washing and elution in 50 µL water was done as per the kit’s instructions. Purified DNA was rendered blunt ended using the NEBnext End Repair Module (New England Biolabs) as per the manufacturer’s instructions.


title: nuclearRNAseq_exercise1

description:
Sample 33
RNAseq_normalizedRPM_CTRLRUN.txt
RNAseq_raw_read_counts_CTRL_RUN.txt
nuclear RNA

source_name: exercise 1


title: Sox15Oct4_coopseq (unbound)


title: Histone_C3_Input_rep2

description: Replicate 2 for Input for C3+SKM iPS cells (histone marks)

value: MEF-derived iPS cells

value: ZFATF1, ZFATF2, ZFATF4, Sox2, Klf4, c-Myc

value: none (input)


title: HA-C2_SKM_rep2

description: Replicate 2 for C2+SKM early cells (HA tag on ATFs)

value: MEF-derived cells

value: ZFATF1, ZFATF2, ZFATF3, Sox2, Klf4, c-Myc

value: HA (abcam #9110)


title: HA-C2_SKM_rep1

description: Replicate 1 for C2+SKM early cells (HA tag on ATFs)


title: H3K27me3_ChIPSeq_sham_operated1

source_name: sham-operated 1

value: PCM1-positive nuclei from left ventricle of the heart

value: Sprague Dawley


title: H3K27me3_ChIPSeq_sham_operated2


title: H3K27me3_ChIPSeq_sham_operated3

source_name: sham-operated 3


title: H3K27me3_ChIPSeq_banded_aorta4

source_name: banded aorta 4


title: Sox5Oct4_coopseq (unbound)


title: Histone_C3_Input_rep1

description: Replicate 1 for Input for C3+SKM iPS cells (histone marks)


title: H3K27me3_ChIPSeq_banded_aorta3

source_name: banded aorta 3


title: Histone_C2_OSKM_Input_rep2

description: Replicate 2 for Input for C2+SKM iPS cells and Oct4+SKM iPS cells (histone marks)

value: ZFATF1, ZFATF2, ZFATF3, Sox2, Klf4, c-Myc/Oct4, Sox2, Klf4, c-Myc


title: nuclearRNAseq_exercise3

description:
Sample 35
RNAseq_normalizedRPM_CTRLRUN.txt
RNAseq_raw_read_counts_CTRL_RUN.txt
nuclear RNA

source_name: exercise 3

value: A solution of at least 100000 sorted nuclei was mixed with 3× its volume of TRIzol LS reagent (Life Technologies) and 2 µL glycogen (Life Technologies), and stored for at most one month at -80°C until further processing according to manufacturer's instructions. Genomic DNA was removed using the TURBO DNA-free kit (Life Technologies) as per the kit’s instructions. RNA was subsequently reverse transcribed using SuperScript III Reverse Transcriptase (Life Technologies) as follows:3 µg Random Primers (mostly hexamers; Life Technologies) were added to 10 µL RNA and 8 µL 5× First-Strand Buffer. This mixture was heated to 94 °C for 3 minutes for RNA fragmentation, and incubated on ice for at least 1 minute. Next 2 µL dNTP Mix (10 mM) (Life Technologies) were added, as well as 4 µl DTT (100 mM), 40U SUPERase.In, 400U SuperScript™ III Reverse Transcriptase and 8 µg Actinomycin D. Water was added to make up the volume to 40 µL, and the sample was incubated at 25 °C for 5 min 50 °C for 30 minutes and inactivated at 70 °C for 15 min. cDNA-RNA duplexes were purified using 72 µL Agencourt AMPure XP (Beckman Coulter) and eluted in 20 µL of the following second-strand synthesis reaction mix:0.33 mM dATP, dCTP and dGTP (Life Technologies), 0.33 mM dUTP (Sigma Aldrich), 0.25 U Ribonuclease H (Life Technologies) and 10 U DNA polymerase I (New England Biolabs). The reaction was allowed to proceed for 2.5 hours at 16°C.


title: Sox2Oct6_coopseq (unbound)

description:
Contains unbound for downstream processing. Contains raw data from 5 replicates.
Processed data file: coopseq_Chang_allData.txt


title: Histone_C2_OSKM_Input_rep1

description: Replicate 1 for Input for C2+SKM iPS cells and Oct4+SKM iPS cells (histone marks)


title: Sox2Brn4_coopseq (unbound)


title: H3K27me3_ChIPSeq_control4

description: H3K27me3_normalized_RPM_in_CBS_bins_CTRL_RUN.txt

source_name: control 4

value: Male Sprague Dawley rats of ~280 g not assigned to the exercise protocol were used as control.


title: H3K27me3_ChIPSeq_banded_aorta1

source_name: banded aorta 1

value: Male Sprague Dawley rats of ~170 g were anesthetized and ventilated through endotracheal intubation with a mixture of 69% N2O, 29% O2 and 2% isoflurane. A silk suture 3-0 was tightened around the ascending aorta proximal to the brachiocephalic trunk. Buprenorphine was given as postoperative analgesia. After 6 weeks, only those rats were retained, that had non-failing hearts with detectable hypertrophy, defined as increased posterior wall thickness in diastole (>1.9 mm), increased left ventricular weight (>0.75 g) and preserved lung weight (<2.0 g).


title: H3K27me3_ChIPSeq_control3

source_name: control 3


title: Sox17Oct4_coopseq (unbound)


title: H3K27me3_ChIPSeq_control1


title: H3K27me3_ChIPSeq_banded_aorta2

source_name: banded aorta 2


title: Sox2Oct4_coopseq (unbound)


title: H3K27me3_ChIPSeq_control2

source_name: control 2


title: H3K9me3-OSKM_rep2

description: Replicate 2 of Oct4+SKM iPS cells (H3K9me3)

source_name: mouse embryonic fibroblasts

value: Oct4, Sox2, Klf4, c-Myc


title: Sox2Brn2_coopseq (unbound)


title: H3K9me3-OSKM_rep1

description: Replicate 1 of Oct4+SKM iPS cells (H3K9me3)


title: H3K9me3-C3_SKM_rep2

description: Replicate 2 of C3+SKM iPS cells (H3K9me3)


title: Sox17E57KOct4_coopseq (Sox bound)

description:
Contains monomer bound for downstream processing. Contains raw data from 2 replicates.
Processed data file: coopseq_Chang_allData.txt


title: H3K27me3_ChIPSeq_exercise2

source_name: exercise 2

value: Male Sprague Dawley rats of ~280 g were assigned to interval training on a treadmill (Columbus Instruments, OH, US) with 25 degree inclination, with 5 days of acclimatization with running velocity for 6 m/min for 30, 45, 60, 75, 90 and 120 min. Thereafter interval training was performed 6 days a week with 12 x 8 min intervals with 2 min resting period (6 m/min) after a warm-up period at 10 m/min. Exercise training was performed over six weeks at a running speed of 15 m/min increasing with 2 m/min each week. Rats not able to fulfill the exercise protocol were withdrawn from the study.


title: H3K27me3_ChIPSeq_exercise3


title: H3K9me3-C3_SKM_rep1

description: Replicate 1 of C3+SKM iPS cells (H3K9me3)


title: Sox17Oct4_coopseq (Sox bound)

value: DNA target oligos


title: Sox18Oct4_coopseq (Sox bound)


title: H3K9me3-C2_SKM_rep2

description: Replicate 2 of C2+SKM iPS cells (H3K9me3)


title: H3K9me2_ChIPSeq_sham_operated2

description: H3K9me2_normalized_RPM_in_CBS_bins_SHAM_AB.txt

value: anti-H3K9me2, clone MABI 0307 (Active Motif, La Hulpe, Belgium)

value: Male Sprague Dawley rats of ~170 g were anesthetized and ventilated through endotracheal intubation with a mixture of 69% N2O, 29% O2 and 2% isoflurane. Sham-operated animals underwent the same surgical procedure as those receiving aortic banding, except tightening the silk suture around the ascending aorta proximal to the brachiocephalic trunk. Buprenorphine was given as postoperative analgesia.


title: H3K9me2_ChIPSeq_sham_operated3

value: A solution of at least 100000 freshly sorted nuclei for nucleosome extraction was topped up with MNase buffer (0.32 M sucrose, 4 mM MgCl2, 1 mM CaCl2 and 1 µL/mL cOmplete PI cocktail) to a final volume of 1.5 mL and spun for 10 minutes (1,000×G, 4°C). All but 100 µL supernatant was removed, and nuclei were gently suspended in an additional 1.4 mL MNase buffer and spun for 10 minutes (1,000×G, 4°C). All but 100 µL supernatant was again removed, and the pelleted nuclei were suspended to a concentration of 4,000 nuclei (as counted by flow cytometry) per µL in MNase buffer. Micrococcal nuclease (Affymetrix USB, Staufen, Germany) was subsequently added (20 U/mL) to the nuclei suspension. In order to digest the chromatin to a predominantly mononucleosomal resolution, the reaction was stopped after 8 minutes of incubation at 37°C by transfer on ice and by the addition of ½ volume of nucleosome extraction buffer (15 mM EDTA, 0.3% Triton X-100, 10 mM Na2HPO4, 3.5 mM KH2PO4, 1.5 M NaCl, 20 µg/mL and 3 µL/mL cOmplete PI cocktail at pH 7.4). Nucleosomes were subsequently extracted at 4°C overnight on a vertical rotor (30 RPM). The next day, nuclear debris was removed by centrifugation for 10 minutes (21,000×G, 4°C), after which 10 µL Protein G Dynabeads (Life Technologies) were added to the supernatant for preclearing the solution and removing the remainder of the antibodies that were used for nuclei labelling. After 1 hour at 4°C on a vertical rotor (30 RPM), the beads were removed by magnetic separation, and the solution split in 2 equal fractions. Per volume, 2.5 volumes of ChIP buffer (3 mM Na2HPO4, 1 mM KH2PO4, 50 mM NaCl, 5 mM EDTA, 2 µg/mL BSA and 1 µL/mL cOmplete PI cocktail at pH 7.4) were added, as well as per fraction either the mouse monoclonal anti-H3K9me2 (clone MABI 0307; Active Motif, La Hulpe, Belgium) or the rabbit polyclonal anti-H3K27me3 (07-449; Millipore, Watford, UK) antibody, each at 1 µg/mL. These solutions were kept overnight on a vertical rotor at 4 °C (5 RPM) to allow for antibody binding. On the next day, 20 µL Protein G Dynabeads were added to each solution, and after mixing on a vertical rotor at 4°C (5 RPM) for 2 hours, beads were magnetically separated and the cleared solution transferred to a new tube to be kept as input fraction. Beads were washed 8 times with RIPA buffer (50 mM Tris-HCl, 1 mM EDTA, 1% NP-40, 0.7% sodium deoxycholate, 0.5 M LiCl and 1 µL/mL cOmplete PI cocktail at pH 7.5) and once with TE buffer (50 mM Tris-HCl, 1 mM EDTA and 1 µL/mL cOmplete PI cocktail at pH 7.5), after which beads were resuspended in 200 µL TE and transferred to a new tube. Proteinase K (2 mg) was subsequently added to each tube (including input fractions), and after incubation at 42°C for 30 minutes, DNA was extracted using the QIAquick PCR purification kit (QIAGEN, Manchester, UK) as follows:binding buffer PB with pH indicator was added directly to the suspension, pH adjusted using Na(CH3COO) and after an incubation of 10 minutes (and magnetic separation of the beads from the ChIP samples), the DNA binding, washing and elution in 50 µL water was done as per the kit’s instructions. Purified DNA was rendered blunt ended using the NEBnext End Repair Module (New England Biolabs) as per the manufacturer’s instructions.


title: H3K9me2_ChIPSeq_sham_operated4


title: H3K9me2_ChIPSeq_sham_operated1


title: Sox15Oct4_coopseq (Sox bound)


title: H3K9me2_ChIPSeq_banded_aorta4


title: H3K27ac-OSKM_rep2

description: Replicate 2 of Oct4+SKM iPS cells (H3K27ac)

value: MEF-derived iPS cells


title: H3K27me3_ChIPSeq_exercise4

source_name: exercise 4

value: anti-H3K27me3, 07-449 (Millipore, Watford, UK)


title: H3K9me2_ChIPSeq_banded_aorta2


title: Sox2Oct6_coopseq (Sox bound)

description:
Contains monomer bound for downstream processing. Contains raw data from 5 replicates.
Processed data file: coopseq_Chang_allData.txt


title: Sox2Brn4_coopseq (Sox bound)


title: H3K9me2_ChIPSeq_banded_aorta1


title: H3K9me2_ChIPSeq_control3

description: H3K9me2_normalized_RPM_in_CBS_bins_CTRL_RUN.txt


title: H3K27me3_ChIPSeq_exercise1


title: H3K27ac-C3_SKM_rep2

description: Replicate 2 of C3+SKM iPS cells (H3K27ac)

value: ZFATF1, ZFATF2, ZFATF4, Sox2, Klf4, c-Myc


title: H3K27ac-OSKM_rep1

description: Replicate 1 of Oct4+SKM iPS cells (H3K27ac)


title: Sox2Brn2_coopseq (Sox bound)


title: H3K9me2_ChIPSeq_control1

source_name: control 1


title: H3K27ac-C3_SKM_rep1

description: Replicate 1 of C3+SKM iPS cells (H3K27ac)


title: H3K9me2_ChIPSeq_control2


title: H3K27ac-C2_SKM_rep2

description: Replicate 2 of C2+SKM iPS cells (H3K27ac)

value: ZFATF1, ZFATF2, ZFATF3, Sox2, Klf4, c-Myc


title: H3K9me2_ChIPSeq_exercise4


title: H3K9me3-C2_SKM_rep1

description: Replicate 1 of C2+SKM iPS cells (H3K9me3)


title: H3K9me2_ChIPSeq_exercise3


title: Sox2Oct4_coopseq (Sox bound)

source_name: Synthesized DNA


title: dcr1D 24h (sRNA-seq)

city: Charlestown

first: Richard

last: Joh

state: MA

department: Center for Cancer Research

institute: Mass General Hospital

description: small RNA

data_processing:
Remove adapter with Cutadapt
Map go S pombe genome with Bowtie2 2.1.0
Select reads that starts with "U"
Select reads between 18 and 25nt
Quantify the number of reads per transcript by HTSeq 0.6.1
Genome_build: S. pombe ASM294v2.22
Supplementary_files_format_and_content: Number of reads per transcript. Txt file

organism: Schizosaccharomyces pombe

source_name: Cells

growth_protocol: Cells grown in EMM 225ug/ml adenine/histidine/leucine/lysine/uracil (OD~1) at 32C, and were washed twice with EMM -N.  Cells were resuspended in EMM -N and incubated at 32C.

extract_protocol:
Total RNA was prepared as described above from which sRNAs (less than 200bp long) were size-selected using the RNAeasy Mini kit (Qiagen). Purified sRNA (20-30nt) samples were obtained by gel extraction.  Gel slices were resuspended in 0.4 M sodium acetate (pH 5.2) overnight at 4oC, and sRNA were collected by ethanol precipitation.
Sequencing libraries were prepared using the Illumina TruSeq Small RNA Library Preparation kit using 25ng of sRNA.

value: dcr1D (975 h+ dcr1D::NatMX6)

tag: treatment

value: N starvation

tag: time

value: 24h after -N


title: H3K27ac-C2_SKM_rep1

description: Replicate 1 of C2+SKM iPS cells (H3K27ac)


title: H3K9me2_ChIPSeq_banded_aorta3

value: PCM1-positive nuclei from left ventricle of the heart

value: Sprague Dawley


title: Sox17E57KOct4_coopseq (POU bound)


title: H3K9me2_ChIPSeq_exercise2


title: C4_SKM_early_rep3

description:
Replicate 3 of C4+SKM early cells transduced with ZFATF1, ZFATF2, ZFATF5, Sox2, Klf4, and c-Myc
processed data file: C4_SKM_early_genes_fpkm.txt, mouse_gene_exp_diff.txt

data_processing:
Illumina Casava 1.8.2 software used for basecalling
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence
Mouse samples were mapped to mm10 with ZFATF1-5 sequences in the reference genome with TopHat 2.1.1.
Transcripts were assembled with Cufflinks 2.2.1.
Differential expression was determined with Cuffdiff, and data were visualized with CummeRbund.
genome build: mm10
processed data files format and content: tab-delimited text files include FPKM values for each sample; tab-delimited files for pairwise comparisons

growth_protocol: Oct4-MerCreMer; Tomato/GFP MEFs were grown in DMEM supplemented with 10% fetal bovine serum on plates coated with 0.1% gelatin. Mouse E14T ES cells and iPS cells were grown in knock-out DMEM supplemented with 15% fetal bovine serum, 1% nonessential amino acids, 2 mM L-glutamine, 1 x 10^3 units/mL leukemia inhibitory factor, 1 mM sodium pyruvate, and 100 µM beta-mercaptoethanol on irradiated MEFs. Prior to harvesting RNA, cells on irradiated MEFs were grown on 0.1% gelatin for one passage. During reprogramming, cells were treated with 100 nM 4-hydroxytamoxifen to induce Cre activation and subsequent expression of GFP. Cells were maintained in a humidified 37°C incubator with 5% CO2.

extract_protocol:
Cells were harvested, RNA was extracted with RNeasy Mini kit (Qiagen #74104), and treated with DNase I.
Libraries for RNA samples were prepared from 50 ng of total RNA and indexed with Illumina’s TruSeq RNA Sample Preparation Kit v2 (RS-122-2001 and RS-122-2002). Multiplexed samples were sequenced on an Illumina HiSeq 2500 with a single 51 bp read and a 7 bp index read.

value: MEF-derived cells

tag: atfs

value: ZFATF1, ZFATF2, ZFATF5


title: Sox5Oct4_coopseq (Sox bound)


title: Sox18Oct4_coopseq (POU bound)


title: H3K9me2_ChIPSeq_control4

value: Male Sprague Dawley rats of ~280 g not assigned to the exercise protocol were used as control.


title: dcr1D 4h (sRNA-seq)

description: small RNA

value: 4h after -N


title: dcr1D 2h (sRNA-seq)

description: small RNA

value: 2h after -N


title: H3K9me2_ChIPSeq_exercise1

source_name: exercise 1

value: anti-H3K9me2, clone MABI 0307 (Active Motif, La Hulpe, Belgium)

value: Male Sprague Dawley rats of ~280 g were assigned to interval training on a treadmill (Columbus Instruments, OH, US) with 25 degree inclination, with 5 days of acclimatization with running velocity for 6 m/min for 30, 45, 60, 75, 90 and 120 min. Thereafter interval training was performed 6 days a week with 12 x 8 min intervals with 2 min resting period (6 m/min) after a warm-up period at 10 m/min. Exercise training was performed over six weeks at a running speed of 15 m/min increasing with 2 m/min each week. Rats not able to fulfill the exercise protocol were withdrawn from the study.


title: dcr1D 1h (sRNA-seq)

description: small RNA

value: 1h after -N


title: C4_SKM_early_rep2

description:
Replicate 2 of C4+SKM early cells transduced with ZFATF1, ZFATF2, ZFATF5, Sox2, Klf4, and c-Myc
processed data file: C4_SKM_early_genes_fpkm.txt, mouse_gene_exp_diff.txt


title: C4_SKM_early_rep1

description:
Replicate 1 of C4+SKM early cells transduced with ZFATF1, ZFATF2, ZFATF5, Sox2, Klf4, and c-Myc
processed data file: C4_SKM_early_genes_fpkm.txt, mouse_gene_exp_diff.txt


title: Sox17Oct4_coopseq (POU bound)

value: DNA target oligos


title: clr4D 24h (sRNA-seq)

description: small RNA

value: clr4D (975 h+ clr4D::NatMX6)


title: C3_SKM_early_rep3

description:
Replicate 3 of C3+SKM early cells transduced with ZFATF1, ZFATF2, ZFATF4, Sox2, Klf4, and c-Myc
processed data file: C3_SKM_early_genes_fpkm.txt, mouse_gene_exp_diff.txt

value: ZFATF1, ZFATF2, ZFATF4


title: clr4D 10h (sRNA-seq)

description: small RNA

value: 10h after -N


title: clr4D 4h (sRNA-seq)

description: small RNA


title: Sox15Oct4_coopseq (POU bound)

description:
Contains monomer bound for downstream processing. Contains raw data from 2 replicates.
Processed data file: coopseq_Chang_allData.txt


title: C3_SKM_early_rep2

description:
Replicate 2 of C3+SKM early cells transduced with ZFATF1, ZFATF2, ZFATF4, Sox2, Klf4, and c-Myc
processed data file: C3_SKM_early_genes_fpkm.txt, mouse_gene_exp_diff.txt


title: dcr1D 0h (sRNA-seq)

description: small RNA

value: dcr1D (975 h+ dcr1D::NatMX6)

value: control

value: 0h after -N


title: clr4D 2h (sRNA-seq)

description: small RNA

value: N starvation


title: Sox5Oct4_coopseq (POU bound)


title: clr4D 1h (sRNA-seq)

description: small RNA


title: dcr1D 10h (sRNA-seq)

description: small RNA


title: C3_SKM_early_rep1

description:
Replicate 1 of C3+SKM early cells transduced with ZFATF1, ZFATF2, ZFATF4, Sox2, Klf4, and c-Myc
processed data file: C3_SKM_early_genes_fpkm.txt, mouse_gene_exp_diff.txt


title: Sox2Oct6_coopseq (POU bound)


title: ago1D 10h (sRNA-seq)

description: small RNA

value: ago1D (975 h+ ago1D::NatMX6)


title: ago1D 24h (sRNA-seq)

description: small RNA

value: 24h after -N


title: Sox2Brn4_coopseq (POU bound)


title: C2_SKM_early_rep3

description:
Replicate 3 of C2+SKM early cells transduced with ZFATF1, ZFATF2, ZFATF3, Sox2, Klf4, and c-Myc
processed data file: C2_SKM_early_genes_fpkm.txt, mouse_gene_exp_diff.txt

value: MEF-derived cells

value: ZFATF1, ZFATF2, ZFATF3


title: ago1D 4h (sRNA-seq)

description: small RNA

value: 4h after -N


title: Sox2Brn2_coopseq (POU bound)


title: ago1D 2h (sRNA-seq)

description: small RNA

value: 2h after -N


title: ago1D 1h (sRNA-seq)

description: small RNA


title: Sox2Oct4_coopseq (POU bound)


title: C2_SKM_early_rep2

description:
Replicate 2 of C2+SKM early cells transduced with ZFATF1, ZFATF2, ZFATF3, Sox2, Klf4, and c-Myc
processed data file: C2_SKM_early_genes_fpkm.txt, mouse_gene_exp_diff.txt


title: ago1D 0h (sRNA-seq)

description: small RNA


title: C2_SKM_early_rep1

description:
Replicate 1 of C2+SKM early cells transduced with ZFATF1, ZFATF2, ZFATF3, Sox2, Klf4, and c-Myc
processed data file: C2_SKM_early_genes_fpkm.txt, mouse_gene_exp_diff.txt


title: wt 24h (sRNA-seq)

description: small RNA


title: Brn4_specseq (unbound)

description:
Contains unbound reads needed to calculate the relative binding energy.
Processed data file: Brn4_specseq_processed.txt

data_processing:
Library strategy: Spec-seq
Extracting binding sites sequence from raw reads. Each read follows the format of GAGTCGTCTCGTCAGCAC-binding site-CCGTAGAGCACTCAGGTC.
Count read numbers for each variant, classified by dimer bound, Sox bound, POU bound, or unbound in coopseq and classfied by bound or unbound in specseq.
For specseq, calculate the bound/unbound ratio for each variant, then rank variants according to their relative affinities.
For coopseq, the cooperativity, omega, is calculated by (Dimer bound x unbound)/(Sox bound x POU bound).
Supplementary_files_format_and_content: Tab delimited text files.  See Processed_data_description.txt.


title: Brn2_specseq (unbound)

description:
Contains unbound reads needed to calculate the relative binding energy.
Processed data file: Brn2_specseq_processed.txt


title: wt 2h (sRNA-seq)

description: small RNA


title: wt 10h (sRNA-seq)

description: small RNA


title: Oct4_SKM_early_rep2

description:
Replicate 2 of Oct4+SKM early cells transduced with an Oct4, Sox2, Klf4, and c-Myc
processed data file: OSKM_early_genes_fpkm.txt, mouse_gene_exp_diff.txt


title: Oct4_SKM_early_rep3

description:
Replicate 3 of Oct4+SKM early cells transduced with an Oct4, Sox2, Klf4, and c-Myc
processed data file: OSKM_early_genes_fpkm.txt, mouse_gene_exp_diff.txt


title: wt 4h (sRNA-seq)

description: small RNA


title: wt 1h (sRNA-seq)

description: small RNA


title: Oct4_SKM_early_rep1

description:
Replicate 1 of Oct4+SKM early cells transduced with an Oct4, Sox2, Klf4, and c-Myc
processed data file: OSKM_early_genes_fpkm.txt, mouse_gene_exp_diff.txt


title: clr4D 0h (sRNA-seq)

description: small RNA


title: clr4D 24h, input for sample 30, rep1 (ChIP-Seq)

description: Sample 56

data_processing:
Map to S pombe genome with Bowtie2 2.2.7
Identify H3K9me2 peaks by MACS2 2.1 (--nomodel --extsize 200 --keep-dup auto)
Identify differentially bound peaks by DiffBind 1.16.3 which uses DESeq2 using replicate 1/3/4/5/6 of wt 0h and 24h
Genome_build: S. pombe ASM294v2.22
Supplementary_files_format_and_content: bedgraph
Supplementary_files_format_and_content: Text.  List of differentially bound peaks of H3K9me2 between wt 0h and wt 24h

growth_protocol: Cells grown in EMM 225ug/ml adenine/histidie/leucine/lysine/uracil (OD~1) at 32C, and were washed twice with EMM -N.  Cells were resuspended in EMM -N and incubated at 32C.

extract_protocol:
Equal number of cells at each time point was cross-linked with 1% formaldehyde at room temperature for 15min. Crosslinking was quenched with 0.125M glycine for 5min and the cells were washed 3 times with cold TBS. Cell pellets (about 2x109 cells) were resuspended in 400μl lysis buffer (20mM Hepes, 0.5M NaCl, 1mM EDTA, 1% Triton-X, 0.1% Na-Deoxycholate, 0.1% SDS, 1mM PMSF and 5mM Benzamidine) and lysed with glass beads. Whole cell extracts (WCEs) were sonicated for 10min using the Q-Sonica waterbath sonicator with final energy output of roughly 16000J producing chromatin 100-400bps in size. 2-4ug of antibody (abcam 1220 for H3K9me2 and active motif 61013 for H3K9me3) plus 10μl Protein A Dynabeads were added to each extract and incubated for 3 hours at 4 deg. Beads were then washed and eluted with TE plus 0.67% SDS at 65ºC for 15min. Samples were incubated at 65 ºC for 8 hours to reverse the crosslinks and then treated with RNaseA and proteinase K to remove RNA and proteins, respectively. Finally, DNA fragments were isolated by Phenol-Chloroform extraction followed by and ethanol precipitation.
Libraries were made with NEBNext Ultra DNA Library rep Kit for Illumina (NEB E7370L)

tag: ip antibody


title: Sox17E57KOct4_coopseq (dimer bound)

description:
Contains dimer bound for downstream processing. Contains raw data from 2 replicates.
Processed data file: coopseq_Chang_allData.txt


title: clr4D 1h, input for sample 29, rep1 (ChIP-Seq)

description: Sample 55


title: Oct4_SKM_rep4

description:
Replicate 4 of Oct4+SKM iPS cells transduced with an Oct4, Sox2, Klf4, and c-Myc
processed data file: OSKM_genes_fpkm.txt, mouse_gene_exp_diff.txt

value: MEF-derived iPS cells


title: clr4D 0h, input for sample 28, rep1 (ChIP-Seq)

description: Sample 54


title: wt 24h, input for sample 27, rep1 (ChIP-Seq)

description: Sample 53


title: Sox18Oct4_coopseq (dimer bound)


title: Oct4_SKM_rep3

description:
Replicate 3 of Oct4+SKM iPS cells transduced with an Oct4, Sox2, Klf4, and c-Myc
processed data file: OSKM_genes_fpkm.txt, mouse_gene_exp_diff.txt


title: Oct4_SKM_rep2

description:
Replicate 2 of Oct4+SKM iPS cells transduced with an Oct4, Sox2, Klf4, and c-Myc
processed data file: OSKM_genes_fpkm.txt, mouse_gene_exp_diff.txt


title: Sox17Oct4_coopseq (dimer bound)


title: wt 0h, input for sample 25, rep1 (ChIP-Seq)

description: Sample 51


title: ago1D 24h, input for sample 24, rep2 (ChIP-Seq)

description: Sample 50


title: Oct4_SKM_rep1

description:
Replicate 1 of Oct4+SKM iPS cells transduced with an Oct4, Sox2, Klf4, and c-Myc
processed data file: OSKM_genes_fpkm.txt, mouse_gene_exp_diff.txt


title: ago1D 0h, input for sample 23, rep2 (ChIP-Seq)

description: Sample 49


title: Sox15Oct4_coopseq (dimer bound)


title: ago1D 24h, input for sample 22, rep1 (ChIP-Seq)

description: Sample 48


title: Oct6_specseq (unbound)

description:
Contains unbound reads needed to calculate the relative binding energy.
Processed data file: Oct6_specseq_processed.txt


title: Sox5Oct4_coopseq (dimer bound)


title: wt 0h (sRNA-seq)

description: small RNA


title: Empty_SKM_rep2

description:
Replicate 2 of Empty+SKM early cells transduced with an Empty control, Sox2, Klf4, and c-Myc
processed data file: Empty_SKM_genes_fpkm.txt, mouse_gene_exp_diff.txt

value: Empty


title: Sox2Oct6_coopseq (dimer bound)

description:
Contains dimer bound for downstream processing. Contains raw data from 5 replicates.
Processed data file: coopseq_Chang_allData.txt


title: clr4D 0h, input for sample 18, rep2 (ChIP-Seq)

description: Sample 44


title: clr4D 24h, input for sample 20, rep2 (ChIP-Seq)

description: Sample 46


title: Sox2Brn4_coopseq (dimer bound)


title: clr4D 24h, input for sample 17, rep1 (ChIP-Seq)

description: Sample 43


title: clr4D 1h, input for sample 19, rep2 (ChIP-Seq)

description: Sample 45

value: 1h after -N


title: Sox2Brn2_coopseq (dimer bound)

value: DNA target oligos


title: clr4D 1h, input for sample 16, rep1 (ChIP-Seq)

description: Sample 42

value: clr4D (975 h+ clr4D::NatMX6)


title: clr4D 0h, input for sample 15, rep1 (ChIP-Seq)

description: Sample 41


title: wt 1h, input for sample 26, rep1 (ChIP-Seq)

description: Sample 52


title: Empty_SKM_rep1

description:
Replicate 1 of Empty+SKM early cells transduced with an Empty control, Sox2, Klf4, and c-Myc
processed data file: Empty_SKM_genes_fpkm.txt, mouse_gene_exp_diff.txt


title: C4_SKM_rep4

description:
Replicate 4 of C4+SKM iPS cells transduced with ZFATF1, ZFATF2, ZFATF5, Sox2, Klf4, and c-Myc
processed data file: C4_SKM_genes_fpkm.txt, mouse_gene_exp_diff.txt

value: ZFATF1, ZFATF2, ZFATF5


title: wt 24h, input for sample 14, rep6 (ChIP-Seq)

description: Sample 40


title: wt 0h, input for sample 13, rep6 (ChIP-Seq)

description: Sample 39


title: C4_SKM_rep3

description:
Replicate 3 of C4+SKM iPS cells transduced with ZFATF1, ZFATF2, ZFATF5, Sox2, Klf4, and c-Myc
processed data file: C4_SKM_genes_fpkm.txt, mouse_gene_exp_diff.txt


title: Brn4_specseq (bound)

description:
Contains bound reads needed to calculate the relative binding energy.
Processed data file: Brn4_specseq_processed.txt


title: wt 0h, input for sample 11, rep5 (ChIP-Seq)

description: Sample 37


title: C4_SKM_rep2

description:
Replicate 2 of C4+SKM iPS cells transduced with ZFATF1, ZFATF2, ZFATF5, Sox2, Klf4, and c-Myc
processed data file: C4_SKM_genes_fpkm.txt, mouse_gene_exp_diff.txt


title: wt 24h, input for sample 12, rep5 (ChIP-Seq)

description: Sample 38


title: wt 24h, input for sample 6, rep2 (ChIP-Seq)

description: Sample 36


title: Brn2_specseq (bound)

description:
Contains bound reads needed to calculate the relative binding energy.
Processed data file: Brn2_specseq_processed.txt


title: ago1D 0h, input for sample 21, rep1 (ChIP-Seq)

description: Sample 47


title: Empty_SKM_rep3

description:
Replicate 3 of Empty+SKM early cells transduced with an Empty control, Sox2, Klf4, and c-Myc
processed data file: Empty_SKM_genes_fpkm.txt, mouse_gene_exp_diff.txt


title: wt 1h, input for sample 5, rep2 (ChIP-Seq)

description: Sample 35


title: wt 0h, input for sample 4, rep2 (ChIP-Seq)

description: Sample 34


title: wt 24h, input for sample 3, rep1 (ChIP-Seq)

description: Sample 33


title: C4_SKM_rep1

description:
Replicate 1 of C4+SKM iPS cells transduced with ZFATF1, ZFATF2, ZFATF5, Sox2, Klf4, and c-Myc
processed data file: C4_SKM_genes_fpkm.txt, mouse_gene_exp_diff.txt


title: C3_SKM_rep3

description:
Replicate 3 of C3+SKM iPS cells transduced with ZFATF1, ZFATF2, ZFATF4, Sox2, Klf4, and c-Myc
processed data file: C3_SKM_genes_fpkm.txt, mouse_gene_exp_diff.txt

value: ZFATF1, ZFATF2, ZFATF4


title: wt 1h, input for sample 2, rep1 (ChIP-Seq)

description: Sample 32


title: C3_SKM_rep2

description:
Replicate 2 of C3+SKM iPS cells transduced with ZFATF1, ZFATF2, ZFATF4, Sox2, Klf4, and c-Myc
processed data file: C3_SKM_genes_fpkm.txt, mouse_gene_exp_diff.txt


title: minus3_rep1

description:
Replicate 1 of HEK293 cells transfected with ATF (3ZF-NLS-VP64) lipofectamine 2000.
processed data file: minus3_fpkm_tracking.txt, human_gene_exp_diff.txt

data_processing:
Illumina Casava 1.8.2 software used for basecalling
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence
Human samples were mapped to hg19 using TopHat 2.0.6.
Transcripts were assembled with Cufflinks 2.1.0.
Differential expression was determined with Cuffdiff, and data were visualized with CummeRbund.
genome build: hg19
processed data files format and content: tab-delimited text files include FPKM values for each sample; tab-delimited files for pairwise comparisons

organism: Homo sapiens

value: 3ZF-NLS-VP64 (EGR1 DBD)


title: plus3_rep1

description:
Replicate 1 of HEK293 cells transfected with ATF (ID-3ZF-NLS-VP64) lipofectamine 2000.
processed data file: plus3_fpkm_tracking.txt, human_gene_exp_diff.txt

value: ID-3ZF-NLS-VP64 (EGR1 DBD)


title: Sox2Oct4_coopseq (dimer bound)


title: wt 0h, input for sample 1, rep1 (ChIP-Seq)

description: Sample 31

value: control

value: 0h after -N


title: C3_SKM_rep1

description:
Replicate 1 of C3+SKM iPS cells transduced with ZFATF1, ZFATF2, ZFATF4, Sox2, Klf4, and c-Myc
processed data file: C3_SKM_genes_fpkm.txt, mouse_gene_exp_diff.txt


title: plus2_rep1

description:
Replicate 1 of HEK293 cells transfected with ATF (ID-2ZF-NLS-VP64) lipofectamine 2000.
processed data file: plus2_fpkm_tracking.txt, human_gene_exp_diff.txt

value: ID-2ZF-NLS-VP64 (EGR1 DBD)


title: wt 24h, H3K9me3, rep1 (ChIP-Seq)

description: Sample 27

value: N starvation

value: 24h after -N


title: clr4D 0h, H3K9me3, rep1 (ChIP-Seq)

description: Sample 28


title: wt 1h, H3K9me3, rep1 (ChIP-Seq)

description: Sample 26


title: wt 0h, H3K9me3, rep1 (ChIP-Seq)

description: Sample 25


title: C2_SKM_rep4

description:
Replicate 4 of C2+SKM iPS cells transduced with ZFATF1, ZFATF2, ZFATF3, Sox2, Klf4, and c-Myc
processed data file: C2_SKM_genes_fpkm.txt, mouse_gene_exp_diff.txt

value: ZFATF1, ZFATF2, ZFATF3


title: C2_SKM_rep3

description:
Replicate 3 of C2+SKM iPS cells transduced with ZFATF1, ZFATF2, ZFATF3, Sox2, Klf4, and c-Myc
processed data file: C2_SKM_genes_fpkm.txt, mouse_gene_exp_diff.txt


title: minus2_rep1

description:
Replicate 1 of HEK293 cells transfected with ATF (2ZF-NLS-VP64) lipofectamine 2000.
processed data file: minus2_fpkm_tracking.txt, human_gene_exp_diff.txt

value: 2ZF-NLS-VP64 (EGR1 DBD)


title: mock-rep1

description:
Replicate 1 of HEK293 cells mock transfected with lipofectamine 2000 (no DNA).
processed data file: mock_fpkm_tracking.txt, human_gene_exp_diff.txt


title: C2_SKM_rep1

description:
Replicate 1 of C2+SKM iPS cells transduced with ZFATF1, ZFATF2, ZFATF3, Sox2, Klf4, and c-Myc
processed data file: C2_SKM_genes_fpkm.txt, mouse_gene_exp_diff.txt


title: Oct6_specseq (bound)

description:
Contains bound reads needed to calculate the relative binding energy.
Processed data file: Oct6_specseq_processed.txt


title: ago1D 0h, H3K9me2, rep2 (ChIP-Seq)

description: Sample 23

value: ago1D (975 h+ ago1D::NatMX6)


title: C2_SKM_rep2

description:
Replicate 2 of C2+SKM iPS cells transduced with ZFATF1, ZFATF2, ZFATF3, Sox2, Klf4, and c-Myc
processed data file: C2_SKM_genes_fpkm.txt, mouse_gene_exp_diff.txt

value: MEF-derived iPS cells


title: ago1D 24h, H3K9me2, rep1 (ChIP-Seq)

description: Sample 22


title: ago1D 24h, H3K9me2, rep2 (ChIP-Seq)

description: Sample 24


title: ago1D 0h, H3K9me2, rep1 (ChIP-Seq)

description: Sample 21


title: RNAseq ms23 maize anthers, line 610_2 (sterile), 2.0 mm

first: Blake

middle: C.

last: Meyers

address: 975 N Warson Road

institute: Donald Danforth Plant Science Center

data_processing:
Small RNA-seq: the adapter sequences were trimmed from raw sequence reads, and then the small RNA sequences were trimmed to the lengths between 15 and 34 nucleotides. Small RNA reads were mapped to the maize genome (Maize Genome Database).
RNA-seq: the adapter sequences were trimmed from raw sequence reads, and then the RNA sequences were trimmed to the lengths of 50 nucleotides. RNA-seq reads were mapped to the maize genome (Maize Genome Database).
Genome_build: ZmaysB73_wgs_1.0 (https://www.ncbi.nlm.nih.gov/assembly/GCA_000275765.1)
Supplementary_files_format_and_content: Text containing distinct smallRNA's and RNA-seq reads with their raw abundance after removing the adapter sequences

organism: Zea mays

source_name: RNAseq ms23 maize anthers, line 610_2

extract_protocol: Small RNA libraries and RNA-seq libraries were constructed from total RNA isolated from anthers of Zea mays using Plant RNA Reagent (Invitrogen, Life Technologies, USA). The sRNA library preparation protocol is based on Illumina’s sRNA Sequencing Sample Preparation Guide (Illumina, San Diego, CA). And the RNA-seq library preparation protocol is based on Illumina’s TruSeq RNA  Sequencing Sample Preparation Guide (Illumina, San Diego, CA) The libraries were sequenced on an Illumina HiSeq at the Delaware Biotechnology Institute (Newark, DE).

tag: line

value: line 610_2 (sterile)

tag: genotype/variation

value: ms23 mutant

tag: tissue

value: anther


title: ES_rep3

description:
Replicate 3 of ES cells
processed data file: ES_genes_fpkm.txt, mouse_gene_exp_diff.txt

source_name: blastocyst-derived embryonic stem cells

tag: cell type

value: ES

tag: strain


title: clr4D 24h, H3K9me3, rep1 (ChIP-Seq)

description: Sample 30

value: clr4D (975 h+ clr4D::NatMX6)


title: ES_rep4

description:
Replicate 4 of ES cells
processed data file: ES_genes_fpkm.txt, mouse_gene_exp_diff.txt


title: clr4D 24h, H3K9me2, rep2 (ChIP-Seq)

description: Sample 20


title: RNAseq ms23 maize anthers, line 610_16 (sterile), 2.0 mm

source_name: RNAseq ms23 maize anthers, line 610_16

value: line 610_16 (sterile)


title: clr4D 1h, H3K9me3, rep1 (ChIP-Seq)

description: Sample 29

value: 1h after -N


title: ES_rep1

description:
Replicate 1 of ES cells
processed data file: ES_genes_fpkm.txt, mouse_gene_exp_diff.txt


title: ES_rep2

description:
Replicate 2 of ES cells
processed data file: ES_genes_fpkm.txt, mouse_gene_exp_diff.txt


title: clr4D 1h, H3K9me2, rep2 (ChIP-Seq)

description: Sample 19


title: MEF_rep4

description:
Replicate 4 of MEF
processed data file: MEF_genes_fpkm.txt, mouse_gene_exp_diff.txt

value: MEF


title: RNAseq ms23 maize anthers, line 610_2 (sterile), 1.5 mm


title: clr4D 0h, H3K9me2, rep2 (ChIP-Seq)

description: Sample 18

value: control


title: RNAseq ms23 maize anthers, line 610_16 (sterile), 1.0 mm


title: RNAseq ms23 maize anthers, line 610_2 (sterile), 1.0 mm


title: MEF_rep3

description:
Replicate 3 of MEF
processed data file: MEF_genes_fpkm.txt, mouse_gene_exp_diff.txt


title: clr4D 1h, H3K9me2, rep1 (ChIP-Seq)

description: Sample 16


title: MEF_rep2

description:
Replicate 2 of MEF
processed data file: MEF_genes_fpkm.txt, mouse_gene_exp_diff.txt


title: Sample_G401_input

city: New York

last: Koche

state: New York

institute: Memorial Sloan Kettering Cancer Center

data_processing:
ChIP-seq reads were filtered for quality and 3' trimmed for adapter sequences.
Reads were aligned to human assembly hg19 using bowtie2 (v2.2.2) with default parameters.
To create density files, the reads were extended to 200 bp, the signal was then normalized to ten million mapped reads and converted to BigWig using BEDTools.
Genome_build: hg19
Supplementary_files_format_and_content: BigWig files containing read densities normalized to 10 million reads

source_name: kidney

growth_protocol: Cells were cultured in 5% CO2 in a humidified atmosphere at 37 ˚C in Dulbecco’s Modified Eagle medium with high glucose (DMEM-HG) supplemented with 10 % FCS and antibiotics (100 U / ml penicillin and 100 µg / ml streptomycin).

extract_protocol:
Extraction protocol not provided.
ChIP-seq libraries were constructed with the NEBNext ChIP-seq library prep kit following the manufacturer’s recommended protocol (New England Biolabs, Ipswich, MA, USA).

treatment_protocol: No treatment

tag: tumor type

value: rhabdoid tumor

value: kidney

tag: chip antibody


title: MEF_rep1

description:
Replicate 1 of MEF
processed data file: MEF_genes_fpkm.txt, mouse_gene_exp_diff.txt


title: wt 24h, H3K9me2, rep6 (ChIP-Seq)

description: Sample 14

value: N starvation


title: clr4D 24h, H3K9me2, rep1 (ChIP-Seq)

description: Sample 17

value: 24h after -N


title: TW-1-M-3-3

city: hangzhou

first: fengjie

last: yuan

state: Zhejiang

address: shiqiao road 198

country: China

institute: Zhejiang Academy of Agricultural Sciences

data_processing:
Illumina Casava1.8 software used for basecalling.
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence by Trimmomatic, then mapped to whole genome using TopHat
Fragments Per Kilobase of exon model per Million mapped reads (FPKM) were calculated.
Genome_build: Glycine_max_v2.0 (ftp://ftp.ncbi.nlm.nih.gov/genomes/all/GCF_000004515.4_Glycine_max_v2.0)
Supplementary_files_format_and_content: gene_expression.txt reports FPKMs

organism: Glycine max

source_name: soybean seed

growth_protocol: 100 seeds of each soybean lpa line were planted in a Petri dish containing B5 agar gel (50 seeds per 15-mm Petri dish) and were placed in a 25°C germination chamber in the dark for 4 d

extract_protocol:
Tissue samples were stored at -80°C prior to use, and RNA was harvested using Trizol reagent. 1 ug of total RNA for the construction of sequencing libraries.
RNA libraries were prepared for sequencing using standard Illumina protocols

value: low phytate soybean mutant TW-1-M

tag: seed field emergence

value: significantly higher rate compared to TW-1

value: germination of soybean seed

value: seed

tag: time of seed harvest

value: emerging primary root reached 1mm in length


title: Sample_G401_Biorbyt_PGBD5

value: Rabbit anti-PGBD5, orb13159,Biorbyt, Berkeley, CA, USA


title: clr4D 0h, H3K9me2, rep1 (ChIP-Seq)

description: Sample 15

value: 0h after -N


title: RNAseq ms23 maize anthers, line 610_1 (sterile), 0.7 mm

source_name: RNAseq ms23 maize anthers, line 610_1

value: line 610_1 (sterile)

value: ms23 mutant

value: anther


title: wt 0h, H3K9me2, rep6 (ChIP-Seq)

description: Sample 13


title: RNAseq ms23 maize anthers, line 610_19 (sterile), 0.4 mm

source_name: RNAseq ms23 maize anthers, line 610_19

value: line 610_19 (sterile)


title: TW-1-M-3-2

tag: developmental stage


title: RNAseq ms23 maize anthers, line 610_16 (sterile), 1.5 mm

value: line 610_16 (sterile)


title: wt 24h, H3K9me2, rep5 (ChIP-Seq)

description: Sample 12


title: sRNA ms23 maize anthers, line 610_16 (sterile), 2.0 mm

source_name: sRNA ms23 maize anthers, line 610_16


title: TW-1-M-3-1


title: YMD3265_saturated_Sau3AI

city: Stanford

first: Seungsoo

last: Kim

state: CA

address: 265 Campus Dr

department: Chemical and Systems Biology

institute: Stanford University

description:
SpSu.32000.bed
Surep-rDNA.bed

data_processing:
Library strategy: Hi-C-seq
Basecalls were converted to FASTQ using bcl2fastq 2.17
Sequencing reads were quality-trimmed (cutadapt -q 20) trimmed of adapter sequences and then trimmed up to the ligation junction (if present), excluding read pairs in which either read was shorter than 20 bp after trimming.
Reads were independently mapped using bowtie2 2.2.3 using the parameter set --very-sensitive. For interspecific hybrids, reads were mapped to a combined reference containing both species references, where if secondary mappings were present the best alignment must have a score ≥ 10 greater than the next best alignment. For intraspecific S. cerevisiae hybrids, reads were mapped separately to both strain references, keeping only read pairs mapping to both—perfectly to one reference and with ≥ 2 mutations including ≥ 1 substitution to the other. In both cases, only read pairs in which both reads mapped with MAPQ ≥ 30 were used.
PCR duplicates (with identical fragment start and end positions) were removed, as were read pairs mapping within 1 kb of each other or in the same restriction fragment, which represent either unligated or invalid ligation products.
The genome was then binned into 32 kb fragments (except the last fragment of each chromosome), and the number of read pairs mapping to each 32 kb genomic bin was counted based on the position of the restriction sites that were ligated together. Due to gaps in the reference genomes of S. uvarum, some repetitive sequences were only represented once and therefore artifactually mapped uniquely; therefore, reads mapping to annotated repetitive sequences were masked from further analysis. Similarly, gaps in the S. paradoxus reference led to mismapping of reads to the corresponding S. cerevisiae sequence; therefore, for S. cerevisiae x S. paradoxus libraries we masked regions in the S. cerevisiae genome where > 1 read from a S. paradoxus Hi-C library mapped, and vice versa. We took a similar approach to mask regions of the Y12 and DBVPG6044 references that were prone to mismapping, as estimated by haploid Y12 and DBVPG6044 Hi-C libraries. For knock-in experiments, the HAS1pr-TDA1pr region was masked to account for its altered genomic location. The resulting matrices were then normalized by excluding the diagonal (interactions within the same genomic bin), filtering out rows/columns with an average of less than 1 read per bin, and then multiplying each entry by the total number of read pairs divided by the column and row sums.
Genome_build: Saccharomyces cerevisiae R64.2.1 from SGD and Saccharomyces cerevisiae and Saccharomyces uvarum (bayanus var. uvarum) from saccharomycessensustricto.org, with minor modifications; Y12 and DBVPG6044 references generated by Pacbio sequencing
Supplementary_files_format_and_content: tab-delimited text files ending in "matrix.txt" include the raw read counts and normalized interaction frequencies for each pair of 32 kb genomic bins; those ending in "rowsums.txt" include the raw sums across each row of these matrices. BED files include either the genomic regions included in each matrix bin or those masked from the matrices.

organism: Saccharomyces paradoxus x Saccharomyces uvarum

growth_protocol: Cells were grown overnight, shaking at 30C (room temperature for S. uvarum) in YPD media (1% yeast extract, 2% peptone, 2% dextrose), or for "raffinose" cultures, YP + 2% raffinose, or for "galactose" cultures, YP + 2% galactose. For non-saturated cultures, fresh cultures were inoculated to OD600=0.1 in the same media and grown until OD600 = 0.6-0.8.

extract_protocol:
Cells were lysed using glass beads and chromatin was extracted, digested using restriction enzyme Sau3AI or HindIII, gap-filled in using biotinylated dCTP, and ligated. Crosslinks were reversed and DNA was purified by phenol/chloroform extraction.
Libraries were prepared according to manufacturer's instructions using the KAPA Hyper Prep kit.

treatment_protocol: Cells were crosslinked by resuspension and incubation in 1% formaldehyde in PBS for 20 minutes at room temperature, and then quenched by addition of 1% solid glycine, incubation for 20 minutes, and a PBS wash. For nocodazole culture, media was supplemented with 15 ug/ml nocodazole and grown for an additional 2 hours prior to crosslinking.

tag: growth conditions

value: saturated

tag: growth medium

value: YPD

tag: growth phase

tag: growth temperature

tag: restriction enzyme


title: TW-1-M-2-3

value: 12h after the seeds finished imbibition


title: YMD3265_exponential_Sau3AI

description:
SpSu.32000.bed
Surep-rDNA.bed

value: exponential


title: wt 24h, H3K9me2, rep4 (ChIP-Seq)

description: Sample 10

tag: treatment

value: N starvation

tag: time

value: 24h after -N


title: YMD3270_saturated_Sau3AI

description:
ScSu.32000.bed
Surep-rDNA-ScYMR290-291.bed

organism: Saccharomyces cerevisiae x Saccharomyces uvarum


title: YMD3270_exponential_Sau3AI


title: sRNA ms23 maize anthers, line 610_2 (sterile), 2.0 mm

source_name: sRNA ms23 maize anthers, line 610_2

tag: line

value: line 610_2 (sterile)


title: RNAseq ms23 maize anthers, line 610_19 (sterile), 0.7 mm

tag: genotype/variation


title: wt 0h, H3K9me2, rep5 (ChIP-Seq)

description: Sample 11

value: control

tag: ip antibody


title: TW-1-M-2-2

value: low phytate soybean mutant TW-1-M

value: significantly higher rate compared to TW-1

value: germination of soybean seed

tag: tissue

value: seed


title: YMD3269_saturated_Sau3AI

description:
ScSu.32000.bed
Surep-rDNA.bed


title: sRNA ms23 maize anthers, line 610_16 (sterile), 1.0 mm


title: TW-1-M-2-1


title: sRNA ms23 maize anthers, line 610_2 (sterile), 1.5 mm


title: YMD3268_saturated_Sau3AI

description:
ScSu.32000.bed
Surep-rDNA.bed


title: TW-1-M-1-3

value: imbibition stage


title: wt 24h, H3K9me2, rep3 (ChIP-Seq)

description: Sample 8

tag: strain


title: TW-1-M-1-2


title: sRNA ms23 maize anthers, line 610_19 (sterile), 0.7 mm

source_name: sRNA ms23 maize anthers, line 610_19

value: ms23 mutant


title: sRNA ms23 maize anthers, line 610_2 (sterile), 1.0 mm

value: anther


title: wt 0h, H3K9me2, rep3 (ChIP-Seq)

description: Sample 7

value: 0h after -N


title: YMD3267_saturated_Sau3AI

description:
ScSu.32000.bed
Surep-rDNA.bed


title: YMD3266_saturated_Sau3AI

description:
ScSu.32000.bed
Surep-rDNA.bed


title: sRNA ms23 maize anthers, line 610_16 (sterile), 1.5 mm

value: line 610_16 (sterile)


title: wt 1h, H3K9me2, rep2 (ChIP-Seq)

description: Sample 5

value: 1h after -N


title: wt 24h, H3K9me2, rep2 (ChIP-Seq)

description: Sample 6


title: YMD3263_exponential_Sau3AI

description:
ScSu.32000.bed
Surep-rDNA.bed

value: YPD


title: TW-1-3-3

value: low phytate soybean mutant TW-1

value: very low rate

value: emerging primary root reached 1mm in length


title: sRNA ms23 maize anthers, line 610_19 (sterile), 0.4 mm

value: line 610_19 (sterile)


title: TW-1-M-1-1


title: wt 0h, H3K9me2, rep2 (ChIP-Seq)

description: Sample 4


title: YMD3263_saturated_Sau3AI

description:
ScSu.32000.bed
Surep-rDNA.bed

value: saturated


title: wt 0h, H3K9me2, rep4 (ChIP-Seq)

description: Sample 9

value: control


title: TW-1-3-2


title: ILY456_nocodazole_Sau3AI

description:
ScSu.32000.bed
Surep-rDNA.bed

value: nocodazole

value: nocodazole-arrested


title: A8_rx3_strong_ZT21_DEX

city: Eggenstein-Leopoldshafen

first: Thomas

last: Dickmeis

address: Hermann-von-Helmholtz-Platz 1

department: ITG

country: Germany

institute: KIT

data_processing:
raw counts
Genome_build: Zv9

organism: Danio rerio

source_name: Larvae Total RNA

growth_protocol: 30  rx3 mutant and wildtype sibling embryos (1 dpf) from pooled clutches were separated and transferred into cell culture flasks containing E3 medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM MgSO4, 0.1% methylene blue). Embryos/larvae were kept at 28°C in an incubator under a 12 h light:12 h dark (LD) exposure.

extract_protocol:
Larvae were sampled in liquid nitrogen and stored at -80°C until further processing. RNA was extracted with TRIzol (Life Technologies) following the manufacturer’s instructions with minor modifications: 30 larvae were sampled in a volume of 1.5 ml TRIzol reagent. Samples were kept at least overnight at -80°C. In order to disrupt the cell membranes, the samples were passed four times through a syringe with a diameter of 0.45 mm.
The cDNA libraries for Sample 1-12 (untreated) were generated using the directional mRNA-seq sample preparation kit (#15018460, Rev.A, October 2010, Illumina) following the manufacturer’s protocol.  mRNAseq libraries of samples 13-20 (treated) were generated following the TruSeq mRNA kit protocol (Illumina). The size and concentration of the generated libraries were evaluated with a Bioanalyzer 2100 and adjusted to a concentration of 10 pM. Cluster and sequencing reactions were performed with the TruSeq SBS kit v3-HS and the paired-end cluster v3-cBot-HS kit.

treatment_protocol: For glucocorticoid treatment, the E3 medium was supplemented with 25 μM dexamethasone (DEX) solved in 0.1% DMSO

value: mixed TU, AB, WIK

value: dexamethasone

tag: age

value: 5 dpf

tag: zeitgeber time

tag: genotype

value: rx3 strong mutant


title: wt 24h, H3K9me2, rep1 (ChIP-Seq)

description: Sample 3

value: N starvation

value: 24h after -N


title: A7_rx3_strong_ZT15_DEX


title: ILY456_galactose_Sau3AI

description:
ScSu.32000.bed
Surep-rDNA.bed

value: galactose

value: YP+galactose

value: exponential


title: A6_rx3_strong_ZT09_DEX


title: TW-1-2-3

value: low phytate soybean mutant TW-1

tag: seed field emergence

value: germination of soybean seed

value: seed

tag: time of seed harvest

value: 12h after the seeds finished imbibition


title: TW-1-2-2

value: very low rate

tag: developmental stage


title: wt 0h, H3K9me2, rep1 (ChIP-Seq)

description: Sample 1

value: control

value: 0h after -N


title: ILY456_exponential_rep2_Sau3AI

description:
ScSu.32000.bed
Surep-rDNA.bed

tag: growth conditions

tag: growth medium

value: YPD


title: wt 1h, H3K9me2, rep1 (ChIP-Seq)

description: Sample 2

value: 1h after -N


title: sRNA ms23 maize anthers, line 610_1 (sterile), 0.7 mm

source_name: sRNA ms23 maize anthers, line 610_1

value: line 610_1 (sterile)

value: ms23 mutant

value: anther


title: A4_wild-type_ZT21_DEX

value: mixed TU, AB, WIK

value: dexamethasone

value: 5 dpf

value: Wild type


title: TW-1-2-1


title: ILY456_exponential_Sau3AI

description:
ScSu.32000.bed
Surep-rDNA.bed

value: exponential

tag: growth phase

tag: growth temperature


title: A5_rx3_strong_ZT03_DEX

tag: treatment

value: rx3 strong mutant


title: YMD3264_exponential_Sau3AI

description:
ScSp.32000.bed
Spgap-rDNA.bed

organism: Saccharomyces cerevisiae x Saccharomyces paradoxus

tag: restriction enzyme


title: ILY456_saturated_HindIII

description:
ScSu.32000.bed
Surep-rDNA.bed

value: saturated

value: HindIII


title: YMD3264_saturated_Sau3AI

description:
ScSp.32000.bed
Spgap-rDNA.bed


title: A3_wild-type_ZT15_DEX


title: A2_wild-type_ZT09_DEX


title: ILY456_saturated_Sau3AI

description:
ScSu.32000.bed
Surep-rDNA.bed


title: FY69_YDG613_exponential_Sau3AI

description:
ScSp.32000.bed
Spgap-rDNA.bed


title: FY69_YZB5-113_saturated_Sau3AI

description:
ScSu.32000.bed
Surep-rDNA.bed


title: TW-1-1-2

value: low phytate soybean mutant TW-1

value: germination of soybean seed

value: seed

value: imbibition stage


title: TW-1-1-3

value: very low rate


title: YZB5-113_exponential_Sau3AI

description:
ScSu.32000.bed
Suva.32000.bed
Surep-rDNA.bed

organism: Saccharomyces uvarum

value: YPD


title: TW-1-3-1

value: emerging primary root reached 1mm in length


title: A1_wild-type_ZT03_DEX


title: TW-1-1-1


title: YDG613_exponential_Sau3AI

description:
ScSp.32000.bed
Spar.32000.bed

organism: Saccharomyces paradoxus


title: Misc_WT rep 4

status: Public on Dec 04 2016

city: La Jolla

first: Kavitha

last: Mukund

state: California

address: 9500 Gilman Drive #0412

institute: University of California, San Diego

description:
processed data column: Sample_PP
DEseq2_ExonCounts.csv

data_processing:
RNA read mapping and the initial quality control was performed using the OSA algorithm
Reads mapped to mouse reference genome version mm10, with Refseq annotation
GenomicFeatures” package was used to build the gene model (corresponding to refgene annotation under version mm10) for counting reads
Count matrix normalization and differential analysis was performed using the “DEseq2”
Genome_build: mm10
Supplementary_files_format_and_content: .csv file of exon counts for 24393 genes

source_name: quadriceps, tibialis anterior and gastrocnemius

extract_protocol: RNA was extracted  using an RNeasy Mini Kit (Qiagen). Following cell lysis with 1.0 mL of Buffer RLT/TRIzol, 0.3 mL of chloroform was added and the sample was vortexed for 15 seconds then centrifuged for 15 minutes at 12,000g. The supernatant was removed and mixed with equal parts 70% ethanol. Samples were then added to an RNeasy Mini spin column and centrifuged. The column was washed with RW1 followed by RPE buffer. After washes, DEPC-treated water was added to the column and RNA was eluted from the columns with centrifugation. RNA concentrations were determined by sample absorbance at 260 nm using an ND-1000 Spectrophotometer (Thermo Scientific; Waltham, MA).

tag: cell type

value: MISC

tag: condition

value: WT


title: Matched control for AMB + LF rep 3

city: Sydney

first: Chi Nam Ignatius

last: Pang

state: NSW

address: School of Biotechnology and Biomolecular Sciences, UNSW Australia

department: The NSW Systems Biology Initiative

country: Australia

institute: The University of New South Wales

description:
4i
Illumina lane: 1

data_processing:
Counting: The number of reads mapped to each gene is counted using HTSeq (0.5.3p9) using the options “-s no -m union -t CDS -i gene_id”. Reads are mapped to the coding sequence (CDS) irrespective of the strand and reads found in ambiguous overlapping CDS were not counted. The GFF gene co-ordinates file was obtained from the Saccharomyces Genome Database and converted to GTF format using custom Perl scripts.
Genome_build: Saccharomyces cerevisiae S288C genome reference (R64-1-1 build)

organism: Saccharomyces cerevisiae S288C

source_name: Saccharomyces cerevisiae S288C

growth_protocol: The S. cerevisiae S288C strain was used for all RNA-seq analyses. Cells were grown in RPMI-1640 at 30 °C in a shaking incubator at 180 rpm. Amphotericin B (Sigma-Aldrich, Australia) was made to 1,600 µg/mL and bovine lactoferrin (MP Biomedical, Australia) was made to 5,120 µg/mL in sterile MilliQ water.   The cells at an ID20 were used for RNA-Seq analysis. At this point, cells are stressed but the confounding effects of cell death are avoided, allowing a direct comparison of the AMB and the AMB + LF treatment. The ID20 for AMB only treatment was 60 minutes. For the  AMB + LF treatment, the ID20 was close to 50 minutes and the cell counts at 60 minutes has dropped beyond the ID20 point, therefore 50 minutes was chosen as the time point for RNA-seq analyses. The same incubation time of 50 minutes and 60 minutes were applied for the matched control samples for AMB only and AMB + LF treatments, respectively. The LF only experiments were grown for 50 min at the ID20 of AMB + LF experiments to enable a comparable analysis between the treatments.

extract_protocol:
Equal volumes of RNAse zap treated 0.4 μm beads and lyophilised cells were bead beaten in a Minilys tissue homogenizer at 5,000 rpm for 2 min. Cells were cooled on ice at 1 min intervals. Total RNA was then isolated with the Qiagen total RNA mini isolation kit. RNA quality was analysed with a Nanodrop (Thermo Scientific) to ensure an OD260/230 and OD260/280 ratio between 1.8 – 2, and a RNA Quality Indicator (RQI) of at least 8 with a Bioanalyser (Bio-Rad). The concentration was checked using the Qubit BR RNA assay (InVitrogen).
For the AMB only treatment, the AMB+LF treatment and their corresponding matching controls, the TruSeq RNA Library prep kit v2 (Illumina) with a poly-A pulldown was used according to the manufacturer’s instructions, with 2 μg of total RNA for each of the three biological triplicate samples as input. The libraries were enriched using 15 cycles of PCR and the insert size ranged from 80 to 330 base pair (bp). The libraries were randomized and sequenced in two separate lanes using HiSeq 2000 with the TruSeq v3 SBS reagents to generate 100 bp paired-end reads. The data was demultiplexed using Casava 1.8.2.     For the LF only treatment and the corresponding matching controls, the TruSeq Stranded mRNA-seq prep kit was used to prepare the RNA samples as per protocol using 1ug of total RNA for each of the three biological triplicate samples as input. The libraries were enriched using 12 cycles of PCR to minimize the number of PCR duplicates. The libraries were multiplexed and sequenced on the same flow cell using NextSeq500 v2 reagents to generate 75 bp single reads. The data was demultiplexed using Casava 1.8.2.

treatment_protocol: The concentration of AMB used for the AMB only experiment was 0.03 µg/mL, the concentration of LF used for the LF only experiment was 2 µg/mL and the concentration of AMB + LF for the synergy experiment was 0.03 µg/mL and 2 µg/mL, for AMB and LF respectively. No drugs were added for the matched control samples.

value: control

tag: growth time

value: 50 minutes at ID20


title: Matched control for AMB + LF rep 2

description:
3g
Illumina lane: 1


title: Misc_DKO rep 4

description:
processed data column: Sample_P
DEseq2_ExonCounts.csv

value: DKO


title: SMP_DKO rep 2

description:
processed data column: Sample_O
DEseq2_ExonCounts.csv

value: SMP


title: AMB + LF rep 3

description:
9d
Illumina lane: 2

value: amphotericin B (0.03 µg/mL) + lactoferrin (2 µg/mL)


title: AMB + LF rep 1

description:
2f
Illumina lane: 1


title: Matched control for AMB only rep 2

description:
5g
Illumina lane: 2

value: 60 minutes at ID20


title: FAP_DKO rep 4

description:
processed data column: Sample_N
DEseq2_ExonCounts.csv

value: FAP


title: FAP_WT rep 4

description:
processed data column: Sample_NN
DEseq2_ExonCounts.csv


title: AMB only rep 3

description:
9e
Illumina lane: 2

value: amphotericin B (0.03 µg/mL)


title: Misc_DKO rep 3

description:
processed data column: Sample_L
DEseq2_ExonCounts.csv


title: FAP_WT rep 3

description:
processed data column: Sample_JJ
DEseq2_ExonCounts.csv


title: Matched control for AMB only rep 1

description:
3f
Illumina lane: 1


title: AMB only rep 2

description:
4e
Illumina lane: 1


title: FAP_DKO rep 3

description:
processed data column: Sample_J
DEseq2_ExonCounts.csv


title: Misc_WT rep 2

description:
processed data column: Sample_HH
DEseq2_ExonCounts.csv


title: Misc_DKO rep 2

description:
processed data column: Sample_H
DEseq2_ExonCounts.csv


title: FAP_WT rep 2

description:
processed data column: Sample_FF
DEseq2_ExonCounts.csv


title: SMP_DKO rep 1

description:
processed data column: Sample_G
DEseq2_ExonCounts.csv


title: SMP_WT rep 1

description:
processed data column: Sample_KK
DEseq2_ExonCounts.csv


title: FAP_DKO rep 2

description:
processed data column: Sample_F
DEseq2_ExonCounts.csv


title: BV2LPS4hr-2

city: Ansan

first: Young Gyu

last: Chai

state: Gyeonggi-do

address: 55 Hanyangdaehak-ro

department: Molecualr & Life Science

country: South Korea

institute: Hanyang Univ.

data_processing:
Illumina CASAVA v1.8.2 software was used for basecalling.
To avoid low-quality data, we clipped and trimmed the reads using Trimmomatic (v0.33)
For the analysis of differentially expressed genes, the data of quality-checked reads for each condition were processed with the TopHat (version 2.0.10) software based on reference genome sequence (Mus musculus UCSC mm10) and the differential gene expressed values of each sample were calculated by Cufflinks(v2.2.1)
the number of reads mapped to gene ID features were counted using HTSeq v0.6.1.
Genome_build: Mus musculus UCSC mm10
Supplementary_files_format_and_content: tab-delimited text files including normalized read counts for each sample

source_name: BV-2 cell line

growth_protocol: BV-2 cells were seeded in poly-L-lysine-coated T-75 flask in DMEM/nutrient mixture F-12 (DMEM/F12, 1:1) containing 20% FBS (catalog # 26140), 100 IU/ml penicillin and 10 µg/ml streptomycin (catalog # 15140) from Invitrogen (CA, USA). The cells were maintained in a humidified incubator with a 95% air/5% CO2 atmosphere at 37°C. The medium was changed every 2-3 days. After two weeks in culture, mixed glial cell cultures are shaken at 150 rpm at 37°C for 45 min, and the glial cell suspension was collected from each flask and seeded on poly-L-lysine coated cell culture plate. PM were sub-plated and used for further experiments.

extract_protocol:
RNA was extracted using RNAiso Plus (Takara, Shiga, Japan) and QIAGEN RNeasy® Mini kit (QIAGEN, Hilden, Germany) according to the manufacturer’s protocol. For RNA-Seq, RNA libraries were created from each group using the NEBNext® Ultra™ Directional RNA Library preparation kit from Illumina® (Illumina, San Diego, CA, USA). The first step in the workflow involved the removal of ribosomal RNA using the RNAMius™ Transcriptome Isolation kit (Life Technologies, Carlsbad, CA, USA).  Following purification, total RNA was fragmented into small pieces using divalent cations at elevated temperature. The cleaved RNA fragments were copied into first-strand cDNA using reverse transcriptase and random primers, followed by second-strand cDNA synthesis using DNA polymerase I and RNase H. The cDNA fragments were then processed through an end-repair reaction by the addition of a single ‘A’ base, followed by ligation of the adapters. The products of these reactions were then purified and enriched by PCR to create the final cDNA library. The cDNA fragments were sequenced using the Illumina HiSeq2000 (101 cycles PE lane)
RNA libraries were prepared for sequencing using standard Illumina protocols

treatment_protocol: BV-2 cells were treated with LPS (10ng/ml) for 4 hrs.

tag: cell line

value: BV-2 cell line

value: treated with LPS (10ng/ml) for 4 hrs


title: BV2LPS4hr-1


title: Misc_WT rep 1

description:
processed data column: Sample_DD
DEseq2_ExonCounts.csv


title: Misc_DKO rep 1

description:
processed data column: Sample_D
DEseq2_ExonCounts.csv


title: SMP_WT rep 2

description:
processed data column: Sample_OO
DEseq2_ExonCounts.csv


title: FAP_WT rep 1

description:
processed data column: Sample_BB
DEseq2_ExonCounts.csv


title: BV2Con4hr-1


title: Matched control for AMB only rep 3

description:
8a
Illumina lane: 2

tag: strain


title: FAP_DKO rep 1

description:
processed data column: Sample_B
DEseq2_ExonCounts.csv


title: AMB + LF rep 2

description:
5c
Illumina lane: 2


title: BV2Con4hr-2


title: Misc_WT rep 3

description:
processed data column: Sample_LL
DEseq2_ExonCounts.csv


title: AMB only rep 1

description:
2i
Illumina lane: 1


title: Matched control for AMB + LF rep 1

description:
1b
Illumina lane: 1


title: PMG-Cont-2

source_name: primary microglia cells

growth_protocol: PM were seeded in poly-L-lysine-coated T-75 flask in DMEM/nutrient mixture F-12 (DMEM/F12, 1:1) containing 20% FBS (catalog # 26140), 100 IU/ml penicillin and 10 µg/ml streptomycin (catalog # 15140) from Invitrogen (CA, USA). The cells were maintained in a humidified incubator with a 95% air/5% CO2 atmosphere at 37°C. The medium was changed every 2-3 days. After two weeks in culture, mixed glial cell cultures are shaken at 150 rpm at 37°C for 45 min, and the glial cell suspension was collected from each flask and seeded on poly-L-lysine coated cell culture plate. PM were sub-plated and used for further experiments.

treatment_protocol: Murine microglial PM, were treated with LPS (10ng/ml) for 4 hrs.

value: primary microglia cells


title: PMG-LPS-1


title: PMG-LPS-2


title: ChIP_SRC1_Veh_rep2

city: Houston

first: kaifu

last: chen

address: 1 Baylor plaza, suite 450A

institute: Baylor College of Medicine

data_processing: Reads are mapped to genome using ELAND. Reads that are uniquely mapped and have no more than 2 mismatches are selected for future analysis. MACS is used to call peaks with p value 1e-10.

source_name: Cell line

extract_protocol: Purified DNA given A overhang by Klenow exo, adapters ligated, gel size selected (300±25bp), PCR amplified and sequenced

value: U2OS-ERalpha (stable)

tag: antibody

value: anti-SRC1 M341

tag: antibody vendor, catalog#

value: Santa Cruz, sc-8995


title: ChIP_ER_E2

value: anti-ERalpha HC20

value: Santa Cruz, sc-543


title: ChIP_ER_Veh


title: ChIP_SRC1_Veh_rep1


title: ChIP_SRC1_Veh_rep3

value: Abcam, ab2859


title: ChIP_IgG

value: normal rabbit IgG

value: Santa Cruz, sc-2027


title: IL01

city: Oxford

first: Helio

last: Pais

address: Jonh Radcliffe Hospital

department: Weatherall Institute of Molecular Medicine

institute: University of Oxford

data_processing:
Sequences were mapped to the genome using Tophat2 with default parameters
Mapped sequences were assigned to genes with in-house script using GenomicFeatures package in Bioconductor
Gene lengths were obtained from Ensembl (version 73)
RPKM values were calculated with in-house R script
Genome_build: hg19
Supplementary_files_format_and_content: comma separated value file contains gene RPKMs

source_name: A673_control shRNA

extract_protocol:
RNA was extracted from cells using the mirVana Paris kit (Life Technologies) according to the manufacturer's protocol.
mRNA fraction was selected from total RNA and converted to cDNA. The cDNA was end-repaired, A-tailed and adapter-ligated. Prior to amplification, samples underwent uridine digestion. The prepared libraries were size-selected, multiplexed and subsequently paired end sequenced over two lanes of a flow cell.

value: Ewing Sarcoma cells

tag: genotype/variation

value: stably expressing shRNA targeting Luciferase gene


title: IL07


title: EF31

source_name: A673_EWS-FLI1 shRNA

value: stably expressing shRNA targeting EWS-FLI1


title: Matched control for LF only rep  1

description:
10h
Illumina lane: 3

value: control

value: 50 minutes


title: Matched control for LF only rep  3

description:
10i
Illumina lane: 3