title: HUVEC 1 Prox1OE Prox1_ChIP

description:
Sample 23
processed data file: Prox1 peak raw reads.txt


title: UT7_ctl_rep1

status: Public on Feb 13 2017

type: SRA

city: Cambridge

first: Gabriela

last: Alexe

state: MA

address: 415 Main St.

department: Computational Biology and Bioinformatics

country: USA

institute: Broad Institute

library_source: transcriptomic

data_processing:
Total RNA was extracted and profiled by RNA sequencing (HiSeq, Illumina) at BioMicroCenter from Massachusetts Institute of Technology (Cambridge, MA, USA). Quality control tests for the unmapped reads were performed using the FASTQC software avaialble from the Babraham Lab http://www.bioinformatics.babraham.ac.uk/projects/fastqc/.
The reads were aligned to the GRCh37 (hg19) annotated human reference genome by using tophat2 (Kim D, Pertea G, Trapnell C, Pimentel H, Kelley R, Salzberg SL. TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions. Genome Biol. 2013 Apr 25;14(4):R36).
The count reads and the FPKM scores for genes were computed based on the RSEM R software package (Li B, Dewey CN. RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome.BMC Bioinformatics. 2011 Aug 4;12:323). Expression data was evaluated as log2 (FPKM). The significance of the differential expression between the control and cyclocreatine treatment phenotypes was estimated using the EBSeq method implemented in R (Bioconductor v.3.3, Leng N, Dawson JA, Thomson JA, Ruotti V, Rissman AI, Smits BM, Haag JD, Gould MN, Stewart RM, Kendziorski C.  EBSeq: an empirical Bayes hierarchical model for inference in RNA-seq experiments. Bioinformatics. 2013 Apr 15;29(8):1035-43), based on the significance cutoff 0.05 for the posterior probability.
Genome_build: GRCh37 (hg19)
Supplementary_files_format_and_content: Tab-delimited text files include gene level read counts values for each sample. The genes are annotated based on the RefSeq hg19 database.

molecule: total RNA

organism: Homo sapiens

source_name: Human bone marrow

extract_protocol:
RNA was extracted from cells with an RNeasy Kit (Qiagen).
RNA libraries were prepared for sequencing using standard Illumina protocols

treatment_protocol: Cyclocreatine was purchased from Sigma-Aldrich (Sigma). TF-1, UT-7 and UCSD-AML1 cells were treated in quadruplicate with either vehicle or 3 mM cyclocreatine for 24 hours.

tag: cell line

tag: tissue

value: Human blood

tag: disease

value: Acute Myeloid Leukemia

tag: treatment

value: Vehicle

tag: time

value: 24 hours


title: HUVEC 5 Flag-Prox1OE Flag_ChIP

city: Leuven

first: Bernard

last: Thienpont

address: herestraat 42

country: Belgium

institute: KULeuven

description:
Sample 27
processed data file: Prox1 peak raw reads.txt

library_source: genomic

data_processing:
FastQ files were mapped to the human genome (build GRCh37/hg19) using bowtie (version 0.12.8; settings -v 2 -m 1 -p --best -S).
Peaks were called in Prox1-FLAG ChIP-seq data by model-based analysis of ChIP-sequencing (MACS), using libraries of FLAG-ChIP on cells transfected with the empty overexpression plasmid as background data set. For calling H3K9ac peaks, MACS was applied on pooled H3K9ac data using the input as background set.
Read counts at H3K9ac peak areas were quantified in each sample using SeqMonk, and background-normalized to enable enrichment assessment.
Differential acetylation at H3K9ac peaks between two groups was assessed in R (version 3.1.0) with EdgeR (version 3.6.8), computing exact tests for differences in the means of negative-binomially distributed counts. We called peak areas as differentially acetylated when the corresponding FDR-adjusted P-value was below 0.05.
Genome_build: hg19
Supplementary_files_format_and_content: Raw read files contain read numbers overlapping H3K9ac and Prox1 peaks. Differential acetylation at H3K9ac peaks was called using EdgeR, comparing GFP-overexpression and Prox1-overexpression HUVECs, and short-hairpin-mediated knockdown of Cpt1a in Prox1-overexpression HUVECs.

molecule: genomic DNA

source_name: human umbilical vein endothelial cells

growth_protocol:
HUVECs between passages 1 and 5 were initially cultured in M199 medium (1 mg/mL D-glucose) supplemented with 20% fetal bovine serum (FBS; Biochrom GmBH, Germany), 2 mM L-glutamine, 30 µg L-1 endothelail cell growth factor supplements (ECGS), 10 units mL-1 heparin, 50 IU mL-1 penicillin and 50 µg mL-1 streptomycin, and switched to growth in endothelial cell growth medium 2 (ECGM2, Promocell, Heidelberg, Germany) for experiment.
Overexpression protocol: Lentiviral expression vector constructs for prospero homeobox protein 1 (Prox1) and GFP control virus were obtained from GeneCopoeia (Rockville, USA) and cloned in the pRRLsinPPT.CMV.MCS.MM W prevector 56. FLAG-tagged Prox1 was also generated and cloned into the pRRL prevector, and an empty vector pRRL virus was used as control.  For Prox1 overexpression experiments, cells were used at least 6 days after transduction to allow stable induction of the lymphatic phenotype.
Knockdown protocol: To generate shRNA vectors against CPT1a, oligonucleotides were cloned into the pLKO.1 vector (Sigma). A nonsense scrambled shRNA sequence was used as negative control. Production of lentiviruses was performed in 293T cells. For transductions, a multiplicity of infection of 20 was used. Cells were transduced overnight in the presence of 0.5 µg/mL polybrene, then rinsed with PBS and refreshed with new media the next day. For shRNA experiments, cells were used 2-4 days post-transduction.

extract_protocol:
Cultured cells were fixed by adding 1% Formaldehyde (16% Formaldehyde (w/v), Methanol-free, Thermo Scientific) directly to the medium and incubating for 8 minutes. Glycine (150 µM) was added for 5 min to quench the formaldehyde. Cells were washed twice with ice-cold PBS 0.5% Triton-X100, scraped and collected by centrifugation (1000 × G for 5 min at 4 °C). The pellet was resuspended in 1400 µL of RIPA buffer (50 mM Tris-HCl pH 8, 150 mM NaCl, 2 mM EDTA pH 8, 1% Triton-X100, 0.5% Sodium deoxycholate, 1% SDS, 1% protease inhibitors) and transferred in a new eppendorf tube. The lysate was homogenized by passing through an insulin syringe, and incubated on ice for 10 min. The chromatin was sonicated for 3 min by using a 250 Digital Sonifier (Branson) with 0.7 s 'On' and 1.3 s 'Off' pulses at 40% power amplitude, yielding a DNA fragment size of 100 to 500 bp. The sample was kept ice-cold at all times during the sonication. The samples were centrifuged (10 min at 16000 × G at 4 °C) and supernatants transferred to a new eppendorf tube. 50 µL of sheared chromatin was set apart as “input”, and 5 µg of either rabbit anti-FLAG (Pierce), rabbit anti-Prox1 (ProteinTech), rabbit anti-p300 antibody (Santa Cruz) or rabbit anti-H3K9ac (Cell Signaling) antibody was added to the remainder of the chromatin. Samples were incubated overnight at 4 °C on a rotator. 20 µL Pierce Protein A/G Magnetic Beads (Life Technologies) were added to the samples and incubated at 4 °C for at least 5 hours. A/G Magnetic Beads were collected and washed 5 times with washing buffer (50 mM Tris-HCl, 200 mM LiCl, 2 mM EDTA, pH 8, 1% Triton, 0.5% Sodium deoxycholate, 0.1% SDS, 1% protease inhibitors), and twice with TE buffer. The A/G magnetic beads were resuspended in 50 µL of TE buffer, and 1.5 µL of RNase A (200 units, NEB, Ipswich, MA, USA) were added to the A/G beads samples and to the input, incubated for 10 minutes at 37 °C. After addition of 1.5 µL of proteinase K (NEB, 200 units) and overnight incubation at 65°C, the DNA was purified using 1.8 × volume of Agencourt AMPure XP (Beckman Coulter) according to the manufacturer’s specifications, and then eluted in 30 µL of TE buffer.
Input and immunoprecipitated DNA was transformed into libraries using the NEBNext Ultra DNA library prep kit for Illumina (NEB), and amplified using KAPA HiFi HotStart ReadyMix for 14 cycles.

tag: cell type

value: human umbilical vein endothelial cells

tag: overexpression

tag: knockdown

tag: antibody

value: rabbit anti-FLAG (Pierce)


title: TF1_ctl_rep4


title: HUVEC 1 Flag-Prox1OE Flag_ChIP

description:
Sample 21
processed data file: Prox1 peak raw reads.txt


title: Norm roots replicate 2

city: Gainesville

first: A.

middle: Mark

last: Settles

state: FL

address: 1301 Fifield Hall

department: Horticultural Sciences

institute: University of Florida

description: Sample 5

data_processing:
The FASTQ/A trimmer from the FASTX toolkit (http://hannonlab.cshl.edu/fastx_toolkit/) trimmed off bases that had a quality score of less than 20. The FASTX quality filter discarded reads if less than 80% of the bases in the read had a quality score of at least 20.
To remove any contaminating reads derived from rRNA, Bowtie software version 0.12.7 (Langmead et al, 2009) aligned reads from all libraries in a non-unique manner to sequences of repetitive elements in the MIPS database (Nussbaumer et al., 2013). The reads that did not align to repetitive elements were kept for downstream analyses.
The six wild-type libraries were uniquely mapped to the Zea mays B73 genome (RefGen_v2) using indel-tolerant Mosaik 2 software version 2.1.33 (https://code.google.com/p/mosaik-aligner/). Mosaik 2 was run with a maximum of 100 hash positions per seed, a hash size of 14, and 2% allowed mismatch between the read and genome.
Duplicate reads were removed using Picard software version 1.54 (http://picard.sourceforge.net).
Freebayes software version 0.9.5 identified W22 variant alleles from the merged wild-type alignments given the parameter of 2% pairwise differences assumed between W22 and B73. The Freebayes parameters used in this study required variant alleles to have more than 10 mapped reads, more than 40% of the reads within each library supporting the variant, at least one read to have a base quality of at least 20 at the site of variation, and a 99% probability that the variant existed.
GSNAP software version 2012-5-24 (Wu and Nacu, 2010), which uniquely aligned wild-type and rgh3 reads to B73 RefGen_v2. During the alignment process, 2% of the read length was permitted to mismatch with the genome, and mate pairs were allowed to deviate 800 bp from the expected paired-end length of 200 bp as a consequence of the large variation in fragment sizes within the dataset. GSNAP was guided by the ZmB73 5b Filtered Gene Set annotations but was permitted to discover novel splicing events.
Transcript isoforms were then assembled from the GSNAP read alignment files. Cufflinks software version 1.3.0 (Trapnell et al., 2010) assembled transcript fragments within each library individually using a minimum intron size of 10 bp and an overlap radius of 10 bp. Cufflinks was restricted to using the ZmB73 5b Filtered Gene Set annotations to estimate isoform expression with the ‘-G’ option. The reference annotation file was merged with the transcript assemblies from all twelve RNA-seq libraries using Cuffmerge. Cuffdiff tested for differential splicing between wild-type and rgh3 roots separately from wild-type and rgh3 shoots. Cuffdiff was run using bias detection and correction algorithm and upper-quartile normalization.
Genome_build: B73 RefGen_v2
Supplementary_files_format_and_content: .gtf files report Cufflinks abundance measurements

organism: Zea mays

source_name: seedling roots

growth_protocol: seedlings grown to 2 inches tall

extract_protocol:
Tissues were separately ground in liquid nitrogen. Total mRNA was extracted using the TRIzol Reagent (Life Technologies) protocol, during which RNAse-free glycogen (Fermentas) was used as a carrier for RNA. The TruSeqTM RNA Sample Preparation Kit (Illumina) was used to build cDNA libraries from 2 ug of input total RNA. The Illumina fragmentation protocol for producing inserts with a median length of 200 bp was used. The KAPA Library Quantification Kit for Illumina Genome Analyzer platform (KAPABIOSYSTEMS) facilitated the accurate pooling of twelve libraries at nearly equal concentrations. The multiplexed pool was sequenced on one lane of the HiSeq 2000 platform to produce 2x100 bp reads.
Illumina TruSeq RNA sample preparation kit

tag: developmental stage

value: seedling

value: roots

tag: genotype/variation

value: wild-type


title: HUVEC 5 empty Prox1 ChIP Input

description:
Sample 31
processed data file: Prox1 peak raw reads.txt

value: empty


title: rgh3 shoots replicate 3

description: Sample 12

source_name: seedling shoot

value: shoot

value: rough endosperm3


title: TF1_ctl_rep3


title: HUVEC 1 Prox1OE Prox1 ChIP Input

description:
Sample 20
processed data file: Prox1 peak raw reads.txt


title: rgh3 shoots replicate 1

description: Sample 4


title: rgh3 roots replicate 1

description: Sample 3


title: TF1_ctl_rep2


title: TF1_ctl_rep1


title: Norm shoots replicate 1

description: Sample 2


title: HUVEC 1 Flag-Prox1OE Prox1 ChIP Input

description:
Sample 18
processed data file: Prox1 peak raw reads.txt


title: HUVEC 1 empty Flag-ChIP Input

description:
Sample 19
processed data file: Prox1 peak raw reads.txt


title: HUVEC 6 ProxOE_ShCpt K9Ac_ChIP_input

description:
Sample 17
processed data file: K9ac raw reads.txt, H3K9ac peaks and their differential acetylation.xlsx

value: shCpt


title: MDA-MB231_MMS_8hr

city: San Antonio

first: Yidong

last: Chen

state: Texas

address: 8403 Floyd Curl Drive, MSC 7784

department: Population Health Sciences

institute: UT Health Science Center at San Antonio

description: 40μg/ml MMS for 8 hours

data_processing:
Illumina Casava v1.8.2 software used for base-calling
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence, then mapped to mm9 whole genome using TopHat v2.0.8b
gene count measurements were obtained using HTSeq-count
Genome_build: UCSC hg19
Supplementary_files_format_and_content: The processed data files are in tabular format.  The first column contains the gene symbols and the second column contain the read counts of the gene using htseq-count

source_name: Breast cancer MDA-MB231

growth_protocol: Cells were passaged in 10cm corningware dishes using media described in Samples section in a 37°C humidified incubator

extract_protocol:
Total RNA was extracted from sub-confluent 10cm dishes using Qiagen RNeasy kit
RNA libraries were prepared for sequencing using standard Illumina protocols

treatment_protocol: Samples were treated with either vehicle (DMSO) or indicated doses of drug for indicated amout of time before extraction of RNA

value: Breast cancer (Basal type)

tag: growth protocol

value: Cells were grown in DMEM supplemented with 10%FBS

value: 40microg/ml MMS for 8 hours


title: MDA-MB231_Ctrl_8hr

description: no treatment

value: DMSO


title: Norm roots replicate 1

description: Sample 1


title: U2OS_Paclitaxel

source_name: Osteosarcoma U2OS

growth_protocol:
Cells were grown in DMEM supplemented with 10%FBS
Cells were passaged in 10cm corningware dishes using media described in Samples section in a 37°C humidified incubator

value: Osteosarcoma

value: 0.2μM Paclitaxel for 8 hours


title: rgh3 roots replicate 2

description: Sample 7


title: HUVEC 1 empty Flag_ChIP

description:
Sample 22
processed data file: Prox1 peak raw reads.txt


title: L8

city: Edmonton

first: Guanxiang

last: Liang

state: Alberta

address: 410 Agriculture/Forestry Centre

country: Canada

institute: University of Alberta

data_processing:
All the reads were demultiplexed according to their index sequences using CASAVA version 1.8 (Illumina)
The small RNAs sequencing reads with good quality were subjected to 3’ adaptor sequence trimming by miRDeep2
The miRNA expression (CPM) were identified by miRDeep2, while piRNA expression (CPM) were identified by costumised Perl program based on method descibed by other studies (Liu et al., 2012.PLoS ONE 7(4): e34770).
Genome_build: Oar_v3.1
Supplementary_files_format_and_content: Tab-delimited text files include CPM values for each Sample

organism: Ovis aries

source_name: Testes tissue

extract_protocol:
Testes tissues were frozen on dry ice, and RNA was extracted using Trizol reagent. Illumina TruSeq samll RNA Sample Prep Kit (Cat#RS-200-0012) was used with 1 ug of total RNA for the construction of sequencing libraries.
RNA libraries were prepared for sequencing using standard Illumina protocols

tag: diet

value: fed for a 10% decrease in body mass over 65 days

value: testis


title: HUVEC 6 NoProx_NoCptSh K9Ac_ChIP_input

description:
Sample 15
processed data file: K9ac raw reads.txt, H3K9ac peaks and their differential acetylation.xlsx

value: human umbilical vein endothelial cells

value: GFP

value: shScramble


title: HUVEC 6 ProxOE_shScramble K9Ac_ChIP_input

description:
Sample 16
processed data file: K9ac raw reads.txt, H3K9ac peaks and their differential acetylation.xlsx


title: L7


title: L6


title: U2OS_Etoposide

value: 20μM Etoposide for 8 hours


title: HUVEC 4 ProxOE_shScramble K9Ac_ChIP_input

description:
Sample 13
processed data file: K9ac raw reads.txt, H3K9ac peaks and their differential acetylation.xlsx


title: HUVEC 4 ProxOE_ShCpt K9Ac_ChIP_input

description:
Sample 14
processed data file: K9ac raw reads.txt, H3K9ac peaks and their differential acetylation.xlsx


title: U2OS_Doxorubicin

value: 1μM Doxorubicin for 8 hours


title: HUVEC 4 NoProx_NoCptSh K9Ac_ChIP_input

description:
Sample 12
processed data file: K9ac raw reads.txt, H3K9ac peaks and their differential acetylation.xlsx


title: L4


title: L5


title: U2OS_Control


title: HUVEC 6 ProxOE_ShCpt K9Ac_ChIP

description:
Sample 11
processed data file: K9ac raw reads.txt, H3K9ac peaks and their differential acetylation.xlsx

value: rabbit anti-H3K9ac (Cell Signaling)


title: L3


title: HUVEC 6 ProxOE_shScramble K9Ac_ChIP

description:
Sample 10
processed data file: K9ac raw reads.txt, H3K9ac peaks and their differential acetylation.xlsx


title: MDA-MB231_Paclitaxel


title: MDA-MB231_Etoposide


title: HUVEC 6 NoProx_NoCptSh K9Ac_ChIP

description:
Sample 9
processed data file: K9ac raw reads.txt, H3K9ac peaks and their differential acetylation.xlsx


title: MDA-MB231_Doxorubicin


title: HUVEC 4 ProxOE_ShCpt K9Ac_ChIP

description:
Sample 8
processed data file: K9ac raw reads.txt, H3K9ac peaks and their differential acetylation.xlsx


title: L1


title: H8

value: fed for a 10% increase in body mass over 65 days


title: MDA-MB231_Control


title: H7


title: HUVEC 4 NoProx_NoCptSh K9Ac_ChIP

description:
Sample 6
processed data file: K9ac raw reads.txt, H3K9ac peaks and their differential acetylation.xlsx


title: H6


title: HUVEC 4 ProxOE_shScramble K9Ac_ChIP

description:
Sample 7
processed data file: K9ac raw reads.txt, H3K9ac peaks and their differential acetylation.xlsx


title: YY1-HeLa

city: Ganzhou

first: Wenjuan

last: Zhang

state: Jiangxi

address: No. 23, Qingnian Road

department: First Affiliated Hospital

country: China

institute: Gannan Medical University

description:
YY1 antibody: sc-7341(Santa Cruz Biotechnology)
YY1 ChIP-seq in HeLa cells

data_processing:
Basecalling was performed using CASAVA version 1.4
Reads were aligned to the hg18 genome assembly using Bowtie version 2.2.1.
The bigwig files were generated by using Homer v4.7, in which the total tags are normalized to 1.00e+07.

source_name: Henrietta Lacks Cells (HeLa cells)

growth_protocol: HeLa cells were cultured inMEM alpha medium supplemented with 10% FBS.

extract_protocol:
For ChIP-seq, fixation was stopped by adding Glycine (0.125M) and incubating for 5 min at RT, followed by washing with PBS twice. Chromatin DNA was sheared to 300–500 bp average in size through sonication. Resultant was immunoprecipitated with control IgG or specific antibodies overnight at 4°C, followed by incubation with protein G magnetic beads (Invitrogen) for an additional 2 h. After washing and elution, the protein–DNA complex was reversed by heating at 65°C overnight. Immunoprecipitated DNA was purified by using QIAquick spin columns (Qiagen).
The libraries were constructed following Illumina’s Chip-Seq Sample prep kit. Briefly, Chip DNA was end-blunted and added with an ‘A’ base so the adaptors from Illumina with a ‘T’ can ligate on the ends. Then 200–400 bp fragments are gel-isolated and purified. The library was amplified by 18 cycles of PCR.

treatment_protocol: Cells grown to ~90% confluent were fixed directly with 1% formaldehyde.

value: HeLa


title: Gro-seq-siYY1-HeLa

description:
Nascent RNA sequencing
Gro-seq in HeLa cells transfected with YY1 siRNA

data_processing:
Library strategy: Gro-seq
Basecalling was performed using CASAVA version 1.4
Reads were aligned to the hg18 genome assembly using Bowtie version 2.2.1.
The bigwig files were generated by using Homer v4.7, in which the total tags are normalized to 1.00e+07.

extract_protocol:
HeLa cells were washed 3 times with cold 1X phosphate buffered saline (PBS) buffer and then incubated in swelling buffer (10mM Tris-Cl pH7.5, 2mM MgCl2, 3mM CaCl2) for 5min on ice and harvested. Cells were first re-suspended and lysed in lysis buffer (swelling buffer with 0.5% IGEPAL and 10% glycerol). The resultant nuclei were washed once again with 10 ml lysis buffer and then re-suspended in 100ml of freezing buffer (50mM Tris-Cl pH 8.3, 40% glycerol, 5mM MgCl2, 0.1mM EDTA).
For run-on assay, resuspended nuclei were mixed with an equal volume of reaction buffer (10mM Tris-Cl pH 8.0, 5mM MgCl2, 1mM DTT, 300mM KCl, 20 units of SUPERase In, 1% sarkosyl, 500mM ATP, GTP, and Br-UTP, 2mM CTP) and incubated for 5 min at 30°C. The nuclear-run-on RNA (NRO-RNA) was then extracted with TRIzol LS reagent (Invitogen) following manufacturer’s instructions. NRO-RNA was then subjected to base hydrolysis on ice for 40 min and followed by treatment with DNase I and antarctic phosphatase. To purify the Br-UTP labeled nascent RNA, the NRO-RNA was immunoprecipitated with an anti-BrdU agarose beads (Santa Cruz Biotech) in binding buffer (0.5XSSPE, 1mM EDTA, 0.05% tween-20) for 1h at 4°C while rotating.  To repair the end, the immunoprecipitated BrU-RNA was re-suspended in 50mL reaction (43mL DEPC water, 5.2mL T4 PNK buffer, 1mL SUPERase In and 1mL T4 PNK (New England BioLabs)) and incubated at 37°C for 1h. RNA was then extracted, precipitated using acidic phenol-chloroform (Ambion) and subjected to poly-A tailing reaction by using poly-A polymerase (New England BioLabs) for 30 min at 37°C. Subsequently, reverse transcription was performed using oNTI223- primers (5’- pGATCGTCGGACTGTAGAACTCT; CAAGCAGAAGACGGCATACGATTTTTTTTTTTTTTTTTTTTVN-3’) where the p indicates 5’ phosphorylation, ‘;’ indicates the abasic dSpacer furan and VN indicates degenerate nucleotides. Specifically, tailed RNA (8.0mL) was subjected to reverse transcription using superscript III (Invitrogen), after which the cDNA products were separated on a 10% polyacrylamide TBE-urea gel. The extended first-strand product (100-500bp) was excised and recovered by gel extraction. After that, the first-strand cDNA was circularized by CircLigase (Epicentre) and re-linearized by Ape1 (New England BioLabs). Re-linearized single strand cDNA (sscDNA) was separated in a 10% polyacrylamide TBE gel as described above and the product of needed size was excised (~170-400bp) for gel extraction. Finally, sscDNA template was amplified by PCR using the Phusion High-Fidelity enzyme (NEB) according to the manufacturer’s instructions. Primers oNTI200 (5’-CAAGCAGAAGACGGCATA-3’) and oNTI201 (5’- AATGATACGGCGACCACCGACAGGTTCAGAGTTCTACAGTCCGACG-3’) were used to generate DNAs for deep sequencing.

treatment_protocol: HeLa cells were subjected to two rounds of siRNAs transfection in 3 days, followed by nuclei isolation.

tag: transfection


title: 14d24h

city: GUANGZHOU

first: HONG

last: ZHU

state: GUANGDONG

address: 723 XINGKE RD.TIANHE DISDRICT

institute: SOUTH CHINA BOTANICAL GARDEN,CHINESE ACADEMY OF SCIENCES

data_processing:
Illumina Casava1.7 software used for basecalling.
sRNA raw reads were preprocessed using Illumina Genome Analyzer Pipeline. After that, all the clean reads were mapped to miRBase 20 (http://www.mirbase.org/), allowing up to two mismatches, to identify known miRNAs in litchi.
MiRCat was applied to fold flanking genome sequence of the unique small RNAs, followed by secondary structure prediction using RNAfold, to identify novel litchi miRNAs. Further criteria for annotation of plant miRNAs according to Meyers et al. (2008) were used to screen for candidate miRNAs.
degradome sequencing reads were analyzed via CleaveLand 3.0 pipeline to detect potentially cleaved miRNA targets.
all the identified targets were annotated by BlastX (E-value<0.00001) to NR and KEGG, followed by GO term analysis.
Genome_build: litchi unigene assembly (submitted & in review)
Supplementary_files_format_and_content: spreasheets include sequncing statistic, known & novel miRNA lists, miRNA target list etc.

organism: Litchi chinensis

source_name: fruit

extract_protocol:
Pericarp tissues were immediately frozen, crushed in liquid nitrogen. Total RNA was isolated using a previously described method (Yang et al., Journal of Experimental Botany, 2013).
Small RNA libraries and a mixed degradome library were prepared for sequencing using standard Illumina protocols.

treatment_protocol: Two groups of litchi fruit were harvested. One group of fruit was stored at ambient temperature (25°C) for 0d and 4d. The other group was stored at cold temperature (1°C) for 2 weeks before transferred to 25°C, and then kept at 25°C for 1d and 2d.

tag: Stage

value: 24h post 14 days of cold storage

value: pericarp

tag: molecule subtype

value: small RNA


title: 14d48h

value: 48h post 14 days of cold storage


title: 14d0h

value: 0h post 14 days of cold storage


title: H4


title: HUVEC 3 ProxOE_ShCpt K9Ac_ChIP_input

description:
Sample 5
processed data file: K9ac raw reads.txt, H3K9ac peaks and their differential acetylation.xlsx

value: shCpt


title: H5


title: Gro-seq-siCTL-HeLa

description:
Nascent RNA sequencing
Gro-seq in HeLa cells transfected with control siRNA

value: control


title: HUVEC 3 NoProx_NoCptSh K9Ac_ChIP_input

description:
Sample 4
processed data file: K9ac raw reads.txt, H3K9ac peaks and their differential acetylation.xlsx


title: L2

value: fed for a 10% decrease in body mass over 65 days


title: 4d

value: 4d at ambient storage


title: HUVEC 3 ProxOE_ShCpt K9Ac_ChIP

description:
Sample 3
processed data file: K9ac raw reads.txt, H3K9ac peaks and their differential acetylation.xlsx

value: human umbilical vein endothelial cells


title: H3

value: testis


title: degradome

value: mixed stages

value: cleaved mRNA


title: H2


title: 0d

value: before storage


title: S03

city: Fuzhou

first: Chen

last: Lisong

address: Shangdian street NO.15

institute: Fujian Agriculture and Forestry University

data_processing: The clean reads were mapped to Citrus clementina genome (JGI version 1.0, http://www.phytozome.org/clementine.php) using SOAP, only perfectly mapped sequences were retained and analyzed further. rRNAs, tRNAs, snRNAs and snoRNAs were removed from the sRNAs sequences through BLASTn search using NCBI Genebank database (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi/) and Rfam (10.1) database (http://www.sanger.ac.uk/resources/databases/rfam.html) (e = 0.01). The remaining sequences were aligned with known plant miRNAs from miRBase 21 (http://www.mirbase.org/).

organism: Citrus sinensis

source_name: B-toxici treated leaves

growth_protocol: 5-week-old seedlings of ‘Xuegan’ (Citrus sinensis) and sour pummelo (C. grandis) were transplanted into 6 L pots containing fine river sand. Seedlings were planted in greehouse under natural light condition.

extract_protocol:
Leaf total RNA was harvested using Trizol reagent. Illumina TruSeq RNA Sample Prep Kit (Cat#FC-122-1001) was used with 1 ug of total RNA for the construction of sequencing libraries.
RNA libraries were prepared for sequencing using standard Illumina protocols

treatment_protocol: Ten weeks after transplanting, each pot was supplied every other day with B-sufficient (10 μM H3BO3, control) or -toxic (400 μM H3BO3) nutrient solution for 15 weeks. There were 20 replications per B treatment with 2 pots in a completely randomized design.

value: matured leaf from 1/3 height of the plants

tag: time point

value: 15 weeks post B treatment

value: 400 μM boron acid


title: HUVEC 5 Prox1OE Prox1 ChIP Input

description:
Sample 29
processed data file: Prox1 peak raw reads.txt


title: S02

organism: Citrus maxima

source_name: Control leaves

value: 10 μM H3BO3, control


title: S01


title: FHHs-1

status: Public on Feb 12 2017

city: Beijing

first: Jinhua

last: Qin

address: Taiping Road

institute: Beijing Institute of Transfusion

data_processing:
Illumina Casava1.7 software used for basecalling.
Paired-end reads were  mapped against the UCSC GRCh38 reference by Tophat with stringent parameters: --coverage-search --microexon-search --b2-very-sensitive. Only unique mapping reads were retained for normalization purposes. Read counts of each gene were summarized using HTSeq version 0.6.1p1. DESeq2 was used to normalize read counts.
Genome_build: hg38
Supplementary_files_format_and_content: tab-delimited text files include raw read counts of each gene for each sample

molecule: polyA RNA

source_name: fetal hepatocytes isolated from fetal liver

growth_protocol: Human ESCs were passaged with Accutase (Sigma) and plated at a density of 100,000 cells/cm2 in mTeSR with 10 μM Y27632 (Selleck) on Matrigel (BD), Laminin (BD), and collagen IV (BD) (3:1:1) mixed gel coated-plate (Corning). In the restriction of definitive endoderm (DEs) stage (S1), cells were cultured for 24 hrs in RPMI with B27 supplement (1:50, Gibco), 100 ng/ml Activin A and 3 μM CHIR99021, and then treated with 100 ng/ml Activin A for 2 days. In the hepatic specification stage to get hepatic stem cells (S2), the culture medium was replaced with RPMI (Gibco) supplemented with B27 supplement (1:50), 20 ng/ml BMP4 and 10 ng/ml FGF2 for 5 days. And in stage III of hepatic maturation, cells were cultured in Hepatocyte Culture Medium (HCM, Lonza) with 20 ng/ml HGF, 10 ng/ml OSM and 1 μM dexamethasone for 10-15 days. During stages II and III, cells are fed every 48 hr. The final stage was also carried out with 10 μM SB203580 (Selleck) or 50μM Vitamin K2 (Sigma).

extract_protocol:
total RNA (Qiagen, Cat#: 74004) were extracted according to manufacturer’s instructions.
mRNA was reverse transcribed and amplified by REPLI-g Single Cell Kit (cat#. 150063). The library was sequenced using Illumina Hiseq platform.

treatment_protocol: hESCs-derived hepatocytes were treated with 10 μM SB203580 or 50μM Vitamin K2.

value: fetal hepatocytes


title: RAI1_4C_Replicate1

city: Lausanne

first: Marion

last: LELEU

address: Station 15

country: Switzerland

institute: EPFL

data_processing:
Library strategy: 4C-seq
De-multiplexing, mapping and 4C-seq were done through HTSstation (David et al, 2014 PLoS ONE and http://htsstation.epfl.ch/).
Reads were mapped to the hg19 Human genome assembly with bowtie2 version 2.2.1with parameters −k 20 −−end−to−end −−sensitive −p 5
Mapped reads were translated to restriction fragments using the HTSstation (David et al, 2014 PLoS ONE and http://htsstation.epfl.ch/).
Reads were normalized to the total number of reads and replicates combined by averaging the resulting signal densities . Smoothed data were obtained by applying a running mean algorithm with 29 fragments per window.
We used a domainogram algorithm to detect significantly interacting regions without imposing a fixed window size as suggested in [de Wit et al, Global chromatin domain organization of the Drosophila genome, PLoS Genet. 2008 Mar 28;4(3)]. We selected BRICKS (Blocks of Regulators In Chromosomal Kontext) with a p-value threshold smaller than 0.01 for both “cis” and “trans” interactions.
Genome_build: hg19
Supplementary_files_format_and_content: Signal/restriction fragment (bigwig) one per replicate.

source_name: EBV transformed mononuclear cells from perypherial blood

growth_protocol: Lymphoblastoid cell lines were grown in RPMI1640 medium supplemented with 10% FCS and 1% PS

extract_protocol: 50 million exponentially growing cells were harvested and crosslinked with 1% formaldehyde, lysed and cut with the restriction enzyme DpnII. After ligation and reversal of the crosslinks, the DNA was purified to obtain the 3C library. This 3C library was further digested with NlaIII and circularized to obtain a 4C library. The inverse PCR primers to make the 4C-seq templates were designed to contain the Illumina adaptor tails, as well as the bait-specific sequences for the locus we interrogated. 4C libraries were multiplexed (two samples per multiplex), and analyzed on a 100-bp single-end Illumina HiSeq flow cell.

value: Lymphoblastoid cells

tag: cell line id


title: FHHs-2


title: RAI1_4C_Replicate2


title: U2OS_MMS

tag: cell line

value: Osteosarcoma

value: 40μg/ml MMS for 8 hours


title: HUVEC 3 NoProx_NoCptSh K9Ac_ChIP

description:
Sample 1
processed data file: K9ac raw reads.txt, H3K9ac peaks and their differential acetylation.xlsx

value: GFP

value: shScramble

value: rabbit anti-H3K9ac (Cell Signaling)


title: batch4-K562-SC-Test8

city: New Haven

first: Xinghua

last: Pan

state: CT

address: 300 Cedar Street, TAC-S320

department: Department of Genetics

institute: Yale University School of Medicine

description: CpG methylome

data_processing:
Library strategy: CGI-Seq
For LQC (Low Quantity of Cells) samples, the raw sequence reads were mapped against the hg19 version of the human reference genome using Bowtie2 with default parameters. Bedtools was then used to obtain the read counts for each RE2 cutting site. Only reads that start from RE2 sites, and covered in a <3500 bp RE1 fragment were defined as informative and thus retained for the subsequent analysis. All reads in both TEST and MC samples were normalized by RPM (Reads per million). We then obtained a CpG methylation score for each RE2 cutting site by using log2 ratio of RPM(TEST)/RPM(MC).
For SC (single cell) samples, raw sequence reads were mapped against the hg19 version of the human reference genome using Bowtie2 with default parameters. Read counts for each informative fragment, instead of each RE2 cutting site, were calculated. Merging reads in each informative fragment generated with RE1 leads to loss of resolution, but provides greater read counts. Based on the experimental design, the read counts from each informative fragment as a whole can represent the associated genomic block. A EM algrithm was thus used to estimate the probability of an informative fragment is methylated.
Genome_build: hg19
Supplementary_files_format_and_content: Bed-like text files with methylation score (Mscore) as the last column. Files with four columns: chromosome, start, end, Mscore. Files with five columns: chromosome, start, end, description, Mscore.

source_name: K562 cell line

growth_protocol: The 4 tyles of cells (or cell lines) were cultured as the standard protocol, without any special treatment.

extract_protocol:
The DNA was released and the protein was lysed with a lysis buffer, which was removed with ethanol participated (removing the supernatant), without conventional DNA extration.
standard library construction, with barcode on one end.

treatment_protocol: DNA was digested with RE1 (a cocktail of 4 MREs), followed by REPLIg amplification, and then subject to digestion with RE2 (2 options: including Option 1 - BstUI, used in Batch 2, or Option 2: ABH, which is AciI + BstUI + HpaII) and generated library for PE sequencing.

tag: group

value: Test

tag: protocol

value: use treatment protocol option 2 (in batch 3 and 4) (i.e. ABH was used as RE2)

tag: cell #


title: S04

tag: tissue

tag: treatment


title: batch4-K562-SC-Test5

description: CpG methylome


title: batch4-K562-SC-Test4

description: CpG methylome


title: batch4-K562-SC-Test6

description: CpG methylome


title: batch4-K562-SC-Test3

description: CpG methylome


title: HUVEC 3 ProxOE_shScramble K9Ac_ChIP

description:
Sample 2
processed data file: K9ac raw reads.txt, H3K9ac peaks and their differential acetylation.xlsx

tag: cell type

value: human umbilical vein endothelial cells

tag: overexpression

tag: knockdown


title: batch4-K562-SC-Test2

description: CpG methylome


title: batch4-K562-SC-Test1

description: CpG methylome


title: batch4-GM12878-SC-Test7

description: CpG methylome

source_name: GM12878 cell line


title: batch4-GM12878-SC-Test4

description: CpG methylome


title: batch4-GM12878-SC-Test5

description: CpG methylome


title: batch4-GM12878-SC-Test6

description: CpG methylome


title: H1

value: fed for a 10% increase in body mass over 65 days

value: testis


title: batch4-GM12878-SC-Test3

description: CpG methylome


title: batch4-GM12878-SC-Test2

description: CpG methylome


title: batch4-GM12878-SC-Test8

description: CpG methylome


title: batch4-GM12878-SC-Test1

description: CpG methylome


title: batch3-K562-10cell-Test4

description: CpG methylome


title: batch4-K562-SC-Test7

description: CpG methylome


title: batch3-K562-10cell-Test1

description: CpG methylome


title: batch3-K562-10cell-MC3

description: CpG methylome

value: Methylation control (MC)


title: batch3-K562-10cell-MC2

description: CpG methylome


title: batch3-K562-10cell-Test2

description: CpG methylome


title: batch3-K562-10cell-Test5

description: CpG methylome


title: batch3-K562-10cell-Test3

description: CpG methylome


title: batch3-GM12878-100cell-Test3

description: CpG methylome


title: batch3-GM12878-100cell-Test4

description: CpG methylome


title: batch3-GM12878-100cell-Test2

description: CpG methylome


title: batch3-K562-10cell-MC1

description: CpG methylome


title: batch3-GM12878-100cell-Test5

description: CpG methylome


title: batch3-GM12878-100cell-MC2

description: CpG methylome


title: batch3-GM12878-100cell-MC1

description: CpG methylome


title: batch3-GM12878-100cell-Test1

description: CpG methylome


title: batch3-GM12878-100cell-MC3

description: CpG methylome


title: batch3-K562-100cell-Test2

description: CpG methylome


title: batch3-K562-100cell-Test4

description: CpG methylome


title: batch3-K562-100cell-Test5

description: CpG methylome


title: batch3-K562-100cell-Test3

description: CpG methylome


title: batch3-K562-100cell-MC3

description: CpG methylome


title: batch3-K562-100cell-Test1

description: CpG methylome


title: batch3-K562-100cell-MC1

description: CpG methylome


title: batch3-iPSC-100cell-Test5

description: CpG methylome

source_name: IPSC


title: batch3-iPSC-100cell-Test4

description: CpG methylome


title: batch3-iPSC-100cell-Test3

description: CpG methylome


title: batch3-iPSC-100cell-Test2

description: CpG methylome


title: batch3-iPSC-100cell-MC1

description: CpG methylome


title: batch3-Fibroblast-100cell-Test5

description: CpG methylome

source_name: Fibroblast cell line


title: batch3-iPSC-100cell-Test1

description: CpG methylome


title: batch3-iPSC-100cell-MC2

description: CpG methylome


title: batch3-iPSC-100cell-MC3

description: CpG methylome


title: batch3-Fibroblast-100cell-Test2

description: CpG methylome


title: batch3-Fibroblast-100cell-Test1

description: CpG methylome


title: batch3-Fibroblast-100cell-Test4

description: CpG methylome


title: batch3-Fibroblast-100cell-MC2

description: CpG methylome


title: batch2-K562-100cell-Test5

description: CpG methylome

value: use treatment protocol option 1 (in batch 2) (i.e. BstUI was used as RE2)


title: batch3-Fibroblast-100cell-MC3

description: CpG methylome


title: batch3-Fibroblast-100cell-Test3

description: CpG methylome


title: batch2-K562-100cell-Test1

description: CpG methylome


title: batch2-K562-100cell-MC2

description: CpG methylome


title: batch2-K562-100cell-MC3

description: CpG methylome


title: batch2-K562-100cell-Test2

description: CpG methylome


title: batch3-Fibroblast-100cell-MC1

description: CpG methylome


title: batch2-K562-100cell-Test4

description: CpG methylome


title: batch2-K562-100cell-Test3

description: CpG methylome


title: batch3-K562-100cell-MC2

description: CpG methylome


title: batch2-K562-100cell-MC1

description: CpG methylome


title: HS-2

source_name: hESCs-derived hepatic stem cells

value: ESC-derived hepatic stem cells


title: SB-D15-2

source_name: hESCs-derived hepatocytes with SB at day 15

value: ESC-derived hepatocytes

value: SB

tag: time

value: day 15


title: SB-D15-1


title: Ctrl-D15-1

source_name: hESCs-derived hepatocytes at day 15


title: VK2-2

source_name: hESCs-derived hepatocytes with VK2


title: Ctrl-D15-2


title: 786O-3

city: Oxfors

first: James

last: Platt

address: Roosevelt Drive

country: United Kingdom

institute: University of Oxford

data_processing:
Library strategy: Capture-C
Reads were trimmed using trim galore and then in silico digested with DpnII using dpnII2E.pl (https://github.com/telenius/captureC/releases)
Resulting reads were then aligned to genome using bowtie 1.0.0
Interaction frequencies were then determined using CCanalyser2.pl (https://github.com/telenius/captureC/releases)
Genome_build: hg19
Supplementary_files_format_and_content: bigwig files contain interaction frequency from all sites

source_name: Renal Carcinoma Cell Line

growth_protocol: Dulbecco Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100U/ml penicillin and 100 μg/ml streptomycin.

extract_protocol: Capture-C libraries were prepared as previously described (Davies et al. 2016 Nature Methods). Cells were fixed with 2% (vol/vol) formaldehyde for 10 min, quenched with 125 mM glycine in PBS and then lysed in cold lysis buffer (10 mM Tris-HCl, pH 8, 10 mM NaCl, 0.2% Igepal, 1× cOmplete Protease Inhibitor Cocktail (Roche). Chromatin was digested with Dpn2 (New England Biolabs) at 37oC overnight.  Fragments were then diluted and ligated with T4 DNA ligase (Thermo Scientific) at 16oC overnight. Crosslinking was reversed by overnight incubation at 60oC with proteinase K (Bioline). The 3C libraries were then purified by phenol-chloroform and chloroform extraction followed by precipitation in ethanol at -80oC overnight. Digestion efficiency was determined by qPCR and gel electrophoresis. Sequencing libraries were prepared from 5 μg of each 3C library by sonication using a S220 focused-ultrasonicator (Covaris) to a average size of 200bp and indexed using NEBnext reagents (New England Bioloabs) according to protocol. Enrichment of 1-2 μg of indexed library incubated with 13 pmol of a pool of biotinylated oligonucleotides (Intergrated DNA technologies or Sigma) was performed using the SeqCap EZ system (#06953212001, Roche/Nimblegen) following the manufacturer’s instructions. Two rounds of capture employing 48-72 hr and 24 hr hybridizations respectively were used. Capture enrichment was determined by qPCR. Correct library size was confirmed using a Bioanalyzer DNA 1000 or Tapestation D1000 kit (Agilent), and DNA concentrations were determined using a Qubit 2.0 Fluorometer (ThermoFisher Scientific).

value: Normoxia

tag: cell line modification


title: WT3

city: Sendai

first: Lei

last: YU

state: Miyagi

address: 2-1 Seiryo-cho, Aoba-ku

department: Department of Medical Biochemistry

country: Japan

institute: Tohoku University School of Medicine

data_processing:
Illumina bcl2fastq v2.15.0 software used for basecalling.
Reads mapped against mouse (mm9) genome using the TopHat application on Illumina BaseSpace.
Differential gene analaysis performed using Cuffdiff v2.1.1 application on Illumina BaseSpace.
Genome_build: mm9
Supplementary_files_format_and_content: tab-delimited text files include FPKM values for each genotype were generated using Cuffdiff.

organism: Mus musculus

source_name: WT_LSK cells

growth_protocol: LSK cells were isolated from bone marrow of 12-15 weeks old WT and MG-G1 mice by flow cytometry

extract_protocol:
Total RNA was prepared by using ISOGEN (Nippon Gene) and 75 ng of total RNA was used for further steps. Isolation of poly(A)-tailed RNA and library construction were performed using SureSelect Strand Specific RNA Sample Prep Kit (Agilent Technologies).
RNA libraries were prepared for sequencing using standard Illumina protocols

tag: genotype/variation

value: wild type

tag: age

value: 12-15 weeks

value: bone marrow

value: LSK cells


title: MG1

source_name: MG-G1_LSK cells

value: GATA1-HSC overexpressing (MG-G1)


title: MG3


title: MG2


title: batch2-K562-100cell-MC4

description: CpG methylome


title: VK2-1


title: 786O-2


title: Ctrl-2

source_name: hESCs-derived hepatocytes


title: 786O-1


title: Ctrl-1


title: WT2


title: 60

city: Urbana

first: Sandra

last: Rodriguez Zas

state: IL - Illinois

address: 1207 W. Gregory Dr.

institute: University of Illinois at Urbana

data_processing:
Quality-scores line in fastq files processed with Casava1.8 use an ASCII offset of 33
Sequences were trimmed for adaptor seuqences and masked for low-complexity and low-quality
100 nt paired-end reads were mapped to the mm10 genome assembly (UCSC; http://genome.ucsc.edu) using TopHat v2.0.13 and default settings (Trapnell et al., 2012; Nature Protocols)
Reads were assembled into transcript isoforms using Cufflinks Version 2.2.0 (Trapnell et al., 2012)
Genome_build: mm10 genome assembly (UCSC)
Supplementary_files_format_and_content: [.txt] tab-delimited file including FPKM values by loci

source_name: High Runner_Blocked_Cerebellum

extract_protocol:
Brain was extracted and the whole cerebellum was dissected, snap frozen on dry ice and stored in a centrifuge tube at -80°C following proven protocols (Gibbons et al. 2014, Behavioural brain research, 272: 131-40.). Tissue was homogenized with an RNase-free disposable pellet pestle (Fisher) and RNA was extracted using the commercially available RNeasy® Lipid Tissue Mini Kit (Qiagen, Valencia, CA). Purification of the isolated RNA included treatment with DNase I (Qiagen, Valencia, CA)\.
RNA libraries were prepared for sequencing using standard Illumina protocols

tag: strain

tag: line

value: High Runner

tag: physical activity

value: Blocked

tag: gender

value: male

value: Cerebellum


title: 48


title: SB-1

source_name: hESCs-derived hepatocytes with SB


title: PHHs-2

source_name: primary human hepatocytes isolated from adult liver

value: primary hepatocytes


title: PHHs-1


title: Mutant Rep 1

city: Norwich

first: John

middle: Tyson

last: Munnoch

address: Norwich Research Park

department: Schol of Biological Sciences

institute: University of East Anglia

data_processing:
library strategy: ChIP-Exo
Alignment of reads to reference genome (NC_018750.1) using bowtie2 (bowtie2-2.2.3) options [bowtie2 -x reference_genome -S output.sam -U read_file]
conversion to .bam format and sorting using samtools (version 1.2).  Commands: samtools view -bS [input.sam] > [output.bam].  Samtools sort [input.bam] [output.sortedbam]
Conversion of sorted bam file to a bedgraph format using bedtools.  Command: genomeCoverageBed -ibam sorted.bam -g reference_genome.fna -bga file.bedgraph
production of a wig file suitable for visualisation in IGB using personal unpublished script file contains header (track   type=wiggle_0   name=WT_16h       graphType=points        visibility=full color=168,130,88  fixedStep       chrom=Sven      start=1 step=1  span=1) with each subsequent line corresponding to the number of times a single base was sequenced with each base represented by a seperate line.
CLC workbench 8 ChIP-Seq protocal was followed using the default settings.  >2700 targets were identified due to the lack of a suitable negative/genomic back ground control.  Arbitrary cut offs of 200 and 500 read depth were used in the publication for defining the regulon.
Additional visual inspection was carried out using the wig files and IGB to validate target peaks.
See dataprocessing_readme.txt
Genome_build: ASM25323v1 (NC_018750.1)
Supplementary_files_format_and_content: wig

organism: Streptomyces venezuelae

source_name: Mutant

growth_protocol: 5-7 ul of current spore stock (OD600 ~0.35 after 8h) was innoculated into 35 ml of MYM-tap growth media, and grown for 16h.

extract_protocol:
[Full] ChIP-Seq was carried out as previously described (28). A spore inoculum (~5-10 ul of 1x108 spores) sufficient to reach an OD600 of 0.35 after 8 hours of growth was added to 35ml of MYM tap media in 250 ml flasks containing springs.  Following growth to the chosen time point, the entire content of the flask was transferred to a 50 ml falcon tube for crosslinking, which was carried out by incubation at 30oC for 30 mins with 1% final concentration of formaldehyde (v/v).  Crosslinking was quenched by incubation at room temperature with glycine (final concentration of 125 mM).  Mycelium was harvested by centrifugation 4000 rpm at 4oC for 10 minutes and washed twice with ice cold PBS before transfer to a 2 ml centrifuge tube.  Pellets were resuspended in 0.75 ml lysis buffer (10 mM Tris-HCl pH 8.0, 50 mM NaCl, 10 mg/ml lysozyme, 1x protease inhibitor-Roche1186170001) and incubate at 37C for 10-25 mins.  Then 0.75 ml 1x IP buffer (100mM tris-HCl pH 8.0, 250 mM NaCl, 0.5% Triton x-100, 0.1% SDS, 1x protease inhibitor (Roche)) was added and samples mixed by pipetting up and down.  Samples were sonicated 7x at 50Hz, 10 sec/cycle with a 1 min incubation on ice after each cycle.  DNA fragmentation was checked by agarose gel electrophoresis following phenol extraction of 25 µl of the crude lysate mixed with 75 µl of TE buffer with 100-200 µl of phenol/chloroform.   Contaminating RNA was removed with 2 µl RNase (1mg/ml) added to extracted DNA followed by an incubation for 30 min at 37°C. A smear of DNA from 200 to 1000 bp with the majority of DNA 200-400 bp should be visible. Crude lysate was centrifuged at 13,000 rpm for 15 minutes at 4°C to clear the sample of cell debris.  M2 magnetic beads (Sigma-Aldrich) were used following manufacturers instructions.  Exonuclease treatment was carried out by penconic genomics as previously desribed (Molecular Mechanisms of Ribosomal Protein Gene Co-regulation", Rohit Reja, Vinesh Vinayachandran, Sujana Ghosh, B. Franklin Pugh.  (2015) Genes & Devel). The cleared lysate was incubated with 40 µl of washed M2 beads and incubated for 4 h at 4C in a vertical rotor.  The lysate was removed and the beads pooled into one 1.5 microfuge tube and washed in 0.5 IP buffer.  The beads were transferred to a fresh microfuge. tube and washed a further 3 times removing as much buffer as possible without disturbing the beads.  The DNA-protein complex was eluted from the beads with 100 µl elution buffer (50 mM Tris-HCl pH7.6, 10mM EDTA, 1% SDS) by incubating at 65°C overnight.  Removing the ~100µl elution buffer, an extra 50 µl of elution buffer was added and further incubated at 65oC for 5 min.   To extract the DNA 150 µl eluate, 2 µl proteinase K (10 mg/ml) was added and incubated 1.5 hrs at 55oC.   To the reaction 150 µl phenol-chloroform was added.  Samples were vortexed and centrifuged at full speed for 10 min.  The aqueous layer was extracted and purified using the Qiaquick column from Qiagen with a final elution using 50 µl EB buffer (Qiagen).   The concentration of samples were determined using Quant-iT™ PicoGreen ® dsDNA Reagent (Invitrogen) or equivalent kit or by nanodrop measurement.
[Brief] Cells were treated with hormaldehyde to crosslink DNA and protein, lysed, sonicated and the tagged protein/DNA complexes were immunoprecipitated using anti-flag antibody bound M2 beads.
DNA libraries were prepared for sequencing using standard Illumina protocols

tag: expressing

value: RsrR::apr pMS82 RsrR 3xFlag

tag: growth time point

tag: chip-antibody

value: ANTI-FLAG M2 Affinity Gel (cat number A2220)


title: 63


title: HS-1

value: ESC-derived hepatic stem cells


title: 28


title: 43

source_name: High Runner_Free_Cerebellum

value: Free


title: 12


title: 2


title: 25

value: Control


title: 40


title: SB-2

value: ESC-derived hepatocytes

value: SB


title: 27


title: 51


title: 30


title: P0 cKO

city: Piscataway

first: Li

last: Cai

address: 599 Taylor Road

institute: Rugers, the Stage U of NJ

description: processed data file: p0wt-ko-gene_exp.diff

data_processing:
SOLiD sequence analysis software
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence, then mapped to mm9 whole genome using bowtie
Reads Per Kilobase of exon per Megabase of library size (RPKM) were calculated using cuffdiff (Trapnell et al., Nature Protocol 2012).
Genome_build: mm9
Supplementary_files_format_and_content: tab-delimited text files include RPKM values for each Sample

source_name: Retina

extract_protocol:
Retinas were removed, flash frozen on dry ice, and RNA was harvested using Trizol reagent. Small RNA Expression Kit (Applied Biosystems) was used with 500 ng of total RNA for the construction of sequencing libraries.
RNA libraries were prepared for sequencing using AB SOLiD protocols

value: post natal day 0

value: retina


title: WT1

value: wild type

value: 12-15 weeks

value: bone marrow


title: P6 WT

description: processed data file: p6wt-ko-gene_exp.diff

value: post natal day 6


title: 15

value: Blocked


title: 11

value: male


title: 49

value: Cerebellum


title: ΔNp63Δ/Δ;p53-/- rep1

city: Tampa

first: Elsa

middle: Renee

last: Flores

address: 12902 USF Magnolia Drive

department: Department of Molecular Oncology

institute: Moffit Cancer Center

description:
small RNA
MN_5

data_processing:
Illumina small RNA adapter sequences were trimmed from the reads.
Reads of length below 10nt or ending in homopolymers of length 9 nt or above were discarded.
Total usable number of reads for each sample was calculated
The reads were mapped to the miRBase reference using BLAST; the abundance of each expressed microRNA was quantified by counting reads mapping to mature mirRNAs +/- 4 nucleotides
Abundance of miRNAs was reported as fraction of usable reads in parts per 10 million and log2 scale
Genome_build: mm10
Supplementary_files_format_and_content: TAB-delimited text format, containing quantile normalized microRNA abundance

source_name: thymic lymphoma tumor

extract_protocol:
Thymic lymphomas were collected 48 hr after the adenoviral injection, and RNA was purified using the miRvana microRNA isolation kit (Ambion) in preparation for smallRNA-Seq.
For the small RNA library construction, RNA samples were prepared using the DGE-Small RNA Sample Prep Kit (Illumina, San Diego, CA).

treatment_protocol: 10 weeks-old mice were injected with either Ad-mCherry or Ad-CRE-mCherry to delete ΔNp63 in the thymic lmyphomas.

value: thymic lymphoma tumor

tag: intratumoral injection

value: Ad-CRE-mCherry

tag: genotype


title: ΔNp63Δ/Δ;p53-/- rep3

description:
small RNA
MN_7

tag: tissue


title: P0 WT


title: p53-/- rep1

description:
small RNA
MN_1

value: Ad-mCherry


title: HFD Thermoneutral 1

city: Cincinnati

first: Rebekah

last: Karns

state: OH

address: 3333 Burnet Ave

department: Gastroenterology, Hepatology, & Nutrition

institute: Cincinnati Children's Hospital Medical Center

description: Total RNA from a C57BL6 WT Mouse on High fat diet for 8 weeks following 2 weeks at Thermoneutral 30-33˚ housing

data_processing:
Standard Illumina software was used for base calling
Sequenced reads were trimmed of barcodes and adaptors, then aligned to the mm10 genome using annotations from Ensembl within Strand NGS software.
Aligned reads were quantified to produce FPKM, which was log2 transformed, normalized using the DESeq algorithm, and baselined to the median of all samples.
Genome_build: mm10
Supplementary_files_format_and_content: Text delimited files

source_name: HFD Thermoneutral_PBMC

extract_protocol:
Ambion mirvana protocol
Standard Illumina protocols were used

treatment_protocol: Two weeks at either standard or thermoneutral housing, followed by 8 weeks on chow or high-fat diet

tag: strain background

value: WT

tag: diet

value: High fat diet

tag: housing

value: Thermoneutral 30-33˚

tag: cell type

value: peripheral blood mononuclear cells (PBMCs)


title: 61

value: Control


title: P6 cKO


title: p53-/- rep4

description:
small RNA
MN_4


title: HFD Standard 2

description: Total RNA from a C57BL6 WT Mouse on Chow for 8 weeks following 2 weeks at Thermoneutral 30-33˚ housing

source_name: HFD Standard_PBMC

value: Standard 22˚


title: Chow Thermoneutral 2

description: Total RNA from a C57BL6 WT Mouse on High fat diet for 8 weeks following 2 weeks at Standard 22˚ housing

source_name: Chow Thermoneutral_PBMC

value: Chow


title: p53-/- rep3

description:
small RNA
MN_3


title: Chow Thermoneutral 1


title: p53-/- rep2

description:
small RNA
MN_2


title: HFD Standard 1


title: Mutant Rep 2

data_processing:
Alignment of reads to reference genome (NC_018750.1) using bowtie2 (bowtie2-2.2.3) options [bowtie2 -x reference_genome -S output.sam -U read_file]
conversion to .bam format and sorting using samtools (version 1.2). Commands: samtools view -bS [input.sam] > [output.bam]. Samtools sort [input.bam] [output.sortedbam]
Conversion of sorted bam file to a bedgraph format using bedtools. Command: genomeCoverageBed -ibam sorted.bam -g reference_genome.fna -bga file.bedgraph
production of a wig file suitable for visualisation in IGB using personal unpublished script file contains header (track type=wiggle_0 name=WT_16h graphType=points visibility=full color=168,130,88  fixedStep chrom=Sven start=1 step=1 span=1) with each subsequent line corresponding to the number of times a single base was sequenced with each base represented by a seperate line.
CLC workbench 8 ChIP-Seq protocal was followed using the default settings. >2700 targets were identified due to the lack of a suitable negative/genomic back ground control. Arbitrary cut offs of 200 and 500 read depth were used in the publication for defining the regulon.
Additional visual inspection was carried out using the wig files and IGB to validate target peaks.
Genome_build: ASM25323v1 (NC_018750.1)
See dataprocessing_readme.txt
Supplementary_files_format_and_content: wig files report target peaks

organism: Streptomyces venezuelae ATCC 10712

extract_protocol:
[Full] ChIP-Seq was carried out as previously described (28). A spore inoculum (~5-10 ul of 1x108 spores) sufficient to reach an OD600 of 0.35 after 8 hours of growth was added to 35ml of MYM tap media in 250 ml flasks containing springs. Following growth to the chosen time point, the entire content of the flask was transferred to a 50 ml falcon tube for crosslinking, which was carried out by incubation at 30oC for 30 mins with 1% final concentration of formaldehyde (v/v). Crosslinking was quenched by incubation at room temperature with glycine (final concentration of 125 mM). Mycelium was harvested by centrifugation 4000 rpm at 4oC for 10 minutes and washed twice with ice cold PBS before transfer to a 2 ml centrifuge tube. Pellets were resuspended in 0.75 ml lysis buffer (10 mM Tris-HCl pH 8.0, 50 mM NaCl, 10 mg/ml lysozyme, 1x protease inhibitor-Roche1186170001) and incubate at 37C for 10-25 mins. Then 0.75 ml 1x IP buffer (100mM tris-HCl pH 8.0, 250 mM NaCl, 0.5% Triton x-100, 0.1% SDS, 1x protease inhibitor (Roche)) was added and samples mixed by pipetting up and down. Samples were sonicated 7x at 50Hz, 10 sec/cycle with a 1 min incubation on ice after each cycle. DNA fragmentation was checked by agarose gel electrophoresis following phenol extraction of 25 µl of the crude lysate mixed with 75 µl of TE buffer with 100-200 µl of phenol/chloroform. Contaminating RNA was removed with 2 µl RNase (1mg/ml) added to extracted DNA followed by an incubation for 30 min at 37°C. A smear of DNA from 200 to 1000 bp with the majority of DNA 200-400 bp should be visible. Crude lysate was centrifuged at 13,000 rpm for 15 minutes at 4°C to clear the sample of cell debris. M2 affinity beads (Sigma-Aldrich #A2220) were prepared by washing in ½IP buffer following manufacturers instructions. The cleared lysate was incubated with 40 µl of washed M2 beads and incubated for 4 h at 4C in a vertical rotor. The lysate was removed and the beads pooled into one 1.5 microfuge tube and washed in 0.5 IP buffer. The beads were transferred to a fresh microfuge. tube and washed a further 3 times removing as much buffer as possible without disturbing the beads. The DNA-protein complex was eluted from the beads with 100 µl elution buffer (50 mM Tris-HCl pH7.6, 10mM EDTA, 1% SDS) by incubating at 65°C overnight. Removing the ~100µl elution buffer, an extra 50 µl of elution buffer was added and further incubated at 65oC for 5 min. To extract the DNA 150 µl eluate, 2 µl proteinase K (10 mg/ml) was added and incubated 1.5 hrs at 55oC. To the reaction 150 µl phenol-chloroform was added. Samples were vortexed and centrifuged at full speed for 10 min. The aqueous layer was extracted and purified using the Qiaquick column from Qiagen with a final elution using 50 µl EB buffer (Qiagen). The concentration of samples were determined using Quant-iT™ PicoGreen ® dsDNA Reagent (Invitrogen) or equivalent kit or by nanodrop measurement.
[Brief] Cells were treated with formaldehyde to crosslink DNA and protein, lysed, sonicated and the tagged protein/DNA complexes were immunoprecipitated using anti-flag antibody bound M2 beads.
DNA libraries were prepared for sequencing using standard Illumina protocols

value: RsrR::apr pMS82 RsrR 3xFlag

value: ANTI-FLAG M2 Affinity Gel (cat number A2220)


title: Mutant Rep 1


title: WT_16h_TAP+

data_processing:
Alignment of reads to reference genome (NC_018750.1) using bowtie2 (bowtie2-2.2.3) options [bowtie2 -x reference_genome -S output.sam -U read_file]
Aligned sam file was split into strand specific files using the below script and adding a suitable sam header:  use strict;  my $file=$ARGV[0];    open (INFIL, "<$file");  open (POS, ">$file.pos.sam");  open (NEG, ">$file.neg.sam");    while (<INFIL>){  chomp;  my @ar = split (/\t/, $_);  if ($ar[0] =~ m/^@/ or $ar[1] == 163){  print NEG "$_\n";  }  if ($ar[0] =~ m/^@/ or $ar[1] == 99){  print POS "$_\n";  }  }
conversion to .bam format and sorting using samtools (version 1.2).  Commands: samtools view -bS [input.sam] > [output.bam].  samtools [input.bam] [output.sortedbam]
Conversion of sorted bam file to a bedgraph format using bedtools.  Command: genomeCoverageBed -ibam sorted.bam -g reference_genome.fna -bga file.bedgraph
production of a wig file suitable for visualisation in IGB using personal unpublished script file contains header (track   type=wiggle_0   name=WT_16h       graphType=points        visibility=full color=168,130,88  fixedStep       chrom=Sven      start=1 step=1  span=1) with each subsequent line corresponding to the number of times a single base was sequenced with each base represented by a seperate line.
CLC workbench 8 RNA-seq protocal was followed using the default settings.
Additional visual inspection was carried out using the wig files and IGB to validate target peaks.
TSS identification was carried out using the TSSAR web server (http://nibiru.tbi.univie.ac.at/TSSAR/) ("TSSAR: TSS Annotation Regime for dRNA-seq data" F. Amman, M.T. Wolfinger, R. Lorenz, I.L. Hofacker, P.F. Stadler and S. Findeiß - BMC Bioinformatics, 2014 15:89 )
Genome_build: NC_018750.1
See dataprocessing_readme.txt
Supplementary_files_format_and_content: TAP+_rsrR_negstrand.wig: wig - a file containg the frequency each base was sequenced for the negative stand of the TAP treated mutant sample
Supplementary_files_format_and_content: TAP+_rsrR_posstrand.wig: wig - a file containg the frequency each base was sequenced for the positive stand of the TAP treated mutant sample
Supplementary_files_format_and_content: TAP-_rsrR_negstrand.wig: wig - a file containg the frequency each base was sequenced for the negative stand of the TAP- mutant sample
Supplementary_files_format_and_content: TAP-_rsrR_posstrand.wig: wig - a file containg the frequency each base was sequenced for the positive stand of the TAP- mutant sample
Supplementary_files_format_and_content: TAP+_wt_negstrand.wig: wig - a file containg the frequency each base was sequenced for the negative stand of the TAP treated WT sample
Supplementary_files_format_and_content: TAP+_wt_posstrand.wig: wig - a file containg the frequency each base was sequenced for the positive stand of the TAP treated WT sample
Supplementary_files_format_and_content: TAP-_wt_negstrand.wig: wig - a file containg the frequency each base was sequenced for the negative stand of the TAP- WT sample
Supplementary_files_format_and_content: TAP-_wt_posstrand.wig: wig - a file containg the frequency each base was sequenced for the positive stand of the TAP- WT sample
Supplementary_files_format_and_content: WT_+ve_strand_TSSAR.txt: TSSAR_output.txt - Contains tab-delimited transcriptional start site data
Supplementary_files_format_and_content: WT_-ve_strand_TSSAR.txt: TSSAR_output.txt - Contains tab-delimited transcriptional start site data
Supplementary_files_format_and_content: Mutant_+ve_strand_TSSAR.txt: TSSAR_output.txt - Contains tab-delimited transcriptional start site data
Supplementary_files_format_and_content: Mutant_-ve_strand_TSSAR.txt: TSSAR_output.txt - Contains tab-delimited transcriptional start site data
Supplementary_files_format_and_content: Expression_values_WT_vs_RsrR_mutant.txt: Expression_values.txt - The data contained within the excel sheet minus the associated calculations
Supplementary_files_format_and_content: Expression_values_WT_vs_RsrR_mutant.xlsx: Expression_values_excel - where the .txt file data came from but this also contains the calculations used to produce the data

source_name: WT

extract_protocol:
Mycelium was harvested at experimentally appropriate time points and immediately transferred to 2 ml round bottom tubes, flash frozen in liquid N2, stored at -80oC or used immediately.  All apparatus used was treated with RNaseZAP (Sigma) to remove RNases for a minimum of 1 hour before use.  RNaseZAP treated mortar and pestles were used, the pestle being placed and cooled on a mixture of dry ice and liquid N2 with liquid N2 being poured into the bowl and over the mortar.  Once the bowl had cooled the mycelium samples were added directly to the liquid N2 and thoroughly crushed using the mortar leaving a fine powder of mycelium.  Grindings were transferred to a pre-cooled 50 ml Falcon tube and stored on dry ice.  Directly to the tube, 2 ml of TRI reagent (Sigma) was added to the grindings and mixed.  Samples are then thawed while vortexing intermittently at room temperature for 5-10 minutes until the solution cleared.   To 1 ml of TRI reagent resuspension, 200 µl of chloroform was added and vortexed for 15 seconds at room temperature then centrifuged for 10 minutes at 13,000 rpm.  The upper, aqueous phase (clear colourless layer) was removed into a new 2 ml tube.  The remainder of the isolation protocol follows the RNeazy Mini Kit (Qiagen) instructions carrying out both on and off column DNase treatments.  On column treatments were carried out following the first RW1 column wash.  DNaseI (Qiagen) was added (10 µl enzyme, 70 µl RDD buffer) to the column and stored at RT for 1 hour.  The column was washed again with RW1 then treated as described in the manufacturer’s instructions.  Once eluted from the column, samples were treated using TURBO DNA-free Kit (Ambion) following manufacturer’s instructions to remove residual DNA contamination.
For TSS cDNA library construction, the samples were split into two halves and poly(A)-tailed using poly(A) polymerase. This was followed by a treatment with Terminator exonuclease (TEX, Epicentre). Each one half of the samples was then treated with RNA 5' Polyphosphatase (+5'PP, Epicentre) in order to convert 5'PPP structures into 5' monophosphate ends. The other half of the samples was left untreated (-5'PP) . Afterwards, an RNA adapter was ligated to the 5'-monophosphate of the RNA. First-strand cDNA synthesis was performed using an oligo(dT)-adapter primer and the M-MLV reverse transcriptase. The resulting cDNAs were PCR-amplified using a high fidelity DNA polymerase. PCR cycles performed and barcode sequences, which are part of the 5' sequencing adapter. The cDNAs were purified using the Agencourt AMPure XP kit (Beckman Coulter Genomics) and were analyzed by capillary electrophoresis.

tag: treatment

value: Tex

value: TAP

tag: growth time point (hours from innuculation)


title: Chow Standard 2

description: Total RNA from a C57BL6 WT Mouse on Chow for 8 weeks following 2 weeks at Standard 22˚ housing

source_name: Chow Standard_PBMC

tag: genotype/variation


title: WT_16h_TAP-


title: ΔNp63Δ/Δ;p53-/- rep2

description:
small RNA
MN_6


title: RsrR_16h_TAP-


title: WT rep 2


title: 26043: WapCre;Rank;Brca1;p53

city: Vienna

first: Verena

last: Sigl

address: Dr. Bohr-Gasse 3

country: Austria

institute: IMBA - Institute of Molecular Biotechnology GmbH

data_processing:
50-bp single-end Illumina mRNA sequencing reads were aligned using Tophat v2.0.10 and bowtie v0.12.9.
aligned reads were counted with HTSeq
FPKM estimation was performed with Cufflinks (v2.1.1) and differential expression analysis with DESeq2 (v1.6.2)
Genome_build: mm10
Supplementary_files_format_and_content: tab-delimited text files include FPKM values for each sample (standard output cufflinks)

source_name: female mouse primary mammary tumor

extract_protocol:
Total RNA was purified using RNAeasy mini Kit (Qiagen). poly-T selected RNA was isolated and dUTP-containing cDNA was generated.
Illumina TruSeq protocol

value: female

value: mammary tumor


title: 26048: WapCre;Brca1;p53


title: 26050: WapCre;Brca1;p53


title: HFD Thermoneutral 2


title: RsrR_16h_TAP+


title: 26044: WapCre;Rank;Brca1;p53


title: WT rep 1


title: TA-F-Ov

status: Public on Feb 11 2017

city: Wuhan

first: Daji

last: Luo

state: Hubei

address: No.185 Donghudong Road, Wuchang distinct

institute: Wuhan University

description: TALENs induced dmy-mutant sex reversal female medaka

data_processing:
Raw Reads(Illumina HiSeq™ 2000),Illumina Casava1.7 software used for basecalling.
Filter Raw Reads(Filter_fq,BGI unpublished software) removed reads with adaptors, unknown nucleotides larger than 5%, and low quality reads which the percentage of low quality bases is more than 20%
Reads Per Kilobase of exon per Megabase of library size (RPKM) were calculated using a protocol from Chepelev et al., Nucleic Acids Research, 2009. In short, exons from all isoforms of a gene were merged to create one meta-transcript. The number of reads falling in the exons of this meta-transcript were counted and normalized by the size of the meta-transcript and by the size of the library.
Assembly and Cluster were used Trinity (release-20130225), TGICL(v2.1) and Phrap (Release 23.0)

organism: Oryzias latipes

extract_protocol:
After the total RNA extraction, magnetic beads with Oligo (dT) are used to isolate mRNA(for eukaryotes). Mixed with the fragmentation buffer, the mRNA is fragmented into short fragments.
cDNA is synthesized using the mRNA fragments as templates. Short fragments are purified and resolved with EB buffer for end reparation and single nucleotide A addition. After that, the short fragments are connected with adapters.The suitable fragments are selected for the PCR amplification as templates. During the QC steps, Agilent 2100 Bioanaylzer and ABI StepOnePlus Real-Time PCR System are used in quantification and qualification of the sample library.The library could be sequenced using Illumina HiSeq™ 2000

value: Ovary


title: WT-M-Te

description: Wildtype male medaka

source_name: Wildtype male medaka

value: Testis


title: shRUNX2_3_repeat1

city: Bethesda

first: Jing

last: Huang

state: MD

institute: National Cancer Institute

description: RNA with Poly A tail

data_processing:
Base calling: RTA 1.12.4.2 (performed at the Next Generation Sequencing Facility at the Center for Cancer Research at the National Cancer Institute)
Demultiplexing: Casava 1.8.2 allowing 1 mismatch
Alignment and filtering: tophat -G hg19_Ensemble_v70.gtf -o ./ -r 10 --mate-std-dev 200 -p 16 read1.fastq read2.fastq
To generate FPKM files: Cufflinks using a gtf file of hg19 (Refseq) downloaded on 08/24/2014 from UCSC browser.
Genome_build: hg19
Supplementary_files_format_and_content: FPKM files were generated using Cufflinks

source_name: SAOS2 cells

growth_protocol: SAOS2 were grown in DMEM, 10% Fetal Bovine Serum, and 1X penicillin-streptomycin (All from Invitrogen).

extract_protocol:
RNAs were extracted using the GenElute Mammalian total RNA Miniprep kit (Sigma, Cat: RTN350-1kt).
Illumina TruSeq RNA, Protocol RS-930-2001 with Illumina TruSeq V3.0

tag: cell line

value: cell culture

tag: passage

value: less than 30 passages

tag: drug resistance

value: puromycin selection for 6 days after lentiviral transduction


title: shLuc, repeat2

description: RNA with Poly A tail


title: shLuc, repeat3

description: RNA with Poly A tail


title: shRUNX2_4_repeat2

description: RNA with Poly A tail


title: shRUNX2_3_repeat3

description: RNA with Poly A tail


title: shRUNX2_4_repeat3

description: RNA with Poly A tail


title: RUNX2 ChIPseq

description: ChIP DNA

data_processing:
Base calling: RTA 1.12.4.2 and sequencing chemistry: Illumina TruSeq 3.0. All raw files were generated using this method.
Alignment: Illumina Casava 1.8.2. All processed data files were generated using this method.
Processed files are peak files. Peak calling was performed using MACS (version 1.4.1 2011_06_27; band width = 300; model fold = 10,30; pvalue cutoff = 1.00e-05)
Genome_build: hg19
Supplementary_files_format_and_content: peak file

growth_protocol: SAOS2 cells were grown in DMEM, 10% Fetal Bovine Serum, 2mM L-glutamine and 1X penicillin-streptomycin (All from Invitrogen) and split every three days.

extract_protocol:
ChIP DNA was extracted by standard phenol approach.
NEB ChIP-seq prep kit

treatment_protocol: No treatment

value: osteosarcoma cell line

tag: passage number

value: less than 20 passages

tag: chip antibody

value: Custom made RUNX2 antibody


title: WT-F-Ov

description: Wildtype female medaka

source_name: Wildtype female medaka


title: shRUNX2_3_repeat2

description: RNA with Poly A tail


title: InvD6-2

city: PORTLAND

first: Qiuchen

last: Guo

state: OREGON

address: 3181 SW SAM JACKSON PARK ROAD

department: CDCB

institute: OHSU

data_processing:
The result data were converted to FASTQ format using Bcl2Fastq software (Illumina)
Reads were aligned to the mouse reference genome, build GRCm38 using STAR 2.4.2a. STAR performed counting of reads per gene as defined in Ensembl build 81 (GENCODE version m6).
Read counts were adjusted to a common non-zero minimum via addition of uniform minor background counts.  Read count distributions were normalized across samples using cyclic Loess normalization by R software
Normalized read counts were log2-transformed for further analysis.
Genome_build: mouse reference genome, build GRCm38
Supplementary_files_format_and_content: Excel data containing log2-transformed normalized read counts for each samples.

source_name: 6 days post-weaning mammary glands

growth_protocol: FACS sorted PDGFRa+ mammary cells were isolated and freezed in -80 for RNA extraction

extract_protocol:
total RNA was extracted using Invitrogen PureLink® RNA Micro Scale Kit
Libraries were prepared according to Illumina's instructions accompanying the TruSeq RNA Sample Preparation V2 kit.

tag: strain

value: Balb/c

value: 4th and 5th mammary glands

tag: reproductive stage

value: InvD6 (6 days post-weaning)

value: PDGFRa+  mammary fibroblasts


title: Chow Standard 1

value: WT

value: Chow

value: Standard 22˚

value: peripheral blood mononuclear cells (PBMCs)


title: shRUNX2_4_repeat1

description: RNA with Poly A tail


title: Input, SAOS2

description: Input DNA

tag: chip antibody vendor

value: No antibody


title: CBFB ChIPseq

description: ChIP DNA

value: CBFB specific antibody

value: Bethyl

tag: chip antibody cat. #


title: FN-1

source_name: nulliparous mouse mammary glands

tag: tissue

value: Nulliparous (N)


title: shLuc, repeat1

description: RNA with Poly A tail

value: cell culture

value: less than 30 passages


title: FN-2


title: WT_AntNPC_ChIP_p300

city: Koeln

first: Milos

last: Nikolic

state: Nordrhein-Westfalen

address: Robert Koch Str. 21

country: Germany

institute: Center for Molecular Medicine Cologne

description: WT (E14)

data_processing:
Basic read quality check was performed using FastQC (Babraham Bioinformatics) and read statistics were obtained with SAMtools
Reads were mapped to the mouse reference assembly, version GRCm38, using TopHat2
Normalized bedGraph files were generated using DeepTools
Peaks were called using MACS2
Genome_build: mm10

source_name: AntNPC (anterior neural progenitors derived from mESC)

growth_protocol: WT (E14) mESC and EED-/- mESC lines  were cultured on gelatin-coated plates using Knock-out DMEM (KO-DMEM, Life Technologies) supplemented with 15% FBS (Life Technologies) and LIF. These mESC lines were differentiated into AntNPC according to a previously described protocol (Gouti et al., 2014), with slight modifications. Briefly, mESC were plated at a density of 10000 cells/cm2 on gelatin-coated plates and grown for three days in N2B27 medium supplemented with 10 ng/ml bFgf (Life Technologies) and without serum or LIF (Advanced Dulbecco’s Modified Eagle Medium F12 (Life Technologies) and Neurobasal medium (Life Technologies) (1:1), supplemented with 1x N2 (Life Technologies), 1x B27 (Life Technologies), 2 mM L-glutamine (Life Technologies), 40 mg/ml BSA (Life Technologies), 0.1 mM 2-mercaptoethanol (Life Technologies)). Subsequently, cells were grown for another two days in N2B27 medium without bFgf (D3–D5).  Moreover, to improve the homogeneity of the differentiation, from D2-D5 the N2B27 medium was also supplemented with 5μM Xav939 (Sigma), a potent WNT inhibitor (Matsuda and Kondoh, 2014). WT mESC (E14) were adapted and subsequently maintained under 2i conditions as previously described (Respuela et al. 2016). Briefly, mESC were adapted for a minimum of five passages and then grown with “2i+LIF” medium (serum-free N2B27 medium supplemented with MEK inhibitor PD0325901 (0.4 μM, Miltenyi Biotec), GSK3β inhibitor CHIR99021 (3 μM, Amsbio) and LIF) in tissue culture dishes pre-treated with gelatin (Sigma).

extract_protocol:
Lysates were clarified from sonicated nuclei and protein-DNA complexes were isolated with antibodies againts the spcific proteins of interest in each case.
DNA libraries were prepared using the Illumina TruSeq RNA sample preparation Kit v2 starting from the end repair step of the protocol. Up to 100 ng ChIP DNA was used as starting material. After end repair and A-tailing, indexing adapters were ligated. The products were then purified and amplified for 18 PCR cycles to create the final libraries.

treatment_protocol: EED-/- mESC were previously generated and characterized (Schoeftner et al., 2006).

tag: genotype

value: mESC differentiation into AntNPC

value: p300 (sc-585, Santa Cruz Biotechnology)


title: WT_2i_mESC_ChIP_p300_1

description: WT (E14)

source_name: mESC

value: mESC

value: 2i conditions


title: WT_2i_ChIP_input

description: WT (E14)


title: WT_2i_ChIP_H3K4me1

description: WT (E14)

value: H3K4me1 (39297, Active Motif)


title: InvD6-1

value: Balb/c

value: 4th and 5th mammary glands

value: InvD6 (6 days post-weaning)


title: WT_2i_ChIP_H3K27ac

description: WT (E14)

value: WT

value: H3K27ac (39133, Active Motif)


title: WT_2i_ChIP_H3K27me3

description: WT (E14)

value: H3K27me3 (39155, Active Motif)


title: WT_AntNPC_ChIP_H3K27me3

description: WT (E14)

value: AntNPC (anterior neural progenitors derived from mESC)


title: WT_2i_mESC_ChIP_p300_2

description: WT (E14)


title: InvD6-3

value: PDGFRa+  mammary fibroblasts


title: WT_mESC_ChIP_H3K27me3_2

description: WT (E14)


title: EEDnull_AntNPC_RNA-seq_2

data_processing:
Basic read quality check was performed using FastQC (Babraham Bioinformatics) and read statistics were obtained with SAMtools
Reads were mapped to the mouse reference assembly, version GRCm38, using TopHat2
gene expression for the individual samples was calculated with Cufflinks as FPKMs.
Read count means, fold-change (FC) and p-values were calculated with DEseq2
Genome_build: mm10

growth_protocol: WT (E14) mESC and EED-/- mESC lines were cultured on gelatin-coated plates using Knock-out DMEM (KO-DMEM, Life Technologies) supplemented with 15% FBS (Life Technologies) and LIF. These mESC lines were differentiated into AntNPC according to a previously described protocol (Gouti et al., 2014), with slight modifications. Briefly, mESC were plated at a density of 10000 cells/cm2 on gelatin-coated plates and grown for three days in N2B27 medium supplemented with 10 ng/ml bFgf (Life Technologies) and without serum or LIF (Advanced Dulbecco’s Modified Eagle Medium F12 (Life Technologies) and Neurobasal medium (Life Technologies) (1:1), supplemented with 1x N2 (Life Technologies), 1x B27 (Life Technologies), 2 mM L-glutamine (Life Technologies), 40 mg/ml BSA (Life Technologies), 0.1 mM 2-mercaptoethanol (Life Technologies)). Subsequently, cells were grown for another two days in N2B27 medium without bFgf (D3–D5).  Moreover, to improve the homogeneity of the differentiation, from D2-D5 the N2B27 medium was also supplemented with 5μM Xav939 (Sigma), a potent WNT inhibitor (Matsuda and Kondoh, 2014).

extract_protocol:
Total RNA was isolated using Tripure reagent according to the manufacturer’s instructions (Roche). Libraries were prepared with the RNA sample preparation kit (TruSeq v2; Ilumina).
RNA libraries were prepared for sequencing using standard Illumina protocols

value: EED-/-


title: EEDnull_AntNPC_RNA-seq_4


title: EEDnull_AntNPC_RNA-seq_3


title: WT_AntNPC_RNA-seq_4


title: WT_mESC_RNA-seq_2


title: WT_AntNPC_RNA-seq_3


title: WT_AntNPC_RNA-seq_2


title: EEDnull_mESC_RNA-seq

data_processing:
Basic read quality check was performed using FastQC (Babraham Bioinformatics) and read statistics were obtained with SAMtools
Reads were mapped to the mouse reference assembly, version GRCm38, using TopHat2
gene expression for the individual samples was calculated with Cufflinks as FPKMs.
Genome_build: mm10
Supplementary_files_format_and_content: Txt file containing FPKMs calculated with Cufflinks for each ENSEMBL mouse gene with a gene symbol.


title: WT_AntNPC_RNA-seq


title: WT_mESC_RNA-seq


title: EED_null_AntNPC_ChIP_H3K27ac

description: EED-/-


title: Wingdisc_GRH_ChIPseq

city: Madison

first: Melissa

middle: M

last: Harrison

email: mharrison3@wisc.edu, ykaur@wisc.edu, torreszelada@wisc.edu, mmfreund2@wisc.edu

state: WI

address: 420 Henry Mall

department: Biomolecular Chemistry

institute: University of Wisconsin Madison

description: GRH ChIP-seq

data_processing:
Basecalls performed using CASAVA1.8.2
All analyses were done on the Galaxy online platform
Raw ChIP reads were trimmed for quality by sliding window with a minimum score of 28
ChIP reads used for analysis were aligned to the dm3 genome assembly using Bowtie with maximum of 2 mismatches and unmapped reads were removed. Additional mapping was done using the dm6 genome and these mapped sequences are provided.
ChIP peaks were called with MACS with a p-value cutoff of 1e-6, MFOLD renrichment ratio of 5, Tag size of 80, and calculating the maximum lambda as local lambda at 1000, 2500, and 5000bp
Raw RNA-seq reads weere trimmed for quality by sliding window with a minimum score of 28
Reads were aligned to the dm3 geneome assembly using TopHat using default settings
Cufflinks was used to assemble transcripts and estimate FPKM for TopHat-aligned data
Cuffmerge was used to merge together resultant Cufflinks files (as GTF files)
Fold change between experimental conditions (with or without GRH) was calculated using CuffDiff, using the TopHat and Cuffmerge files produced in previous steps.
Genome_build: dm3 or dm6
Supplementary_files_format_and_content: BED: File produced by MACS peak calling software. File contains genomic regions corresponding to ChIP peaks (mapped to dm3)
Supplementary_files_format_and_content: WIG: File produced by MACS peak calling software. Contains profile data of genome from ChIP-seq experiment (mapped to dm3)
Supplementary_files_format_and_content: TABULAR: File produced by CuffDiff softare for assessing RNA-seq data. Fold change, p-value, and q-values are given for each gene or locus that describes the change in gene expression between experiment conditions.
Supplementary_files_format_and_content: BAM: File produced by Bowtie mapping software. File is mapped to "dm6" Drosophila genome

organism: Drosophila melanogaster

source_name: larval wing disc

growth_protocol: For ChIP-seq experiments, wild type OreR embryos at various stages were collected. To deplete zygotic GRH, two mutants (grhIM and grhB37) were balanced over CyO, sChFP and embryos were scored for fluorescence. To deplete maternal GRH, germline clones were produced as described in Harrison et al., 2010 using the FLP-FRT system described in Chou and Perrimon, 1996

extract_protocol:
ChIP-seq: Embryos were collected, fixed in formaldehyde, chromatin was isolated, and the chromatin was immunoprecipitated as described in Li et al., 2008. Immunoprecipitation was done using two antibodies against Grainy head and an input sample was also collected for sequencing. RNA-seq: Embryos depleted of either maternal or zygotic GRH as well as sibling controls were collected and examined to determine the stage. 7 embryos were collected for each set and lysed in TRIzol reagent (Life Technologies) supplemented with 150 µg/ml glycogen. RNA was extracted and cDNA libraries were prepared using the Illumina Truseq RNA sample prep kit.
ChIP-seq libraries were prepared using standard Illumina protocols and RNA-seq libraries were prepared using Illumina Truseq RNA sample prep kit according to manufacterer's instructions

value: wild type

tag: antibody

value: GRH

tag: Stage

value: 3rd instar larval wing disc


title: Archer_T47D_RNASeq_Rep3

city: RTP

first: Christopher

last: Lavender

state: NC

address: 111 T. W. Alexander Drive

institute: NIEHS

description: Poly-A enriched RNA-Seq

data_processing:
Start-Seq: Start-Seq reads were first filtered by quality score; reads with an average Sanger score less than 20 were removed from analysis. Following quality filtering, Cutadapt (version 1.2.1) was used to remove adapter sequences. Alignment of Start-Seq reads was performed using Bowtie (version 0.12.8) to the hg19 genome assembly. From each uniquely mapped Start-Seq fragment, the 5’ end was taken forward into the TSS calling procedure. TSSs were called using the higher depth Replicate 1 and validated by Replicate 2. Gene TSS and uaTSS calling procedures were described previously (Scruggs et al., Mol Cell 2015).  To call daTSS positions, a method analogous to uaTSS identification was used. daTSS search space was defined as 1 to 2000 nt downstream of an obsTSS in the antisense orientation.  To ensure that identified daTSSs were not simply mis-called gene TSSs or uaTSSs, additional criteria were used to filter daTSS calls. A daTSS call was filtered if (1) it was within 1000 nt upstream of the daTSS there was a genomic position with Start-Seq reads greater than or equal to 10% of reads at the TSS, implicating the daTSS as a potential uaTSS for an uncalled TSS, or (2) it was within 1000 nt of an annotated TSS on the same strand, implicating the daTSS as a potential gene TSS.
RNA-Seq: Prior to alignment, RNA-Seq reads were filtered based on quality; reads with an average Sanger quality score less than 20 were removed from analysis. Following quality filtering, Cutadapt was used to remove adapter sequences. Insert lengths were estimated by transcriptome alignment using Bowtie.  Sequence alignment was then performed using TopHat (version 2.0.4) [35]. Following de-duplication by Picard, alignments from individual replicates were merged. FPKM values were calculated using Cufflinks (version 2.2.1).
Genome_build: hg19
Supplementary_files_format_and_content: Start-Seq: bigWig files describe the stranded coverage of the 5’ end of Start-Seq reads. For each uniquely mapped Start-Seq read, the 5’ end of the alignment was considered.  TSS calls from the Start-Seq data are reported as BED files.  The TSS calling procedure has been described previously (Scruggs et al., Mol Cell 2015). All calls are compiled in a XLSX spreadsheet, annotated by Start-Seq read coverage and RNA-Seq FPKM values for associated genes.
Supplementary_files_format_and_content: RNA-Seq: Gene FPKM values are reported in CSV format. FPKM values were calculated using Cufflinks (version 2.2.1).

source_name: T-47D cells

growth_protocol: T-47D cells were cultured in DMEM containing 10% FBS.  Prior to harvest, cells were cultured in medium supplemented with 5% charcoal dextran-treated serum for at least 24 hours.

extract_protocol:
Start-Seq: Start-Seq experiments were performed as previously described (Nechaev et al., Science 2010). RNA-Seq: Total RNA was harvested using an RNeasy kit (Qiagen) with on-column DNase treatment.  RNA quality was validated by Bioanalyzer (Agilent). Paired-end strand-specific poly-A enriched RNA-seq libraries were generated by the NIH Intramural Sequencing Center (NISC). MNase-Seq: Nuclei were harvested from T-47D cells and digested for 5 minutes at 37C with a range of MNase (Worthington) concentrations. Reactions were stopped by addition of EDTA and then treated with RNase and proteinase K. Digested DNA was isolated by phenol/chloroform extraction and ethanol precipitation. FAIRE-Seq: FAIRE-seq experiments were performed as previously described (Burd et al., Mol Cell Biol 2012).
Start-Seq: Libraries were constructed as previously described (Nechaev et al., Science 2010) using an Illumina TruSeq small RNA kit. RNA-Seq: Libraries were constructed using an Illumina TruSeq Stranded mRNA kit. MNase-Seq: Libraries were constructed using an Illumina TruSeq sample preparation kit. FAIRE-Seq: Libraries were constructed as previously described (Burd et al., Mol Cell Biol 2012).
RNA-Seq: Prior to alignment, RNA-Seq reads were filtered based on quality; reads with an average Sanger quality score less than 20 were removed from analysis. Following quality filtering, Cutadapt was used to remove adapter sequences. Insert lengths were estimated by transcriptome alignment using Bowtie. Sequence alignment was then performed using TopHat (version 2.0.4) [35]. Following de-duplication by Picard, alignments from individual Replicates were merged. FPKM values were calculated using Cufflinks (version 2.2.1).


title: PELhx5_null_mESC_ChIP_H3K27me3

description: PE Lhx5(-109) -/-

tag: cell type


title: EEDnull_AntNPC_RNA-seq

tag: treatment

value: mESC differentiation into AntNPC


title: Archer_T47D_RNASeq_Rep1

description: Poly-A enriched RNA-Seq


title: Wingdisc_GRH_input

description: GRH input


title: WT_mESC_ChIP_p300

description: WT (E14)

data_processing:
Supplementary_files_format_and_content: bedGraph
Basic read quality check was performed using FastQC (Babraham Bioinformatics) and read statistics were obtained with SAMtools
Reads were mapped to the mouse reference assembly, version GRCm38, using TopHat2
Normalized bedGraph files were generated using DeepTools
Peaks were called using MACS2
Genome_build: mm10

growth_protocol: WT (E14) mESC and EED-/- mESC lines  were cultured on gelatin-coated plates using Knock-out DMEM (KO-DMEM, Life Technologies) supplemented with 15% FBS (Life Technologies) and LIF. These mESC lines were differentiated into AntNPC according to a previously described protocol (Gouti et al., 2014), with slight modifications. Briefly, mESC were plated at a density of 10000 cells/cm2 on gelatin-coated plates and grown for three days in N2B27 medium supplemented with 10 ng/ml bFgf (Life Technologies) and without serum or LIF (Advanced Dulbecco’s Modified Eagle Medium F12 (Life Technologies) and Neurobasal medium (Life Technologies) (1:1), supplemented with 1x N2 (Life Technologies), 1x B27 (Life Technologies), 2 mM L-glutamine (Life Technologies), 40 mg/ml BSA (Life Technologies), 0.1 mM 2-mercaptoethanol (Life Technologies)). Subsequently, cells were grown for another two days in N2B27 medium without bFgf (D3–D5).  Moreover, to improve the homogeneity of the differentiation, from D2-D5 the N2B27 medium was also supplemented with 5μM Xav939 (Sigma), a potent WNT inhibitor (Matsuda and Kondoh, 2014).

value: p300 (sc-585, Santa Cruz Biotechnology)


title: Archer_T47D_RNASeq_Rep2

description: Poly-A enriched RNA-Seq


title: PESox1_null_mESC_ChIP_H3K27me3

description: PE Sox1(+35) -/-

value: mESC


title: EEDnull_mESC_RNA-seq_2


title: WT_mESC_ChIP_H3K27me3

description: WT (E14)


title: WT_mESC_ChIP_input

description: WT (E14)


title: WT_mESC_ChIP_H3K27ac

description: WT (E14)


title: WT_AntNPC_ChIP_input

description: WT (E14)


title: WT_mESC_ChIP_H3K4me1

description: WT (E14)


title: EED_null_mESC_ChIP_input


title: WT_AntNPC_ChIP_H3K27ac

description: WT (E14)


title: WT_mESC_ChIP_H3K4me3

description: WT (E14)

value: H3K4me3 (39159, Active Motif)


title: EED_null_mESC_ChIP_H3K27me3


title: PE_Six3_EEDnull_mESC

description: EED-/-

data_processing:
Library strategy: 4C-seq
Supplementary_files_format_and_content: bedGraph
Reads were assigned to samples based on the first 10 bases of the read. Subsequently, the primer sequence was removed from the read and the remaining sequence was trimmed to 36 bases. These 36 bases were aligned to the mouse reference genome mm9 using Bowtie 0.12.7.
The resulting mapped reads were analyzed with R3C-seq (Thongjuea et al., 2013) in order to generate RPM (reads per million) normalized bedgraph files for downstream visualization and analysis.
Genome_build: mm9

extract_protocol:
4C-seq assays were performed as previously described (Splinter et al., 2012; Stadhouders et al., 2013) with slight modifications. 10 million mESC or AntNPC were crosslinked with 1% formaldehyde during 20 minutes and quenched with 0.125M glycine for 10 min. Cells were washed with PBS and resuspended in lysis buffer (50 mM Tris–HCl pH 7.5, 150 mM NaCl, 5 mM EDTA, 0.5% NP-40, 1% TX-100 and 1X protease inhibitors) during 10 min on ice.  Following centrifugation for 5 min at 650g (4 °C), nuclei were re-suspended in 0.5 ml of 1.2X restriction buffer with 0.3% SDS and incubated at 37 °C and 900 rpm for 1h. After that, Triton X-100 was added to a final concentration of 2% followed by 1h incubation at 37 °C and 900 rpm. Afterwards, chromatin was digested overnight at 37 °C and 900 rpm with 400 U of NlaIII (R0125L, NEB). NlaIII was inactivated by adding SDS to a final concentration of 1.6% and incubating the mixture for 20 min at 65 °C while shaking (900 rpm). The digested chromatin was transferred to 50-ml tubes and 6.125 ml of 1.15X ligation buffer (50 mM Tris-HCl pH 7.6, 10 mM MgCl2, 1 mM ATP, 1mM DTT) were added. Triton X-100 was also added to a final concentration of 1% and the resulting solution was incubated for 1 h at 37 °C while shaking gently. After that, digested chromatin was ligated with 100 U of T4 DNA ligase (15224-041, Life Technologies) for 8 h at 16 °C, followed by RNAse A treatment (300 μg, Peqlab) for 45 min at 37 °C. Subsequently, chromatin was de-crosslinked with 300 μg of Proteinase K (Peqlab) and overnight incubation at 65 °C. DNA was then purified by phenol/chloroform extraction followed by ethanol precipitation and re-suspension in 100 μl of water. At this step, the digestion and ligation efficiencies were evaluated by analysing a small fraction of the purified DNAs by agarose electrophoresis. The remaining DNA was digested with 50U of DpnII (R0543M, NEB) in 500 μl of 1x NEBuffer DpnII at 37 °C overnight. DNA samples were purified by phenol/chlorophorm extraction, followed by ethanol precipitation. Once purified, DNA samples were re-suspended in 500 μl of water and a second ligation was performed by adding 200 U of T4 DNA Ligase into a final volume of 14 ml 1X ligation buffer followed by overnight incubation at 16 °C. DNA samples were subjected to another round of phenol/chlorophorm extraction and ethanol precipitation, re-suspended in 100 μl of water and purified with a QIAgen PCR purification column (28104, QIAgen). The efficiencies of the second digestion and ligation were also evaluated by agarose electrophoresis.
4C samples were amplified by inverse PCR using primers located within selected poised enhancers and that included illumina adaptor sequences and short barcodes. The inverse PCRs were performed with the expand long template PCR system (11681842001, Roche) and 30 amplification cycles (94°C 2 min, 30x [94°C 10 sec, 60°C 1 min, 68°C 3 min], 68°C 5 min). The inverse PCR primers were designed as previously described (Stadhouders et al., 2013). Once amplified, 4C-seq libraries were sequenced on the HiSeq2500, generating reads of either 74 or 100 bp in length.


title: EED_null_mESC_ChIP_H3K27ac


title: PE_Six3_WT_AntNPC

description: WT (E14)


title: PE_Lmx1b_WT_mESC

description: WT (E14)


title: PE_Six3_WT_mESC

description: WT (E14)


title: PE_Sox1_EEDnull_mESC

description: EED-/-


title: PE_Lmx1b_EEDnull_mESC

description: EED-/-


title: PE_Sox1_WT_AntNPC

description: WT (E14)


title: PE_Lmx1b_WT_AntNPC

description: WT (E14)