title: UP09005213


title: UP09003135

status: Public on Jul 04 2014

type: SRA

city: Helsinki

first: Panu

last: Somervuo

address: Viikinkaari 4

country: Finland

institute: University of Helsinki

library_source: transcriptomic

data_processing:
Illumina Casava 1.7 and 1.8 software used for basecalling.
Reads were trimmed by finding longest subsequence with all bases>=Phred20. PolyA tail was removed and reads with minimum length of 50 bp were mapped with TopHat2 (version 2.0.5) taking into account strand specificity (forward strand).
Mapped read counts were collected within 16667 predicted gene models.
Genewise read counts were normalized using TMM method in R package edgeR. Normalized counts were obtained by dividing unnormalized counts by effective library size.
Genome_build: GenBank APLT01000000 (submitted)
Supplementary_files_format_and_content: text file including unnormalized read counts of 16667 genes is linked to each sample records and 'NormalizedCounts.txt' is linked to Series records.

molecule: total RNA

organism: Melitaea cinxia

extract_protocol:
Total RNA was extracted from frozen butterfly thorax with Trizol (Life Technologies Corporation, CA, USA) followed by chloroform, phenol-chloroform-isoamylalcohol and second chloroform purification. RNA was precipitated with isopropanol and washed with ethanol. RNA quality was verified with NanoDrop measurement (Thermo Fischer Scientific Inc., MA, USA) and Bioanalyzer run (Agilent Technologies, CA, USA).
An in-house strand-specific library construction protocol based on polyA-anchoring.

treatment_protocol: The material was collected in the autumn of 2009 as diapausing larvae from four different regional populations in northern Europe: Åland (Finland), Saaremaa (Estonia), Uppland (Sweden) and Öland (Sweden). The larvae overwintered in mixed (within population) groups in a growth chamber under constant temperature of +5°C and 85% relative humidity. In spring 2010, larvae were reared singly under common garden conditions (12:12; L/D; 28/15°C) with fresh leaves of the host plant Plantago lanceolata.  After eclosion the adults were held in the same common garden conditions for three days.

tag: population

value: UP

tag: gender

value: Female


title: UP09005210

value: Male


title: OL09005214

value: OL


title: UP09004156


title: OL09005234


title: UP09004161


title: SA09005234

value: SA


title: OL09004199


title: SA09003111


title: SA09004159


title: OL09005216


title: OL09004159


title: SA09003124


title: OL09004153


title: SA09005210


title: UP09004160


title: SA09005239


title: SA09004153


title: SA09004174


title: UP09003111


title: SA09005233


title: SA09004163


title: UP09004158


title: SA09003114


title: OL09003120


title: UP09003115


title: SA09003138


title: SA09005202


title: OL09003138


title: OL09003133


title: SA09003145


title: OL09003104


title: OL09005227


title: SA09003118


title: SA09005201


title: SA09003115


title: OL09004162


title: OL09004172


title: SA09005241


title: SA09004161


title: OL09004173


title: OL09003134


title: OL09003147


title: OL09004165


title: OL09004192


title: OL09004152


title: OL09003114


title: OL09003131


title: OL09004182


title: OL09004198


title: SA09005206


title: OL09004160


title: OL09004163


title: OL09004175


title: OL09005206


title: OL09004185


title: OL09004184


title: OL09005222


title: AL0900050062

value: AL


title: OL09005250


title: OL09004177


title: OL09005235


title: OL09003135


title: OL09004183


title: OL09005328


title: AL0902620131


title: AL0990010013


title: AL0910110012


title: AL0990290271


title: AL0906890083


title: AL0900070061


title: AL0917670013


title: AL0902370252


title: OL09005221


title: AL0906620011


title: AL0901590042


title: OL09003128


title: AL0902090043


title: AL0911750442


title: AL0900040262


title: AL0934080112


title: AL0902420051


title: AL0990290301


title: AL0904280011


title: AL0935520051


title: AL0985440022


title: OL09005219


title: AL0905620173


title: AL0902280171


title: AL0916760292


title: AL0904560012


title: AL0900290221


title: AL0937370012


title: AL0911750441


title: AL0900360082


title: AL0901930052


title: AL0906080013


title: AL0900080112


title: AL0924990073


title: AL0913930172


title: AL0915850102


title: AL0925010051


title: AL0919560031


title: AL0913930142


title: AL0910040862


title: AL0912000043


title: AL0905620243


title: AL0902700033


title: AL0916760271


title: AL0902370052


title: AL0905110081


title: AL0907470012


title: AL0914230011


title: AL0911750292


title: AL0933890032


title: THP1

status: Public on Jul 03 2014

city: Beijing

first: yadong

last: yang

address: No.1-104 Beichen West Road, Chaoyang

country: China

institute: Beijing Institute of Genomics

data_processing:
filter reads with low quality using perl script
alignment and annotation with DSAP (http://dsap.cgu.edu.tw/)
Normalize the expression abundance with perl script
Genome_build: hg19
Supplementary_files_format_and_content: txt, containing the expression abundance of HL60, K562 and THP1

organism: Homo sapiens

source_name: peripheral blood

growth_protocol: K562, HL-60 and THP-1 were cultured in RPMI1640 Medium supplied with 10% fetal bovine serum and penicillin (100 U/ml)-streptomycin (0.1 mg/ml).

extract_protocol:
Total RNA was isolated from K562, HL-60 and THP-1 cells using TRIZOL® Reagent.
The approximately 18-30 nucleotide RNAs were excised from denaturing polyacrylamide gel, ligated to sequencing adaptors on both ends, and reverse transcribed using small RNA sample prep kit (Illumina). The resulting cDNA libraries were PCR-amplified for 15 cycles and purified on acrylamide gel.

treatment_protocol: normal

tag: disease

value: acute monocytic leukemia

tag: tissue

value: peripheral blood

tag: cell line


title: HL60

value: acute promyelocytic leukemia


title: K562

source_name: bone marrow

value: chronic myelogenous leukemia (CML)

value: bone marrow


title: TI5-001_blood (mRNA)

city: Meath

first: Nathan

last: Lawless

address: Grange

country: Ireland

institute: Teagasc

data_processing:
Illumina CASAVA
FASTQC v0.10.0
Fastq Illumina filter v0.1
Cutadapt v1.2
Fastq quality filter package v0.0.13.2
TopHat v 2.0.8
HTSeq-count v 0.5.3p3
Genome_build: UMD3.1 assembly
Supplementary_files_format_and_content: All files processed as fastq files, converted to SAM format using samtools  v 0.1.18

organism: Bos taurus

source_name: Monocyte

growth_protocol: Milk and blood derived CD14+ monocytes were isolated by FACS, cells were labelled with monoclonal anti-bovine CD14 IgG1 and a PE-conjugated anti-mouse IgG antibody. Labelled cells were separated based on fluorescence intensity using the BD FACS Aria Cell Sorting System. Cells with greater than 95% purity were isolated from milk from the infected/control gland and peripheral blood of each animal .

extract_protocol:
The mirVana RNA Isolation Kit (Ambion, Austin, TX, USA.) was used to extract total RNA from FACS-isolated cell populations, following the manufacturer's instructions
One-hundred indexed mRNA libraries were prepared for cluster generation using the TruSeq RNA sample preparation kit v2 (50 cycles) (Illumina, California, USA.). Procedures were performed according to the manufacturer’s protocol

treatment_protocol: An inoculation load of 500 CFU of S. uberis in 1 mL of saline was administered through the teat canal of the front right quarter. One control animal (inoculated with saline) was run alongside the infected animal under the same conditions as described above. Milk and blood samples were obtained from each animal at 0, 12, 24, 36 and 48 hrs post-infection (or mock infection).

tag: breed

value: Holstein Friesian

tag: cell type

value: CD14 + blood derived monocytes

tag: preperation kit

value: Indexed TruSeq RNA sample preparation kit v2

tag: infection

value: S. uberis

tag: time

value: 0 h


title: TI5-005_blood (mRNA)


title: TI5-004_blood (mRNA)


title: TI5-002_blood (mRNA)


title: TI4-005_blood (mRNA)


title: AL0948150012


title: TI4-001_blood (mRNA)


title: TI4-003_blood (mRNA)


title: TI4-002_blood (mRNA)


title: TI-3-002_blood (mRNA)


title: TI-3-001_blood (mRNA)


title: TI-3-004_blood (mRNA)


title: TI-3-003_blood (mRNA)


title: TI-1-005_blood (mRNA)


title: TC5-005_blood (mRNA)

value: Control


title: TI-2-003_blood (mRNA)


title: TI-1-004_blood (mRNA)


title: TI-2-005_blood (mRNA)


title: TI-3-005_blood (mRNA)


title: TI-1-001_blood (mRNA)


title: TI-2-004_blood (mRNA)


title: TI-1-003_blood (mRNA)


title: TI-2-001_blood (mRNA)


title: TI-1-002_blood (mRNA)


title: TC5-003_blood (mRNA)


title: TC5-002_blood (mRNA)


title: TI-2-002_blood (mRNA)


title: TC5-004_blood (mRNA)


title: TI4-004_blood (mRNA)


title: TC5-001_blood (mRNA)


title: TI5-003_blood (mRNA)


title: TC4-003_blood (mRNA)


title: TC4-004_blood (mRNA)


title: TC4-002_blood (mRNA)


title: TC4-005_blood (mRNA)


title: TC4-001_blood (mRNA)


title: TC-3-004_blood (mRNA)


title: TC-3-005_blood (mRNA)


title: TC-3-003_blood (mRNA)


title: TC-3-001_blood (mRNA)


title: TC-2-003_blood (mRNA)


title: TC-2-005_blood (mRNA)


title: TC-2-004_blood (mRNA)


title: TI5-004_milk (mRNA)

value: CD14 + milk derived monocytes


title: TC-2-002_blood (mRNA)


title: TC-1-003_blood (mRNA)


title: TC-1-001_blood (mRNA)


title: TC-1-002_blood (mRNA)


title: TI5-005_milk (mRNA)


title: TC-1-004_blood (mRNA)


title: TI5-001_milk (mRNA)


title: TI4-005_milk (mRNA)


title: TI5-003_milk (mRNA)


title: TI5-002_milk (mRNA)


title: TI4-004_milk (mRNA)


title: TI4-003_milk (mRNA)


title: TI4-001_milk (mRNA)


title: TI-3-005_milk (mRNA)


title: TI-3-001_milk (mRNA)


title: TI-3-002_milk (mRNA)


title: TI-3-004_milk (mRNA)


title: TI-3-003_milk (mRNA)


title: TI-2-004_milk (mRNA)


title: TI-2-003_milk (mRNA)


title: TI-2-005_milk (mRNA)


title: TC-3-002_blood (mRNA)


title: TC-2-001_blood (mRNA)


title: TC-1-005_blood (mRNA)


title: TI-2-002_milk (mRNA)


title: TI-1-005_milk (mRNA)


title: TI-1-004_milk (mRNA)


title: TI-2-001_milk (mRNA)


title: TI-1-002_milk (mRNA)


title: TI4-002_milk (mRNA)


title: TC5-005_milk (mRNA)


title: TC5-002_milk (mRNA)


title: TC5-003_milk (mRNA)


title: TC5-001_milk (mRNA)


title: TC4-001_milk (mRNA)


title: TC4-002_milk (mRNA)


title: TC4-003_milk (mRNA)


title: TC4-004_milk (mRNA)


title: TC-3-002_milk (mRNA)


title: TC-3-003_milk (mRNA)


title: TC-3-001_milk (mRNA)


title: TC-3-005_milk (mRNA)


title: TC-3-004_milk (mRNA)


title: TC-2-004_milk (mRNA)


title: TC-2-005_milk (mRNA)


title: TC-2-001_milk (mRNA)


title: TC-2-002_milk (mRNA)


title: TC-1-005_milk (mRNA)


title: TC-1-004_milk (mRNA)


title: TI-1-003_milk (mRNA)


title: TI-1-001_milk (mRNA)


title: TC-1-003_milk (mRNA)


title: TC-1-001_milk (mRNA)


title: TC5-004_milk (mRNA)


title: TC-2-003_milk (mRNA)


title: TI5-001_blood

data_processing:
Illumina CASAVA 1.4
FASTQC v0.10.0
Fastq Illumina filter v0.1
Cutadapt v1.2
Fastq quality filter package v0.0.13.2
novoalign v2.08.03
HTSeq-count v 0.5.3p3
Genome_build: UMD3.1 assembly
Supplementary_files_format_and_content: All files processed as fastq files, converted to SAM format using samtools v 0.1.18

extract_protocol:
MicroRNA was extracted using mirPremierTM microRNA Isolation Kits (Sigma-Aldrich, Steinheim, Germany.) following the manufacturer's instructions
One-hundred indexed miRNA libraries were prepared for cluster generation using the TruSeq Small RNA sample preparation kit (50 cycles). Procedures were performed according to the manufacturer’s protocol.

value: Indexed TruSeq small RNA sample preparation kit (50 cycles)


title: TI5-005_blood


title: TI5-002_blood


title: TI4-003_blood


title: TI5-004_blood


title: TI-3-004_blood


title: TI4-005_blood


title: TI-3-005_blood


title: TI4-001_blood


title: TI4-004_blood


title: TI-3-003_blood


title: TI5-003_blood


title: TI4-002_blood


title: TI-3-002_blood


title: TI-2-005_blood


title: TI-3-001_blood


title: TI-2-002_blood


title: TI-2-001_blood


title: TI-2-004_blood


title: TI-1-005_blood


title: TI-1-004_blood


title: TC-1-002_milk (mRNA)


title: TI-1-003_blood


title: TI-1-002_blood


title: TI-1-001_blood


title: TC5-005_blood

value: control


title: TC4-005_milk (mRNA)


title: Mmus E14-WT ChIP Input serum

city: Copenhagen

first: Kristian

last: Helin

address: Ole Maaloes Vej 5

country: Denmark

institute: BRIC - University of Copenhagen

library_source: genomic

data_processing:
Sequencing of ChIP-seq samples produced single-end 42bp reads after removal of multiplex index sequence tags. Reads were trimmed for low quality nucleotides and mapped to the GRCm38 (mm10) mouse genome sequence using bowtie (Langmead et al., 2009), with parameters “-m1 --strata --best”. Mapped reads were de-duplicated by keeping a single read in each group of reads mapping the exact same genomic sequence. Pre-processing, mapping and de-duplication were performed on the public server of the Galaxy Project.
MACS (Zhang et al., 2008) version 1.4 was used to call for Suz12 peaks using the input sample as control and a p-value cut-off of 1E-15. This stringent cut-off was based on the observation that, when plotting the number of called peaks as a function of decreasing p-values, their relative proportion between all conditions were not changing anymore from around 1E-15 and lower.
Genome_build: mm10
Supplementary_files_format_and_content: Genomic track representing ChIP-seq read coverage

molecule: genomic DNA

organism: Mus musculus

source_name: Embryonic Stem Cells

growth_protocol: mESCs cultured in serum conditions were grown in G-MEM (Sigma) supplemented with 15% FBS (Hyclone), PenStrep (Gibco), Glutamax (Gibco), non-essential amino acids (Gibco), Sodium pyruvate (Gibco), beta-mercaptoethanol (Gibco) and leukemia inhibitory factor (LIF) (produced and tested in our lab). Cells were cultured on 0.1% gelatin-coated plates (Sigma) at 37° and 5% CO2.

extract_protocol:
Cells were cross-linked with 1% formaldehyde in PBS for 10 min at room temperature and the reaction stopped with addition of glycine to a final concentration of 0.125M. Cells were harvested in SDS buffer (50 mM Tris at pH 8.1, 0.5% SDS, 100 mM NaCl, 5 mM EDTA). Pelleted cells were resuspended in IP buffer (100 mM Tris at pH 8.6, 0.3% SDS, 1.7% Triton X-100, and 5 mM EDTA) and sonicated to produce DNA fragments <1000 bp with an average size around 300 bp using a Diagenode Bioruptor. Lysates were precleared with protein A sepharose beads (GE Healthcare). Cleared samples (0.5 mg of chromatin) were immunoprecipitated O/N in IP buffer with the indicated antibodies: anti-Suz12 (D39F6 XP Cell Signaling), anti-Ezh2 (D2C9 XP Cell Signaling), anti-H3K27me3 (C36B11 Cell Signaling), anti-histone H3 (made in our lab, peptide purified by standard methods), anti-RNAPII N-20 (Santa Cruz, sc-899), IgG from rabbit (Sigma). Protein A beads were blocked O/N in 1X TE with 0,2 mg/mL sonicated herring sperm (Sigma) and 0,5 mg/mL BSA (New England Biolabs) before incubation with antibody-chromatin complexes for 3h at 4°C. Washes were performed first 3 times with Mixed Micelle buffer (150 mM NaCl, 20 mM Tris-Cl, pH 8.1, 5 mM EDTA, pH 8.0, 5% w/v sucrose, 1% Triton X-100, 0.2% SDS, 0.02% NaN3), twice with Buffer 500 (0.1% (w/v) deoxycholic acid, 1 mM EDTA, 50 mM HEPES, pH 7.5, 500 mM NaCl, 1% (v/v) Triton X-100, 0.02% NaN3), twice with LiCl/detergent buffer (0.5% (w/v) deoxycholic acid (sodium salt), 1 mM EDTA, 250 mM LiCl, 0.5% (v/v) Igepal, 10 mM Tris-Cl, pH 8.0, 0.02% NaN3) and one time in 1X TE. Immune complexes were eluted and reverse cross-linked in 1% SDS, 0.1 M NaHCO3 O/N at 65°C. DNA was recovered and purified using Qiagen PCR purification kit.
ChIP was performed as described above with 1 mg of chromatin except that Protein A beads for precipitation were not blocked with BSA and herring sperm. The immunoprecipitated DNA was quantified by real-time qPCR and Qubit fluorometer (Life Technologies). DNA library for Illumina sequencing was prepared using the NEBNext® ChIP-Seq Library Prep Master Mix Set for Illumina (New England Biolabs) using 10 ng DNA as input and following manufacturer’s instructions. Library cDNA was quantified and the quality assessed using the High Sensitivity DNA Kit with a 2100 Bioanalyzer (Agilent). Equimolar amounts of samples, with compatible indexes, were pooled for multiplex sequencing. Samples were sequenced at the National High-throughput DNA Sequencing Centre of the University of Copenhagen for 50 bp HiSeq Single Read sequencing using Illumina Sequencing Technology.

tag: strain

tag: genotype

value: wild type

tag: culture condition

value: serum


title: Mmus Suz12-KO ChIP Differentiation Suz12 0h  rep. 1

data_processing:
Sequencing of ChIP-seq samples produced single-end 42bp reads after removal of multiplex index sequence tags. Reads were trimmed for low quality nucleotides and mapped to the GRCm38 (mm10) mouse genome sequence using bowtie (Langmead et al., 2009), with parameters “-m1 --strata --best”. Mapped reads were de-duplicated by keeping a single read in each group of reads mapping the exact same genomic sequence. Pre-processing, mapping and de-duplication were performed on the public server of the Galaxy Project.
MACS (Zhang et al., 2008) version 1.4 was used to call for Suz12 peaks using the input sample as control and a p-value cut-off of 1E-15. This stringent cut-off was based on the observation that, when plotting the number of called peaks as a function of decreasing p-values, their relative proportion between all conditions were not changing anymore from around 1E-15 and lower.
Genome_build: mm10
Supplementary_files_format_and_content: bed files of MACS peaks and bigWig representing ChIP-seq read coverage on the genome

treatment_protocol: To induce differentiation, medium was replaced with  an equivalent medium containing 1 µM all-trans-retinoic acid (ATRA) (Sigma) and no LIF. During differentiation, ATRA-medium was changed every 24 hours.

tag: differentiation timepoint

tag: chip antibody

value: anti-Suz12 (D39F6 XP Cell Signaling)


title: TC5-004_blood


title: TC5-003_blood


title: TI-2-003_blood


title: Mmus E14-WT ChIP Suz12 serum Tripto 9h  rep. 2

description: Mmus_E14-WT_ChIP_Suz12_serum_Tripto_9h_r2_peaks.bed

data_processing:
Sequencing of ChIP-seq samples produced single-end 42bp reads after removal of multiplex index sequence tags. Reads were trimmed for low quality nucleotides and mapped to the GRCm38 (mm10) mouse genome sequence using bowtie (Langmead et al., 2009), with parameters “-m1 --strata --best”. Mapped reads were de-duplicated by keeping a single read in each group of reads mapping the exact same genomic sequence. Pre-processing, mapping and de-duplication were performed on the public server of the Galaxy Project.
MACS (Zhang et al., 2008) version 1.4 was used to call for Suz12 peaks using the input sample as control and a p-value cut-off of 1E-15. This stringent cut-off was based on the observation that, when plotting the number of called peaks as a function of decreasing p-values, their relative proportion between all conditions were not changing anymore from around 1E-15 and lower.
Genome_build: mm10
Supplementary_files_format_and_content: bed files of MACS peaks, Excel files for the table of Suz12 and H3K27me3 fold changes over Non-target CGIs at 0h and the table of RNAPII fold changes over TSS and TTS regions

treatment_protocol: To inhibit transcription, cells were treated with DRB and Triptolide (Sigma) at a final concentration of 100 and 10 µM respectively.

tag: treatment

value: Triptolide

tag: treatment timepoint


title: Mmus E14-WT ChIP Suz12 serum Tripto 3h

description: ChIP-seq_drugs_Non-target-CGIs-at-0h_FoldChange.xlsx


title: Mmus E14-WT ChIP Differentiation Suz12 72h  rep. 1


title: TC5-002_blood


title: Mmus E14-WT ChIP Suz12 serum Tripto 0h  rep. 1

description: Mmus_E14-WT_ChIP_Suz12_serum_Tripto_0h_r1_peaks.bed


title: TC4-002_blood


title: Mmus E14-WT ChIP Suz12 serum DRB 12h  rep. 2

description: Mmus_E14-WT_ChIP_Suz12_serum_DRB_12h_r2_peaks.bed

value: DRB


title: TC4-004_blood


title: Mmus E14-WT ChIP Suz12 serum Tripto 0h  rep. 2

description: Mmus_E14-WT_ChIP_Suz12_serum_Tripto_0h_r2_peaks.bed


title: Mmus E14-WT ChIP Suz12 serum DRB 12h  rep. 1

description: Mmus_E14-WT_ChIP_Suz12_serum_DRB_12h_r1_peaks.bed


title: TC4-003_blood


title: TC-3-004_blood


title: TC-3-003_blood


title: Mmus E14-WT ChIP Suz12 serum Tripto 9h  rep. 1

description: Mmus_E14-WT_ChIP_Suz12_serum_Tripto_9h_r1_peaks.bed


title: Mmus E14-WT ChIP Suz12 serum DRB 0h  rep. 2

description: Mmus_E14-WT_ChIP_Suz12_serum_DRB_0h_r2_peaks.bed


title: Mmus E14-WT ChIP RNAPII serum Tripto 9h

value: anti-RNAPII N-20 (Santa Cruz, sc-899)


title: Mmus E14-WT ChIP Suz12 serum DRB 0h  rep. 1

description: Mmus_E14-WT_ChIP_Suz12_serum_DRB_0h_r1_peaks.bed


title: TC4-005_blood


title: Mmus E14-WT ChIP RNAPII serum Tripto 0h


title: TC4-001_blood


title: TC5-001_blood


title: TC-2-005_blood


title: Mmus E14-WT ChIP Suz12 serum DRB 6h


title: TC-2-003_blood


title: TC-3-005_blood


title: TC-2-001_blood


title: TC-1-005_blood


title: Mmus E14-WT ChIP H3K27me3 serum Tripto 9h

value: anti-H3K27me3 (C36B11 Cell Signaling)


title: TC-1-004_blood


title: TC-1-003_blood


title: Mmus E14-WT ChIP H3K27me3 serum Tripto 3h


title: Mmus E14-WT iTC serum Tripto 2h Ctr  rep. 2

data_processing:
Pre-processing and read mapping  Reads were trimmed for low quality nucleotides and mapped to the GRCm38 (mm10) mouse genome sequence using bowtie with parameters “-m1 --strata --best”. Mapped reads were de-duplicated by keeping a single read in each group of reads mapping the exact same genomic sequence.
Quantification of iTC-seq read density on genes  Reads from duplicate experiments were merged together and processed as follows. In order to allow background correction and comparison between the different conditions, reads in each sample were randomly selected with proportions given by the ratio between the minimum number of total reads in all samples and the number of total reads in each sample. Quantification of the number of iTC-seq reads overlapping with genes annotated in Ensembl GRCm38.72 were performed with seqMINER 1.3.3e using the Enrichment Based Method with Density array calculation option set to “peak interval”. Read densities of the unlabeled sample were subtracted from read densities of the labeled sample. Negative values resulting from this background subtraction were set to zero. The total read density per gene was normalized by the gene length and expressed in reads per kb (RPK).
Genomic profiles  Reads from duplicate experiments were merged together and coverage levels were computed at base pair resolution, combining read counts from both strands into a single profile. These coverage levels were scaled by a constant factor of 1/(million of total reads). The genomic profile in each condition was then calculated by subtracting scaled coverage levels of the unlabeled sample from scaled coverage levels of the labeled sample.
Genome_build: mm10
Supplementary_files_format_and_content: Table of gene transcription levels

extract_protocol:
mESCs were grown in 10 cm plates as described above. Labeling was performed by replacing the old medium with new medium pre-equilibrated at 37°C, 5% CO2 and either freshly supplemented with 150 µM of 4-thiouridine or not (background control). After 10 min of incubation at 37°C, 5% CO2, cells were washed once with 1X PBS, lysed with 4 mL of TRIzol Reagent and split in 4 x 1 mL. Each aliquot was mixed with 200 µL of CI and centrifuged at full speed for 15 min at 4°C. Supernatants were extracted twice with 500 µL of PCI, pH4.5, once with CI and ethanol precipitated with 0.3 M NaCl, 40 μg of RNAse-free glycogen at -80°C for 15 min. After centrifugation at 20,000 x g for 90 min at 4°C, pellets were washed with 70% ethanol, air dried, re-suspended with 10 mM Tris-HCl, pH 8.0. Aliquots from the same samples were pooled. RNA purity and amount was checked using a NanoDrop and RNA concentration was adjusted to 1 µg/µL. 75 µg were further purified using the Micro Bio-Spin P-30 Gel Columns (RNase-free) Kit (Bio-Rad) according to the manufacturer's instructions.     RNA fragmentation was performed using the NEBNext Magnesium RNA Fragmentation Module as follows. 60 µg was mixed with 75 µL of 1X RNA Fragmentation Buffer, incubate at 94C for 20 min, transferred on ice and mixed with 7.5 µL of 10X RNA Fragmentation Stop Solution. RNA integrity (before fragmentation) and fragmentation efficiency were checked using the RNA 6000 Nano Kit with a 2100 Bioanalyzer. After fragmentation, the size of RNA fragments should have a normal distribution peaking at 60 nt. Samples were purified using the Micro Bio-Spin P-30 Gel Columns (RNase-free) Kit as before.    RNA end-repair was performed as follows. Samples were incubated at 65C for 5 min to disrupt RNA secondary structure and transferred on ice, mixed with 90 µL of H2O, 20 µL of 10X T4 Polynucleotide Kinase Buffer (New England Biolabs), 10 µL of 10U/µL T4 Polynucleotide Kinase Enzyme, incubated at 37C for 1 hour, mixed with 5 µL of 100 mM ATP and incubated at 37°C for 1 hour. Each sample was split in 2 x 100 µL aliquots before purification with the RNeasy MinElute Cleanup Kit with the following modified procedure to keep small RNAs. Aliquots were mixed with 350 μL of Buffer RLT (without β-Mercaptoethanol), 550 μL of 100% Ethanol and run through an RNeasy MinElute spin column. Columns were washed once with 500 µL of Buffer RPE (containing Ethanol) and once with 500µL of 80% Ethanol, dried and eluted twice with 14 µL of H2O. Eluates from the same samples were pooled and volumes were adjusted 52.5 µL with H2O. Biotinylation of the 4sU-labeled RNAs was performed by mixing samples with 7.5 µL of 10X Labeling Buffer (100 mM Tris-HCl pH7.5, 10 mM EDTA), 15 µL of 1 mg/mL EZ-Link HPDP-Biotin (Pierce) dissolved in dimethylformamide and incubating at 25°C for 2 hours. Samples were purified with the Micro Bio-Spin P-30 Gel Columns (RNase-free) Kit as before, mixed with 2 µL of 5M NaCl, incubated 65°C for 5 min and placed on ice. After a second purification using the Micro Bio-Spin P-30 Gel Columns (RNase-free) Kit, volumes were adjusted to 200 µL with H2O, samples were incubated at 65°C for 5 min and placed on ice. Biotinylated RNA pull-down was achieved as follows. For each sample, 1 mg of Dynabeads MyOne Streptavidin C1 (Invitrogen) was washed twice with 190 µL of Solution A (0.1 M NaOH, 0.05 M NaCl) for 2 min, twice with 190 µL of Solution B (0.1 M NaCl) and resuspended in 200 µL of 2X SBW buffer (20 mM Tris-HCl pH7.5, 1M NaCl, 0.2 % SDS). Bead suspensions were then mixed with the denatured RNA samples, incubated at 25°C for 1 hour, washed 3 times with 900 µL of 1X SBW buffer, washed 3 times with 900 µL of 10 mM Tris-HCl pH 7.5 and eluted twice with 100 µL of SE Buffer (10 mM Tris-HCl pH7.5, 5 mM EDTA, 0.1 % SDS) at 99°C for 5 min. Eluates were centrifuged at full speed to remove traces of beads, pooled and ethanol precipitated with 0.3 M NaCl, 40 μg of RNAse-free glycogen at -80°C for 15 min. After centrifugation at 20,000 x g for 90 min at 4°C, pellets were washed with 70% ethanol, air-dried and resuspended with 7µL H2O. RNA amount and size was checked using the RNA 6000 Pico Kit. Each sample was supplemented with 10 ng of inactivated carrier RNA consisting of an equimolar mix of both HPLC-purified RNA oligonucleotides 5'-GAUGAAACGAGUACUACCGACC-3' and 5'PHO-GAUGAAACGAGUACUACCGACC-3'ddC (with 5’PHO and 3'ddC stating respectively for 5’ Phosphate and 2',3' dideoxyC modifications).
RNA library for Illumina sequencing was prepared using the NEBNext Multiplex Small RNA Library Prep Set for Illumina® (1–12) (New England Biolabs). Multiplex 3’ SR Adaptor ligation was performed as follows. 6 µL containing either 200 ng or 30 ng of RNA (for either RNA-seq or iTC-seq samples respectively) were mixed with 1 µL of 1:5 diluted Multiplex 3’ SR Adaptor, incubated at 70°C for 2 min and transferred to ice. Samples were then supplemented with 10 µL of 2X 3’ Ligation Reaction Buffer and 3 µL of 3’ Ligase Enzyme Mix and incubated at 25°C for 1 hour. RT primer hybridization was performed by mixing samples with 4.5 µL of H2O, 1 µL of 1:5 diluted Multiplex SR RT Primer and by incubating successively at 75C for 5 min, 37C for 30 min and 25C for 15 min. Multiplex 5’ SR Adaptor ligation was done by mixing samples with 1 µL of denatured 1:5 diluted Multiplex 5’ SR Adaptor, 1 µL of 5’ Ligation Reaction Buffer, 1 µL of 5’ Ligase Enzyme Mix and incubating at 25°C for 1 hour. cDNA synthesis was performed by mixing samples with 8 µL of First Strand Synthesis Reaction Buffer, 1 µL of Murine RNase Inhibitor, 1 µL M-MuLV Reverse transcriptase (RNase H-) and incubating successively at 50°C for 1 hour, 70 °C for 15 min and on ice. cDNA samples were purified using the QIAquick PCR Purification Kit (Qiagen) according to the manufacturer's instructions and eluted in 40 µL. cDNA amplification was performed as follows. Samples were mixed with 50 µL of LongAmp Taq® 2X Master Mix, 5 µL of 1:5 diluted Multiplex SR Primer and 5 µL of 1:5 diluted Multiplex Index (X) Primer. PCR was performed in a Thermocycler with the following program: 1 cycle for the initial denaturation step (94°C for 60 s), either 12 or 15 cycles (for either RNA-seq or iTC-seq samples respectively) for the amplification step (94°C for 10 s, 60°C for 30 s, 65°C for 30 s), 1 cycle for the final extension step (65°C for 7 min) and 1 cycle for the cooling step (4°C). PCR product purification was performed using AMPure XP system (Agencourt) as follows. Samples were split in 2 x 50 µL aliquot. Each aliquot was mixed with 90 µL of AMPure XP beads and incubate at room temperature (RT) for 15 min. Beads were washed twice with 200 µL of 80% ethanol for 30 s, air dried and resuspended with 10 µL of H2O. Supernatants from same sample were pooled and DNA amount was quantified using the dsDNA HS Assay Kit with a Qubit Fluorometer (Invitrogen). Expected amount range from either 300 to 450 ng or from 20 to 40 ng (for either RNA-seq or iTC-seq samples respectively). Library purity and integrity was checked using the DNA 1000 Kit with a 2100 Bioanalyzer (Agilent). DNA sizes should have a normal distribution peaking at 180 nt. Equimolar amounts of either 4 RNA-seq samples or 2 iTC-seq samples, with compatible indexes, were pooled for multiplex sequencing. Pool concentration was adjusted between samples and 0.1 to 0.4 pmols were sent to the National High-throughput DNA Sequencing Centre of the University of Copenhagen for 50 bp HiSeq Single Read sequencing using Illumina Sequencing Technology.

tag: labeling


title: Mmus E14-WT ChIP H3K27me3 serum DRB 0h


title: TC-1-002_blood


title: TC-3-002_blood


title: Mmus E14-WT iTC serum Tripto 2h Ctr  rep. 1


title: TC-3-001_blood


title: Mmus E14-WT iTC serum Tripto 2h 4sU  rep. 2


title: TI5-005_milk


title: Mmus E14-WT ChIP RNAPII serum DRB 12h


title: TI5-004_milk


title: TC-1-001_blood


title: Mmus E14-WT iTC serum Tripto 2h 4sU  rep. 1


title: Mmus E14-WT iTC serum Tripto 1h Ctr  rep. 2


title: Mmus E14-WT ChIP RNAPII serum DRB 0h


title: TI5-003_milk


title: Mmus E14-WT iTC serum Tripto 1h Ctr  rep. 1


title: TC-2-004_blood


title: TI5-002_milk


title: Mmus E14-WT ChIP H3K27me3 serum Tripto 0h


title: TC-2-002_blood


title: TI5-001_milk


title: Mmus E14-WT ChIP H3K27me3 serum DRB 12h


title: TI4-005_milk


title: Mmus E14-WT iTC serum Tripto 1h 4sU  rep. 2


title: TI4-004_milk


title: Mmus E14-WT iTC serum Tripto 1h 4sU  rep. 1


title: TI4-003_milk


title: Mmus E14-WT iTC serum NoDrug 0h 4sU  rep. 2

value: No drug


title: Mmus E14-WT iTC serum NoDrug 0h Ctr  rep. 1


title: Mmus E14-WT iTC serum DRB 5h Ctr  rep. 2


title: Mes_CCA

city: Saint Louis

first: Weixiong

last: Zhang

state: MO

address: Campus Box 1045 One Brookings Drive

country: USA

institute: Washington university in St Louis

description: small RNA-Seq

data_processing:
The raw sequence reads were first processed to remove reads with no 3' sequencing adaptor, of low quality, or shorter than 17 nt. The sequence adaptor trimming was done by an in-house program that recursively searches for the longest subsequence of the adaptor appearing within a sequence read. If a raw sequence read did not have a subsequence of the adaptor that was longer than 4 nt, it was considered to have no adaptor.
Genome_build: n/a
Supplementary_files_format_and_content: txt format. Content: 1st column is SEQUENCE;  2nd column is COUNT

organism: Manihot esculenta

growth_protocol: Stem segments with three nodes of Cassava (Manihot esculenta Crantz) cultivar, were cut from 8-month-old plants, and inclined in 3-L pots filled with barren red soil: vermiculite (1:1, v/v), fertilized with Hoagland’s solution, to propagate and generate well-balanced seedlings. The solution was renewed with 300ml quarter-strength solution once a week. After 2 months of planting, the uniform seedlings were subjected to cold stress treatment. All plants were field grown at the Institute of Tropical Bioscience and Biotechnology (ITBB), Chinese Academy of Tropical Agricultural Sciences (CATAS), Haikou, during April and June of natural conditions (11h light, 13h dark and 25°C during the day and 18°C at night).

extract_protocol: Total RNA of each sample was isolated individually, and then pooled with an equal amount from each sample into one for profiling. As a result, four mRNA libraries and four small-RNA libraries, corresponding to the conditions of CA, CCA,CS and NC, were constructed.

treatment_protocol: Three organs/tissues (folded leaf, fully expanded leaf and roots) of Cassava cultivar SC124 harvested at 6h, 24h and 5d for three cold treatments of CA, CCA and CS, for profiling genes at the stages of initial response, secondary response, and functional adaption to cold stresses.

value: Cassava under stress acclimation Cold stress after cold acclimation (CCA)


title: Mes_CS

description: small RNA-Seq

source_name: folded leaf, fully expanded leaf and roots

value: Cassava under cold shock (CS)


title: Mmus E14-WT iTC serum NoDrug 0h 4sU  rep. 1


title: Mes_CA

description: small RNA-Seq

value: folded leaf, fully expanded leaf and roots

value: Cassava under cold acclimation (CA)


title: TI4-001_milk

value: Holstein Friesian

value: S. uberis

value: 0 h


title: Mes_NC

description: small RNA-Seq

value: Cassava under normal condition (NC)


title: Mmus E14-WT iTC serum DRB 5h 4sU  rep. 1


title: Mmus E14-WT iTC serum DRB 5h Ctr  rep. 1

value: Control


title: Mmus E14-WT iTC serum DRB 5h 4sU  rep. 2

value: wild type


title: Mmus E14-WT iTC serum DRB 2h Ctr  rep. 2

value: serum


title: TI4-002_milk

value: CD14 + milk derived monocytes

value: Indexed TruSeq small RNA sample preparation kit (50 cycles)


title: TI-3-005_milk


title: Input for HP1a RNA Illumina random priming r2

city: Boston

first: Peter

last: Kharchenko

state: MA

address: 10 Shattuck St.

department: DBMI

institute: Harvard Medical School

data_processing:
Illumina base-calling pipeline; Heclios base-calling pipeline
for Illumina data: aligned to dm3 using bowtie (-t 1 -k 1); Helicos data was aligned using helisphere pipeline with default parameters, and uniquely aligned reads only.
smoothed enrichment calculated using SPP (http://compbio.med.harvard.edu/Supplements/ChIP-seq/), get.smoothed.enrichment.mle().
Genome_build: dm3 for D.melanogaster
Supplementary_files_format_and_content: strand-specific smoothed read density; 'pos.bedGraph' for positive strand density;  'neg.bedGraph' for negative strand density

organism: Drosophila melanogaster

source_name: S2 cell line

growth_protocol: Drosophila S2 cells were grown in four 50 ml large T-flasks (225 cm2) in 10% FBS –supplemented Schneider’s Drosophila medium (Invitrogen Cat # 11720) to a density of ~5x106 cells/ml. Cell cultures were split in half by adding an equal volume of HyClone CCM3 serum-free medium (Thermo Scientific, Cat No.SH30065) and grown in eight T-flasks (225 cm2) to a density of ~5x106 cells/ml. Cells were transferred into four 2.8L Fernbach glass flasks (Bellco, Glass Inc.). To generate 100 g of 6-20 hr embryos, flies were grown at 25°C with 65% humidity using large embryo collection cages (Flystuff cat. N. 59-101) containing molasses plates with yeast paste. 100 g of 3rd instar larvae or adult flies were used. Larvae were separated from the food by washing in 20% sucrose (see Ashburner, 1989). Flies were collected 1-12 hours after eclosion.

extract_protocol:
Soluble chromatin was incubated with IgG agarose beads (Sigma, Cat # A2909). For every 10 ml of sonicated chromatin, 0.5-1 ml beads were added (for 50 ml of 3rd instar larvae or adult fly chromatin 1 ml beads was used). The mixture was rotated end-over end in the 15 ml Falcon tubes for 12-16 h at +4°C. Beads were then washed 3 times for 10 min at +4°C with 15 bead volumes of RIPA buffer and spun down for 5 min at 1000g, +4°C between washes. Beads were then washed for 10 min with 15 bead volumes of buffer TEN 140 buffer with 0.1% Triton X-100 at +25°C and spun down for 5 min at 1000 g, +25°C. In order to elute the complexes (protein-DNA-RNA), 12-15 bead volumes of IgG Elution buffer were added to the beads. The slurry was mixed by inversion for 1 hour at +25°C. This elution step was repeated one more time. In order to eliminate urea from the samples, the eluates were first concentrated  in 10K Amicon Ultra-15 columns (3000g, 15min at +25°C) and then washed/buffer exchanged 3 times with 15 ml of TEN 140 buffer with 0.1% Triton X100 (3000g, 5-15min at +25°C) in a fresh Amicon column. The resulting 0.5 ml concentrate was diluted with 2500 ul of RIPA buffer and transferred into two Eppendorf tubes.  Biotin-Streptavidin affinity purification   150-300 ul of Streptavidin Agarose beads (Thermo Scientific, cat # 20349) were added to each tube.  The mixture was rotated end-over-end for 12-16 hours at +16°C. Beads from both tubes were pooled into one 15 ml Falcon tube washed once with RIPA buffer, once with TEN 140 buffer with 0.1% Triton X100, twice with IgG Elution buffer and twice with IgG Elution buffer without SDS. For each washing step, beads were mixed with 12 ml of a given buffer on a rotating wheel for 10 min at +25°C and spun down for 5 min at 1000 g, +25°C. Beads were re-suspended in 10 ml of TEN 140 buffer and divided into three tubes: 6 ml for protein work, 2 ml for DNA and 2 ml for RNA analysis.
Libraries were prepared according to Illumina's instructions accompanying the DNA Sample Kit. Briefly, DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (32 to 52 exo minus) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3’ end. After adapter ligation DNA was PCR amplified with Illumina primers for 12 cycles and library fragments from 300-700 bp (insert plus adaptor and PCR primer sequences) were band isolated from an agarose gel. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Genome Analyzer following the manufacturer's

treatment_protocol: Harvested S2 cells were homogenized by using a 100 ml Dounce homogenizer (Bellco, Glass Inc.), 10 strokes of each of A and B pestles. For every 4-5 ml of cell pellet volume 100 ml of NEB buffer+0.1 mM PMSF pre-chilled on ice were added. Without delay, 100 ml of cell/nuclear homogenate was poured into a T-225 flask containing a room-temperature mixture of 360 ml of PBS and 40 ml of 37% formaldehyde and incubated for 30 min at 25°C on a orbital shaker platform with vigorous shaking (100rpm). Fixed nuclei were pelleted by spinning for 10 min at 4000 g, +4°C. The supernatant was carefully decanted and the nuclear pellet was washed four times with 100 ml of ice-cold PBS with 0.1 mM PMSF. Nuclei were pelleted between washes for 10 min at 4000g, +4°C. Nuclei were resuspended in glycerol buffer and snap-frozen in liquid N2 prior to further processing. Frozen nuclear extracts from Drosophila S2 cells, embryos, 3rd instar larvae or adult flies were thawed and spun down for 10 min at 4000 g, +4°C. Pellets were washed with 10-20 volumes of TE buffer with 0.1 mM PMSF and spun down for 10 min at 4000g, +4°C. Pellets were again re-suspended with 10 volumes of TE buffer with 0.1 mM PMSF by pipetting up and down (for 3rd instar larval or adult fly chromatin, a motorized Teflon pestle was used). SDS was added to the mixture to a final concentration of 1%. The mixture was inverted in the tube 10 times and spun down for10 min at 4000 g, +4°C. The supernatant was carefully removed (Note: the pellet may be quite loose) and the pellet was re-suspended with 10 volumes of TE buffer with 0.1 mM PMSF by pipetting and further spun down for 10 min at 4000 g, +4°C. This washing step was repeated twice. The pellet was re-suspended with 1.5 volumes of TE buffer with 0.1 mM PMSF by pipetting up and down (for 3rd instar larvae or adult flies motorized Teflon pestle had been used). SDS was added to a mixture to a final concentration of 0.1%. The resulting viscous mixture was sonicated in 4.5 ml aliquots using a Misonix Sonicator 3000 with Microtip power output level 7 and total sonication processing time of 3.5 min, 15 sec pulse “on” and 45 sec “off” time to generate DNA fragments in the range of 300-2000 bp.   Triton X-100 (1% final) and NaCl (140 mM final) were added to the sonicated samples. Samples were mixed on a rotating wheel for 5 min  at +4C and spun down for 10 min at 10000 g, +4°C. The supernatant containing the soluble chromatin was collected. Note: In order to control for chromatin input composition and quality (for protein, DNA and RNA), a 1 ml aliquot is reserved before proceeding with the next step.

tag: cell line background

tag: pull-down tag

tag: platform

value: Illumina


title: Mmus Suz12-KO RNA Differentiation 72h rep. 2

data_processing:
Reads were pre-processed to remove adapter sequences (AGATCGGAAGAGCACACGTCTGAACTCCAGTCACNNNNNNATCTCGTAGCCGTCTTCTGCTTG) and mapped to the Genome Reference Consortium Mouse Build 38 (GRCm38, also referred to as mm10) using Tophat (Trapnell et al., 2009). Pre-processing and mapping were performed on the public Galaxy server (https://usegalaxy.org).
Gene expression levels were quantified using the featureCounts program (Liao et al., 2013) by counting the number of reads unambiguously mapped within exons of annotated transcripts (Ensembl GRCm38.72).
Differential expression analyses were performed as pairwise comparisons between biological conditions using the DESeq2 package (Anders and Huber, 2010) with default parameters. For each comparison, up- and down-regulated genes were identified according to an FDR of 5% and a minimum fold change of 2, and non-detected genes were defined as genes associated to null read counts in both of the compared conditions.
Genome_build: mm10
Supplementary_files_format_and_content: Table of gene expression levels in log10 (rpkm) including annotations for significant up- or down-regulation

extract_protocol:
mESCs were grown in 10 cm plates as described above, washed once with 1X PBS, lysed with 4 mL of TRIzol Reagent (Ambion) and split in 4 x 1 mL. Each aliquot was mixed with 200 µL of Chloroform:Isoamyl alcohol (24:1) (CI) and centrifuged at full speed for 15 min at 4°C. Supernatants were extracted twice with 500 µL of Phenol:CI (125:24:1) (PCI), pH4.5, once with CI and ethanol precipitated with 0.3 M NaCl, 40 μg of RNAse-free glycogen at -80°C for 15 min. After centrifugation at 20,000 x g for 90 min at 4°C, pellets were washed with 70% ethanol, air dried, resuspended with 10 mM Tris-HCl, pH 8.0. Aliquots from the same samples were pooled. RNA purity and amount was checked using a NanoDrop spectrophotometer. Expected yields range from 100 to 200 µg. 30 µg were further purified using the RNeasy MinElute Cleanup Kit (Qiagen) according to the manufacturer's instructions to remove small RNA (i.e. < 200 nt). RNA integrity was checked using the RNA 6000 Nano Kit with a 2100 Bioanalyzer (Agilent).    Depletion of 18S and 28S rRNA was performed on 5 µg of each sample using the RiboMinus Human/Mouse Transcriptome Isolation Kit (Ambion) according to the manufacturer's instructions. RNA fragmentation was performed using the NEBNext Magnesium RNA Fragmentation Module (New England Biolabs) as follows. 600 ng was mixed with 50 µL of 1X RNA Fragmentation Buffer, incubate at 94C for 20 min, transferred on ice and mixed with 5 µL of 10X RNA Fragmentation Stop Solution. Depletion in 18S and 28S rRNA (before fragmentation) and fragmentation efficiency was checked using the RNA 6000 Pico Kit with a 2100 Bioanalyzer. After fragmentation, the size of RNA fragments should have a normal distribution peaking at 60 nt. Samples were purified using the RNA Clean & Concentrator-5 Kit (Zymo Research) using the manufacturer protocol for small fragments and eluted in 14.5 µL.    RNA end-repair was performed by mixing samples with 0.5 µL of Murine RNase Inhibitor, 2 µL of 10X T4 Polynucleotide Kinase Buffer (New England Biolabs), 1 µL of 10U/µL T4 Polynucleotide Kinase Enzyme, incubating at 37C for 30 min, mixing with 2 µL of 10 mM ATP and incubating at 37°C for 30 minutes. Samples were purified using the RNA Clean & Concentrator-5 Kit as before and eluted in 8 µL. RNA amount was quantified using a NanoDrop spectrophotometer (40 to 50 ng/µL).
RNA library for Illumina sequencing was prepared using the NEBNext Multiplex Small RNA Library Prep Set for Illumina® (1–12) (New England Biolabs). Multiplex 3’ SR Adaptor ligation was performed as follows. 6 µL containing either 200 ng or 30 ng of RNA (for either RNA-seq or iTC-seq samples respectively) were mixed with 1 µL of 1:5 diluted Multiplex 3’ SR Adaptor, incubated at 70°C for 2 min and transferred to ice. Samples were then supplemented with 10 µL of 2X 3’ Ligation Reaction Buffer and 3 µL of 3’ Ligase Enzyme Mix and incubated at 25°C for 1 hour. RT primer hybridization was performed by mixing samples with 4.5 µL of H2O, 1 µL of 1:5 diluted Multiplex SR RT Primer and by incubating successively at 75C for 5 min, 37C for 30 min and 25C for 15 min. Multiplex 5’ SR Adaptor ligation was done by mixing samples with 1 µL of denatured 1:5 diluted Multiplex 5’ SR Adaptor, 1 µL of 5’ Ligation Reaction Buffer, 1 µL of 5’ Ligase Enzyme Mix and incubating at 25°C for 1 hour. cDNA synthesis was performed by mixing samples with 8 µL of First Strand Synthesis Reaction Buffer, 1 µL of Murine RNase Inhibitor, 1 µL M-MuLV Reverse transcriptase (RNase H-) and incubating successively at 50°C for 1 hour, 70 °C for 15 min and on ice. cDNA samples were purified using the QIAquick PCR Purification Kit (Qiagen) according to the manufacturer's instructions and eluted in 40 µL. cDNA amplification was performed as follows. Samples were mixed with 50 µL of LongAmp Taq® 2X Master Mix, 5 µL of 1:5 diluted Multiplex SR Primer and 5 µL of 1:5 diluted Multiplex Index (X) Primer. PCR was performed in a Thermocycler with the following program: 1 cycle for the initial denaturation step (94°C for 60 s), either 12 or 15 cycles (for either RNA-seq or iTC-seq samples respectively) for the amplification step (94°C for 10 s, 60°C for 30 s, 65°C for 30 s), 1 cycle for the final extension step (65°C for 7 min) and 1 cycle for the cooling step (4°C). PCR product purification was performed using AMPure XP system (Agencourt) as follows. Samples were split in 2 x 50 µL aliquot. Each aliquot was mixed with 90 µL of AMPure XP beads and incubate at room temperature (RT) for 15 min. Beads were washed twice with 200 µL of 80% ethanol for 30 s, air dried and resuspended with 10 µL of H2O. Supernatants from same sample were pooled and DNA amount was quantified using the dsDNA HS Assay Kit with a Qubit Fluorometer (Invitrogen). Expected amount range from either 300 to 450 ng or from 20 to 40 ng (for either RNA-seq or iTC-seq samples respectively). Library purity and integrity was checked using the DNA 1000 Kit with a 2100 Bioanalyzer (Agilent). DNA sizes should have a normal distribution peaking at 180 nt. Equimolar amounts of either 4 RNA-seq samples or 2 iTC-seq samples, with compatible indexes, were pooled for multiplex sequencing. Pool concentration was adjusted between samples and 0.1 to 0.4 pmols were sent to the National High-throughput DNA Sequencing Centre of the University of Copenhagen for 50 bp HiSeq Single Read sequencing using Illumina Sequencing Technology.


title: Input for HP1a RNA Illumina random priming r1


title: Mmus Suz12-KO RNA Differentiation 72h rep. 1


title: TI-3-004_milk


title: Mmus E14-WT ChIP H3K27me3 serum DRB 6h

value: DRB


title: Mmus E14-WT iTC serum DRB 2h Ctr  rep. 1


title: TI-3-003_milk


title: Mmus E14-WT iTC serum DRB 2h 4sU  rep. 2


title: TI-3-002_milk


title: Mmus Suz12-KO RNA Differentiation 24h rep. 2


title: Mmus Suz12-KO RNA Differentiation 48h rep. 1


title: Mmus E14-WT iTC serum DRB 2h 4sU  rep. 1


title: Input for MSL3 RNA Illumina random priming r2


title: Mmus Suz12-KO RNA Differentiation 24h rep. 1


title: TI-2-005_milk


title: Mmus Suz12-KO RNA Differentiation 0h rep. 2


title: Mmus Suz12-KO RNA Differentiation 0h rep. 1


title: TI-2-004_milk


title: Input for MSL3 RNA Illumina random priming r1


title: MSL3 RNA Illumina random priming r2

value: MSL3 BioTAP


title: Mmus E14-WT RNA Differentiation 72h rep. 2


title: Mmus E14-WT RNA Differentiation 48h rep. 2


title: Mmus E14-WT RNA Differentiation 72h rep. 1


title: Mmus E14-WT iTC serum NoDrug 0h Ctr  rep. 2

value: No drug


title: Mmus E14-WT RNA Differentiation 48h rep. 1


title: Mmus E14-WT RNA Differentiation 24h rep. 2


title: TI-2-003_milk


title: TI-2-002_milk


title: Mmus E14-WT RNA Differentiation 24h rep. 1


title: HP1a RNA Illumina ligation

source_name: embryo

value: HP1a BioTAP


title: TI-2-001_milk


title: Mmus E14-WT RNA Differentiation 0h rep. 2


title: HP1a RNA Illumina random priming r2


title: TI-1-005_milk


title: Mmus E14-WT RNA 2i  rep. 2

data_processing:
Reads were pre-processed to remove adapter sequences (AGATCGGAAGAGCACACGTCTGAACTCCAGTCACNNNNNNATCTCGTAGCCGTCTTCTGCTTG) and mapped to the Genome Reference Consortium Mouse Build 38 (GRCm38, also referred to as mm10) using Tophat (Trapnell et al., 2009). Pre-processing and mapping were performed on the public Galaxy server (https://usegalaxy.org).
Gene expression levels were quantified using the featureCounts program (Liao et al., 2013) by counting the number of reads unambiguously mapped within exons of annotated transcripts (Ensembl GRCm38.72).
Differential expression analyses were performed as pairwise comparisons between biological conditions using the DESeq2 package (Anders and Huber, 2010) with default parameters. For each comparison, up- and down-regulated genes were identified according to an FDR of 5% and a minimum fold change of 2, and non-detected genes were defined as genes associated to null read counts in both of the compared conditions.
Genome_build: mm10
Supplementary_files_format_and_content: Table of gene expression fold changes from DESeq2 analyses including annotations for significant up- or down-regulation

growth_protocol: mESCs cultured in 2i conditions were grown in 50% D-MEM/F12 (Gibco), 50% Neurobasal media (Gibco), N-2 supplement (Gibco), B27 supplement (Gibco), MEK inhibitor PD0325901 (1 µM), GSK3 inhibitor CH99021 (3 µM), PenStrep (Gibco), Glutamax (Gibco), non-essential amino acids (Gibco), Sodium pyruvate (Gibco), beta-mercaptoethanol (Gibco) and leukemia inhibitory factor (LIF) (produced and tested in our lab). Cells were cultured on 0.2% gelatin-coated plates (Sigma) at 37° and 5% CO2. To induce differentiation, medium was replaced with an equivalent medium containing 15% FBS serum and 1 µM ATRA (Sigma) and no N-2/B27, MEK or GSK3 inhibitor and no LIF.

extract_protocol:
mESCs were grown in 10 cm plates as described above, washed once with 1X PBS, lysed with 4 mL of TRIzol Reagent (Ambion) and split in 4 x 1 mL. Each aliquot was mixed with 200 µL of Chloroform:Isoamyl alcohol (24:1) (CI) and centrifuged at full speed for 15 min at 4°C. Supernatants were extracted twice with 500 µL of Phenol:CI (125:24:1) (PCI), pH4.5, once with CI and ethanol precipitated with 0.3 M NaCl, 40 μg of RNAse-free glycogen at -80°C for 15 min. After centrifugation at 20,000 x g for 90 min at 4°C, pellets were washed with 70% ethanol, air dried, resuspended with 10 mM Tris-HCl, pH 8.0. Aliquots from the same samples were pooled. RNA purity and amount was checked using a NanoDrop spectrophotometer. Expected yields range from 100 to 200 µg. 30 µg were further purified using the RNeasy MinElute Cleanup Kit (Qiagen) according to the manufacturer's instructions to remove small RNA (i.e. < 200 nt). RNA integrity was checked using the RNA 6000 Nano Kit with a 2100 Bioanalyzer (Agilent).    Depletion of 18S and 28S rRNA was performed on 5 µg of each sample using the RiboMinus Human/Mouse Transcriptome Isolation Kit (Ambion) according to the manufacturer's instructions. RNA fragmentation was performed using the NEBNext Magnesium RNA Fragmentation Module (New England Biolabs) as follows. 600 ng was mixed with 50 µL of 1X RNA Fragmentation Buffer, incubate at 94C for 20 min, transferred on ice and mixed with 5 µL of 10X RNA Fragmentation Stop Solution. Depletion in 18S and 28S rRNA (before fragmentation) and fragmentation efficiency was checked using the RNA 6000 Pico Kit with a 2100 Bioanalyzer. After fragmentation, the size of RNA fragments should have a normal distribution peaking at 60 nt. Samples were purified using the RNA Clean & Concentrator-5 Kit (Zymo Research) using the manufacturer protocol for small fragments and eluted in 14.5 µL.    RNA end-repair was performed by mixing samples with 0.5 µL of Murine RNase Inhibitor, 2 µL of 10X T4 Polynucleotide Kinase Buffer (New England Biolabs), 1 µL of 10U/µL T4 Polynucleotide Kinase Enzyme, incubating at 37C for 30 min, mixing with 2 µL of 10 mM ATP and incubating at 37°C for 30 minutes. Samples were purified using the RNA Clean & Concentrator-5 Kit as before and eluted in 8 µL. RNA amount was quantified using a NanoDrop spectrophotometer (40 to 50 ng/µL).
RNA library for Illumina sequencing was prepared using the NEBNext Multiplex Small RNA Library Prep Set for Illumina® (1–12) (New England Biolabs). Multiplex 3’ SR Adaptor ligation was performed as follows. 6 µL containing either 200 ng or 30 ng of RNA (for either RNA-seq or iTC-seq samples respectively) were mixed with 1 µL of 1:5 diluted Multiplex 3’ SR Adaptor, incubated at 70°C for 2 min and transferred to ice. Samples were then supplemented with 10 µL of 2X 3’ Ligation Reaction Buffer and 3 µL of 3’ Ligase Enzyme Mix and incubated at 25°C for 1 hour. RT primer hybridization was performed by mixing samples with 4.5 µL of H2O, 1 µL of 1:5 diluted Multiplex SR RT Primer and by incubating successively at 75C for 5 min, 37C for 30 min and 25C for 15 min. Multiplex 5’ SR Adaptor ligation was done by mixing samples with 1 µL of denatured 1:5 diluted Multiplex 5’ SR Adaptor, 1 µL of 5’ Ligation Reaction Buffer, 1 µL of 5’ Ligase Enzyme Mix and incubating at 25°C for 1 hour. cDNA synthesis was performed by mixing samples with 8 µL of First Strand Synthesis Reaction Buffer, 1 µL of Murine RNase Inhibitor, 1 µL M-MuLV Reverse transcriptase (RNase H-) and incubating successively at 50°C for 1 hour, 70 °C for 15 min and on ice. cDNA samples were purified using the QIAquick PCR Purification Kit (Qiagen) according to the manufacturer's instructions and eluted in 40 µL. cDNA amplification was performed as follows. Samples were mixed with 50 µL of LongAmp Taq® 2X Master Mix, 5 µL of 1:5 diluted Multiplex SR Primer and 5 µL of 1:5 diluted Multiplex Index (X) Primer. PCR was performed in a Thermocycler with the following program: 1 cycle for the initial denaturation step (94°C for 60 s), either 12 or 15 cycles (for either RNA-seq or iTC-seq samples respectively) for the amplification step (94°C for 10 s, 60°C for 30 s, 65°C for 30 s), 1 cycle for the final extension step (65°C for 7 min) and 1 cycle for the cooling step (4°C). PCR product purification was performed using AMPure XP system (Agencourt) as follows. Samples were split in 2 x 50 µL aliquot. Each aliquot was mixed with 90 µL of AMPure XP beads and incubate at room temperature (RT) for 15 min. Beads were washed twice with 200 µL of 80% ethanol for 30 s, air dried and resuspended with 10 µL of H2O. Supernatants from same sample were pooled and DNA amount was quantified using the dsDNA HS Assay Kit with a Qubit Fluorometer (Invitrogen). Expected amount range from either 300 to 450 ng or from 20 to 40 ng (for either RNA-seq or iTC-seq samples respectively). Library purity and integrity was checked using the DNA 1000 Kit with a 2100 Bioanalyzer (Agilent). DNA sizes should have a normal distribution peaking at 180 nt. Equimolar amounts of either 4 RNA-seq samples or 2 iTC-seq samples, with compatible indexes, were pooled for multiplex sequencing. Pool concentration was adjusted between samples and 0.1 to 0.4 pmols were sent to the National High-throughput DNA Sequencing Centre of the University of Copenhagen for 50 bp HiSeq Single Read sequencing using Illumina Sequencing Technology.


title: Mmus E14-WT RNA Differentiation 0h rep. 1


title: Mmus E14-WT RNA 2i  rep. 3


title: TI-1-004_milk


title: HP1a RNA Illumina random priming r1


title: TI-1-003_milk


title: TI-3-001_milk


title: Mmus Suz12-KO RNA 2i  rep. 2


title: Mmus Suz12-KO RNA 2i  rep. 3


title: Mmus E14-WT RNA 2i  rep. 1


title: TI-1-002_milk


title: TI-1-001_milk


title: Ez RNA Illumina ligation

value: Ez BioTAP


title: Mmus Ring1A+B-DKO RNA 2i  rep. 2


title: Mmus Ring1A+B-DKO RNA 2i  rep. 1


title: TC5-005_milk


title: Mmus Ezh2-WT RNA 2i  rep. 3


title: Mmus Ring1A+B-DKO RNA 2i  rep. 3


title: Mmus Suz12-KO RNA Differentiation 48h rep. 2


title: Mmus Ezh2-WT RNA 2i  rep. 1


title: MSL3 RNA Illumina random priming r1


title: Mmus Ezh2-WT RNA 2i  rep. 2


title: Mmus Ezh2-KO RNA 2i  rep. 3


title: Mmus Ezh2-KO RNA 2i  rep. 2


title: Mmus Ezh2-KO RNA 2i  rep. 1


title: TC5-004_milk


title: Mmus Suz12-KO RNA 2i  rep. 1


title: Mock FLAG ChIP

city: Los Angeles

first: Kevin

last: Peterson

state: CA

address: 1425 San Pablo St.

department: Department of Stem Cell Biology and Regenerative Medicine

institute: Eli and Edythe Broad-CIRM Center for Regenerative Medicine, University of Southern California Keck School of Medicine

data_processing:
Base pair calling was performed using Illumina pipeline RTA Version # 1.13.48 against a phix control-generated matrix.
50 bp reads were mapped using bwa v0.6.1-r104.
Reads mapping to Ptch1 (chr13:63,112,841-64,166,828) were filtered using samtools.
Normalized wig file was generated using QuEST.
Genome_build: mm9
Supplementary_files_format_and_content: Processed data is wig file corresponding to region: chr13:63,112,841-64,166,828.

source_name: E11.5 limb bud, mock ChIP

extract_protocol:
Chromatin precipitation protocol was previously described (Vokes et al. 20078). Briefly, embryonic limbs were dissociated in trypsin for 5' and then crosslinked in DMEM/10%FBS/1% formaldehyde at room-temperature for 30 minutes. Cells were lysed in lysis buffer (140mM NaCl, 0.5% NP40, 0.25% Triton-X 100, 10% glycerol, 1mM EDTA, 50mM HEPES-KOH, pH7.5) at 4˚C with agitation for 10 minutes then washed with Buffer 2 (200mM NaCl, 1mM EDTA, 0.5mM EGTA, 10mM Tris-Cl pH8.0) for 10 minutes at room-temperature. Chromatin was fragmented by sonication (Branson digital sonifier with microtip) in Buffer 3 (1mM EDTA, 0.5mM EGTA, 10mM Tris-Cl pH8.0). Lysate was cleared by centrifugation and supplemented with NaCl, NaDeoxycholate, and TritonX 100 to the final concentrations of 100mM, 0.1%, and 1%, respectively. Magnetic beads (Dynal/Invitrogen, sheep anti-mouse IgG) were blocked with a blocking buffer (5mg/ml BSA in PBS) then incubated with an antibody in the blocking buffer overnight. Unbound antibody was washed away by extensive wash with the blocking buffer. Beads/antibody complex was incubated with the lysate overnight at 4°C. Beads were washed 5 times with RIPA buffer (1% NP40, 0.7% NaDeoxycholate, 0.5M LiCl, 1mM EDTA, 50mM HEPES-KOH, pH7.5). DNA was eluted with elution buffer (1% SDS, 1mM EDTA, 50mM Tris-Cl, pH8.0) at 65˚C for 15 min, and the eluate was incubated further overnight at 65˚C to reverse crosslinking. DNA was purified using PCR purification kit (Qiagen). All buffers except RIPA buffer was supplemented with protease inhibitor cocktail mix (Roche).
Libraries were prepared according to Illumina protocols.

tag: strain/background

value: Swiss Webster

tag: tissue

value: limb bud

tag: age

value: FLAG-M2 monoclonal (Sigma, F3165)


title: H3K4me3_ChIP-seq

city: Nijmegen

last: Stunnenberg

address: Geert Grooteplein 28

department: Department of Molecular Biology (274)

institute: Radboud University

data_processing:
Cluster generation and sequencing were performed using the Illumina GAIIx or HiSeq2000 genome analyzer. The 36 (GAIIx) or 43 bp tags (HiSeq2000) were mapped to the reference human genome hg19 (NCBI build 37), using the BWA allowing one mismatch. The image files were processed to extract DNA sequence data. Only uniquely mapped-reads were used for data analysis and visualization. The output data were converted to bam files for downstream analysis and bigWig (bw) files for viewing the data in the UCSC Genome Browser.
Genome_build: hg19
Supplementary_files_format_and_content: BigWig were generated by counting the number of sequence reads (directionally extended to a final 300 bp) per 10-bp genomic window. Methylation density represents methylation level per 50-bp genomic windows
Supplementary_files_format_and_content: Cytosine methylation levels within CpG context are present in the MCF7_CpG_methcounts.bed.gz
Supplementary_files_format_and_content: Peak-calling was performed with MACS 2.0 tool against a reference input sample from the cell line expressing TTE-MBD2

source_name: MCF-7 WT

extract_protocol:
Cells were fixed for 10 minutes at room temperature by the addition of formaldehyde to a final concentration of 1%, after which glycine was added to a concentration of 100 mM. Cells were then washed twice with PBS and collected into 2 ml of lysis buffer (150 mM NaCl, 20 mM Tris pH 8.0, 2 mM EDTA, 1% triton X-100, protease inhibitor [complete EDTA free, Roche, 04 693 132 001], 100 mM PMSF). The lysate was sonicated to an average of 300-500bp fragments The resulting sonicate was centrifuged at 4000×g for 5 minutes, an aliquot of 10% retained for input and the remaining material transferred to a fresh tube.
5-10 ng DNA was subjected to end repair using Klenow DNA polymerase, T4 ligase and T4 polynucleotide kinase (T4 PNK). A 3′ protruding A base was generated using Taq polymerase and Illumina adapters were ligated. The DNA was loaded on E-gel for size selection (300 bp), further amplified by PCR and used for cluster generation on the Illumina GAIIx.

tag: antibody/details

value: H3K4me3 (pAb-MEHAHS-024, rabbit polyclonal, HC-0010), Diagenode


title: Gli3FLAG

description:
Gli3FLAG mice were maintained on mixed C57BL/6J;129/SvJ background and crossed to Swiss Webster females.
Embryonic forelimbs and hindlimbs were pooled and processed for ChIP-seq analysis.

source_name: E11.5 limb bud, FLAG-Gli3 ChIP

value: mixed


title: MCF7_RNA-Seq

extract_protocol:
Total RNA was extracted with the RNeasy kit and on-column DNase treatment (Qiagen). 250 ng of total RNA was treated by Ribo-Zero rRNA Removal Kit (epicentre) to remove  ribosomal RNAs  according to manufacturer instructions. 16 µl of purified RNA were fragmented by addition of  4 µl 5x fragmentation buffer (200 mM Tris acetate pH 8.2, 500 mM potassium acetate and 150 mM magnesium acetate) and incubated at 94°C for exactly 90 s. After ethanol precipitation, fragmented RNA  was mixed with 5 μg random hexamers, followed by incubation at 70 °C for 10 min and chilling on ice.  First-strand cDNA was synthesized with this primer mix by adding 4 μl 5× first-strand buffer, 2 μl 100 mM DTT, 1 μl 10 mM dNTPs, 132 ng of actinomycin D, 200 U SuperScript III, followed by 2 h at 48 °C. First strand cDNA was purified by Qiagen mini elute coloum and eluted  in 34 μl elution buffer. Second-strand cDNA was synthesized by adding 91.8 μl, 5 μg random hexamers, 4 μl of 5× first-strand buffer, 2 μl of 100 mM DTT, 4 μl of 10 mM dNTPs with dTTP replaced by dUTP, 30 μl of 5× second-strand buffer, 40 U of Escherichia coli DNA polymerase, 10 U of E. coli DNA ligase and 2 U of E. coli RNase H, and incubated at 16 °C for 2 h followed by incubation with 10 U T4 polymerase at 16 °C for 10 minutes. Double stranded cDNA was purified by Qiagen mini elute column and used for sample preparation and sequencing. We incubated 1 U USER (NEB) with 250 bp size-selected, adaptor-ligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR.
5-10 ng DNA was subjected to end repair using Klenow DNA polymerase, T4 ligase and T4 polynucleotide kinase (T4 PNK). A 3′ protruding A base was generated using Taq polymerase and Illumina adapters were ligated. The DNA was loaded on E-gel for size selection (300 bp), further amplified by PCR and used for cluster generation on the Illumina GAIIx.

value: Ribozero processed and fragmented RNA


title: H2A.Zac_ ChIP-seq

value: H2A.Zac (acetyl K4+K7+K11, ab18262, sheep polyclonal, 659355) Abcam


title: TC5-003_milk

tag: breed

value: Holstein Friesian

tag: cell type

tag: preperation kit

tag: infection

value: Control


title: TC5-001_milk

value: CD14 + milk derived monocytes

tag: time

value: 0 h


title: TC4-005_milk

value: Indexed TruSeq small RNA sample preparation kit (50 cycles)


title: nuclear RNA Illumina

data_processing:
Illumina base-calling pipeline; Heclios base-calling pipeline
for Illumina data: aligned to dm3 using bowtie (-t 1 -k 1); Helicos data was aligned using helisphere pipeline with default parameters, and uniquely aligned reads only.
smoothed enrichment calculated using SPP (http://compbio.med.harvard.edu/Supplements/ChIP-seq/), get.smoothed.enrichment.mle().
Genome_build: dm3 for D.melanogaster
Supplementary_files_format_and_content: bedGraph files containing either smoothed enrichment MLE (*IP.bedGraph), or strand-specific smoothed read density (*pos.bedGraph; *neg.bedGraph)

value: Illumina

tag: molecule subtype

value: nuclear RNA


title: MSL3 RNA Illumina

value: MSL3 BioTAP

value: RNA fraction of the BioTAP-XL pulldowns


title: CG3680 r2 pull-down

value: CG3680 BioTAP


title: TC4-004_milk


title: Patient 2 premalignant 1

first: Adam

middle: C

last: Gower

address: 72 East Concord Street, E632

department: Department of Medicine

institute: Boston University School of Medicine

description: Laser-microdissected premalignant lesion

data_processing:
Single-end sequencing was performed for 36 cycles (patients 1 and 2) or 50 cycles (patients 3 and 4).  Basecalling was performed for all cycles for patients 1, 3 and 4 and for the first 35 cycles for patient 2.  Illumina CASAVA software version 1.6.0 (patients 1 and 2) or 1.7.0 (patients 3 and 4) was used for basecalling.  Reads that failed Illumina's chastity filter [brightest intensity / (brightest intensity + second brightest intensity) < 0.6 for at least two of the first 25 cycles] were automatically removed during preprocessing.
After chastity filtering, the first 35 bases of each read were aligned to the human genome (build hg19) using Bowtie v0.12.7 (the first base of reads from patient 1 was omitted during alignment due to a technical issue).  Only unique alignments were allowed, with up to two mismatches per read, with the command: 'bowtie -p 2 -a --best --strata -v 2 -m 1 -S --fullref -t'
Coverage estimates were computed by merging exons from all isoforms of each of 55,841 Ensembl Gene (ENSG) loci (Ensembl build 69) to create a single meta-transcript for each ENSG, and counting the number of reads falling in the exons of each meta-transcript using the BEDTools software suite.
Reads aligning to the mitochondrial genome were removed from further analysis. Reads per kilobase per million aligned reads (RPKM) were computed for each remaining ENSG in each sample by normalizing counts to the size of each meta-transcript and to the number of reads aligned to the nuclear genome in each sample.  RPKM values were seventh-root-transformed prior to analysis to produce an approximately normal distribution of (nonzero) gene expression values.
Genome_build: hg19
Supplementary_files_format_and_content: Tab-delimited text file including the number of reads (counts) overlapping each gene and sample.
Supplementary_files_format_and_content: Tab-delimited text file including RPKM values for each gene and sample.

source_name: Premalignant lesion

extract_protocol:
Tissues were sectioned at a 7-micron thickness and mounted on regular uncharged glass slides for patients 1, 2, and 3, and on polyethylene napthalate (PEN) membrane slides (Leica) for patient 4, followed by H&E staining. LCM was performed using the Arcturus eIIx for patient 1 and 2, Zeiss PALM for patient 3, and Leica LMD7000 for patient 4. A tissue area of 800,000 to 1,200,000 μm2 was dissected and collected from each lesion.  RNA was extracted from laser-microdissected cells using the RNeasy Micro Kit (QIAGEN).
The cDNA was generated using the Ovation RNA-Seq System (NuGEN) for patients 1 and 2 and the 15 Ovation RNA-Seq V2 System (NuGEN) for patients 3 and 4. For patients 1 and 2, cDNA of ~200 bp was selected by gel purification. For patients 3 and 4, the cDNA was sheared to 140-180 bp using the Covaris focused-ultrasonicator with the following settings: duty cycle 10%; intensity 5; cycles per burst 200; total time 6 minutes.

tag: patient

value: Patient 2

tag: pathology

value: Premalignant

value: Premalignant lesion


title: Patient 1 premalignant 1

value: Patient 1


title: Patient 2 premalignant 2


title: CG3680 r1 input


title: CG3680 r1 pull-down


title: TC4-002_milk


title: Patient 1 tumor 2

description: Laser-microdissected squamous cell carcinoma

source_name: Squamous cell carcinoma

value: Tumor

value: Squamous cell carcinoma


title: TC4-003_milk


title: Patient 2 normal 1

description: Laser-microdissected normal basal cells

source_name: Normal basal cells

value: Normal

value: Normal basal cells


title: Patient 1 tumor 1


title: TC4-001_milk

value: Holstein Friesian

value: Control


title: Patient 2 tumor 3


title: TC5-002_milk

value: CD14 + milk derived monocytes


title: CG3680 r2 input


title: Patient 2 tumor 1


title: Patient 1 normal 2

description: Laser-microdissected normal basal cells


title: Patient 1 normal 4

description: Laser-microdissected normal basal cells


title: TC-3-004_milk

value: Indexed TruSeq small RNA sample preparation kit (50 cycles)


title: TC-3-005_milk


title: TC-3-003_milk


title: CG8290 r2 pull-down

value: CG8290 BioTAP

value: Illumina


title: CG8290 r2 input


title: CG8290 r1 input


title: CG8290 r1 pull-down


title: HP1a input

value: embryo


title: TC-3-002_milk


title: TC-3-001_milk

value: 0 h


title: TC-2-005_milk


title: TC-2-004_milk


title: MSL3 pull-down

value: MSL3 BioTAP


title: MSL3 input


title: Patient 1 normal 1

description: Laser-microdissected normal basal cells


title: Knockout

city: Taipei City

first: Shau-Ping

last: Lin

state: select a state

address: 4F, No. 81, Chang-Xing St.,

department: Institute of Biotechnology

country: Taiwan

institute: National Taiwan University

description: Sample 2

data_processing:
Basecalls performed using CASAVA version 1.9
Sequenced reads were mapped to mm10 whole genome using TopHat v2.0.8 with parameters -p 4 -G genes.gtf -r 50.
Reads Per Kilobase of exon per Megabase of library size (RPKM) were calculated using Cufflinks v2.1.1.
The statistics significance was filtered with a 2-fold changed selection criterion of RPKM from the corresponding genes.
Genome_build: mm10
Supplementary_files_format_and_content: GTF files include FPKM values for each Sample.

source_name: Spermatogonial progenitor cells

extract_protocol:
Postnatal SPCs were isolated from 8dpp testes. RNA was extracted using Ambion Tri-Reagent and two microgram RNA was used to create the construction of sequencing libraries.
Two microgram RNA was purified by using poly-T oligo-attached magnetic beads and mRNA was fragmented into small pieces using divalent cations under elevated temperature. The cleaved RNA fragments were copied into first strand cDNA using reverse transcriptase and random primers, followed by second strand cDNA synthesis using DNA Polymerase I and RNase H. These cDNA fragments were end-repaired, a single A base addition, and adapter ligation. The products were then purified and enriched with PCR to create the final cDNA library. Paired-end 100 nucleotide reads from each mRNA library were obtained using HiScanSQ (Illumina Inc., CIC bioGUNE, Bilbao, Spain).

tag: strain

value: Testis

value: Postnatal SPCs

tag: genotype

value: Dnmt3l knockout


title: F6_blasts

city: Seattle

first: Stephen

last: Tapscott

state: WASHINGTON

address: 1100 Fairview N. Ave

department: Human Biology

institute: Fred Hutch Cancer Research Center

data_processing:
Base calls and quality scores were generated using Illumina's Real-Time Analysis software v1.8.7.  Binary base call files were converted to text formatted qseq files via OLB 1.9.0, which were then filtered and converted to fastq format by CASAVA 1.7, using the default Chastity threshold of 0.6.
Read counts for the genes are collected by Bioconductor Biostrings and IRanges packages.  Reads that are mapped ambiguously to M locations are counted as 1/M at each occurrence.
Genome_build: hg19
Supplementary_files_format_and_content: biopsy.gene.counts.csv.gz contains the read counts for each gene for FSHD/control biopsy samples
Supplementary_files_format_and_content: cultured.gene.counts.csv.gz contains the read counts for each gene for myotubes and myoblasts from FSHD/control samples

source_name: Female FSHD1 myoblasts F6

growth_protocol: Muscle cell cultures: Muscle cells derived from two FSHD1 and three FSHD2 were cultured and RNA was harvested from both myoblasts and myotubes. These cultures were described previously (24). As previously described, Primary myoblast cell lines were received from the University of Rochester biorepository (http://www.urmc.rochester.edu/fields-center) and were cultured in DMEM/F-10 media (Gibco) in the presence of 20% heat-inactivated fetal bovine serum (Gibco), 1% penicillin/streptomycin (Gibco). Media was supplemented with 10ng/ml rhFGF (Promega) and 1μM dexamethasone (SIGMA). Myoblasts were fused at 80% confluence in DMEM/F-12 Glutamax media containing 2% KnockOut serum replacement formulation (Gibco) for 36 hours.

extract_protocol:
Muscle biopsy samples were obtained under local anesthesia by the minimally needle biopsy technique utilizing the modified Bergstrom needle. RNA was extracted, poly(A) selected, and subjected to Illumina sequencing using standard protocols to generate 100bp  single-end reads.
Sequencing libraries were prepared from total RNA using the TruSeq RNA Sample Prep Kit (Illumina) according to the manufacturer's instructions.

tag: gender

value: Female

value: FSHD1 myoblasts


title: F6_tubes

source_name: Female FSHD1 myotubes F6

value: FSHD1 myotubes


title: F4_blasts

source_name: Male FSHD1 myoblasts F4

value: Male


title: Wild-type

description: Sample 1

value: wild-type


title: F20_tubes

source_name: Female FSHD2 myotubes F20

value: FSHD2 myotubes


title: F20_blasts

source_name: Female FSHD2 myoblasts F20

value: FSHD2 myoblasts


title: F14_tubes

source_name: Female FSHD2 myotubes F14


title: TC-2-002_milk

source_name: Monocyte

value: Holstein Friesian

value: CD14 + milk derived monocytes

value: Control


title: TC-2-003_milk

value: Indexed TruSeq small RNA sample preparation kit (50 cycles)


title: TC-1-003_milk


title: TC-2-001_milk


title: TC-1-002_milk


title: TC-1-005_milk


title: HP1a pull-down

value: embryo

tag: pull-down tag

value: HP1a BioTAP

value: Illumina


title: TC-1-004_milk


title: F14_blasts

source_name: Female FSHD2 myoblasts F14


title: F12_tubes

source_name: Male FSHD2 myotubes F12


title: roundspd dKO rep1

city: reno

first: Wei

last: Yan

state: NV

address: 1664 N. Virginia Street

department: Physiology

institute: University of Nevada

data_processing:
Illumina Casava1.7 software used for basecalling.
Sequenced reads were mapped to mm9 whole genome using TOPHAT
Fragments Per Kilobase of exon per Megabase of library size (RPKM) were calculated and isoforms were assembled by using a protocol from Cufflinks
Genome_build: mm9

source_name: Round spermatids

growth_protocol: Normal breeding

extract_protocol:
Using mirVana RNA isolation Kit extract total RNAs
RNA libraries were prepared for sequencing using standard Illumina protocols

treatment_protocol: No treatment

value: Round spermatids

value: Adult

value: dKO


title: roundspd wt rep1

value: wild type


title: roundspd wt rep2


title: Pol2_Ser2P_WT_ChIP-seq

data_processing:
Cluster generation and sequencing were performed using the Illumina GAIIx or HiSeq2000 genome analyzer. The 36 (GAIIx) or 43 bp tags (HiSeq2000) were mapped to the reference human genome hg19 (NCBI build 37), using the BWA allowing one mismatch. The image files were processed to extract DNA sequence data. Only uniquely mapped-reads were used for data analysis and visualization. The output data were converted to bam files for downstream analysis and bigWig (bw) files for viewing the data in the UCSC Genome Browser.
FASTQ sequence files were aligned using RMAPBS (Smith et al., 2009; Smith et al., in prep) allowing a maximum of 10 mismatches for efficient mapping of reads containing bisulfite converted unmethylated cytosines. Cytosine methylation levels were determined using the standard new pipeline within RMAPBS (Methpipe; Andrew Smith, in prep). In short, both mates of the paired-end sequencing were mapped separately by RMAPBS. If mated reads mapped within 300 bp, the reads were merged and included for analysis. Mated reads mapping with a distance of more than 300 bp were excluded from further analysis. Mated reads mapped within 300 bp, but mapping equally well on multiple positions on the genome, were discarded. If multiple mated reads mapped on exactly the same genomic coordinates, all but one (at random) were discarded. Symmetric cytosines (within a CpG context) on both forward and reverse strands were combined. Cytosine methylation level was called per individual C as: #C/(#C+#T).
Reads were aligned to hg19 using BWA. Details about aligner version and parameters used are stored in the headers of the BAM files. PCR duplicates were removed, therefore the final BAM files only contain uniquely mapped reads.
Genome_build: hg19
Supplementary_files_format_and_content: BigWig were generated by counting the number of sequence reads (directionally extended to a final 300 bp) per 10-bp genomic window. Methylation density represents methylation level per 50-bp genomic windows
Supplementary_files_format_and_content: Cytosine methylation levels within CpG context are present in the MCF7_CpG_methcounts.bed.gz
Supplementary_files_format_and_content: Peak-calling was performed with MACS 2.0 tool against a reference input sample from the cell line expressing TTE-MBD2

extract_protocol:
Cells were fixed for 10 minutes at room temperature by the addition of formaldehyde to a final concentration of 1%, after which glycine was added to a concentration of 100 mM. Cells were then washed twice with PBS and collected into 2 ml of lysis buffer (150 mM NaCl, 20 mM Tris pH 8.0, 2 mM EDTA, 1% triton X-100, protease inhibitor [complete EDTA free, Roche, 04 693 132 001], 100 mM PMSF). The lysate was sonicated to an average of 300-500bp fragments The resulting sonicate was centrifuged at 4000×g for 5 minutes, an aliquot of 10% retained for input and the remaining material transferred to a fresh tube.
5-10 ng DNA was subjected to end repair using Klenow DNA polymerase, T4 ligase and T4 polynucleotide kinase (T4 PNK). A 3′ protruding A base was generated using Taq polymerase and NEXTflex adapters were ligated. The DNA was subjected to 4 cycles PCR amplification using Kappa polymerase and purified DNA was loaded on E-gel for size selection (300 bp). The DNA was isolated, further amplified by PCR and used for cluster generation on the HiSeq2000 genome analyzer

value: Anti-RNA polymerase II subunit B1 (phospho CTD Ser-2), clone 3E10, Millipore (Cat 04-1571)


title: C22_blasts

source_name: Female Control myoblasts C22

value: Female

value: Control myoblasts


title: C22_tubes

source_name: Female Control tubes C22

value: Control myotubes


title: Pol2_Ser5P_WT_ChIP-seq

source_name: MCF-7 WT

value: Anti-RNA polymerase II subunit B1 (phospho-CTD Ser-5), clone 3E8, Millipore (Cat 04-1572)


title: Pol2_N20_WT_ChIP-seq

value: Pol II (N-20):sc-899X SantaCruz


title: C21_tubes

source_name: Male Control myotubes C21

value: Male


title: roundspd dKO rep2


title: F4_tubes

source_name: Male FSHD1 myotubes F4

value: FSHD1 myotubes


title: C21_blasts

source_name: Male Control myoblasts C21


title: Pol2_Ser5P_TTE-MBD2_ChIP-seq

source_name: MCF-7 TTE-MBD2

tag: genetic manipulation

value: MCF7 cell line has been stably trasfected with TTE-MBD2 construct.


title: C20_tubes

source_name: Female Control myotubes C20


title: F7

source_name: Female FSHD1 biopsy F7

value: FSHD1 Muscle biopsy


title: PoL2_CTD_WT_ChIP-seq

value: RNA Polymerase II 8WG16 Monoclonal Antibody, Catalog Number MMS-126R, Covance


title: MBD2_ChIP-seq replicate

extract_protocol:
Cells were trypsinized and re-suspended with PBS to a final concentration of 8x106 cells/ 500 μl PBS. Cell suspensions were crosslinked with 1.5mM DSG (disuccinimidyl glutarate, Thermo,  #20593) for 45’ at room temperature with gentle rotation. After two washes with 500 μl PBS, cell pellets were re-suspended in 1ml PBS and 1% formaldehyde was added for 10’ at room temperature.  Cross-linking was quenched with 125mM glycine and after two times ice-cold PBS washes, pellets were resuspended in 270 μl lysis buffer (50mM Tris pH8.0, 1%SDS, 10mMEDTA protease inhibitor) and incubated 5’ on ice.  Sonication was performed for 15’-20’ with Bioruptor sonicator (NGS, Diagenode) and lysates were centrifuged at 13000rpm 4°C, for 5min.
5-10 ng DNA was subjected to end repair using Klenow DNA polymerase, T4 ligase and T4 polynucleotide kinase (T4 PNK). A 3′ protruding A base was generated using Taq polymerase and NEXTflex adapters were ligated. The DNA was subjected to 4 cycles PCR amplification using Kappa polymerase and purified DNA was loaded on E-gel for size selection (300 bp). The DNA was isolated, further amplified by PCR and used for cluster generation on the HiSeq2000 genome analyzer

value: Rabbit anti-MBD2 Antibody, Bethyl Laboratories , (A301-632)


title: MCF7_BisulfiteSeq

data_processing:
Cluster generation and sequencing were performed using the Illumina GAIIx or HiSeq2000 genome analyzer. The 36 (GAIIx) or 43 bp tags (HiSeq2000) were mapped to the reference human genome hg19 (NCBI build 37), using the BWA allowing one mismatch. The image files were processed to extract DNA sequence data. Only uniquely mapped-reads were used for data analysis and visualization. The output data were converted to bam files for downstream analysis and bigWig (bw) files for viewing the data in the UCSC Genome Browser.
FASTQ sequence files from bisulfite-seq were aligned using RMAPBS (Smith et al., 2009; Smith et al., in prep) allowing a maximum of 10 mismatches for efficient mapping of reads containing bisulfite converted unmethylated cytosines. Cytosine methylation levels were determined using the standard new pipeline within RMAPBS (Methpipe; Andrew Smith, in prep). In short, both mates of the paired-end sequencing were mapped separately by RMAPBS. If mated reads mapped within 300 bp, the reads were merged and included for analysis. Mated reads mapping with a distance of more than 300 bp were excluded from further analysis. Mated reads mapped within 300 bp, but mapping equally well on multiple positions on the genome, were discarded. If multiple mated reads mapped on exactly the same genomic coordinates, all but one (at random) were discarded. Symmetric cytosines (within a CpG context) on both forward and reverse strands were combined. Cytosine methylation level was called per individual C as: #C/(#C+#T).
Genome_build: hg19
Supplementary_files_format_and_content: BigWig were generated by counting the number of sequence reads (directionally extended to a final 300 bp) per 10-bp genomic window. Methylation density represents methylation level per 50-bp genomic windows
Supplementary_files_format_and_content: Cytosine methylation levels within CpG context are present in the MCF7_CpG_methcounts.bed.gz
Supplementary_files_format_and_content: Peak-calling was performed with MACS 2.0 tool against a reference input sample from the cell line expressing TTE-MBD2

extract_protocol:
Pelleted cells were resuspended in digestion buffer (100mM NaCl, 10mM Tris-HCl pH8, 15mM EDTA pH8, 0.5% SDS, 0.1mg/ml Proteinase K) and incubated at 55°C for 12-18 hr. DNA was isolated by two times extraction with 5 ml phenol/chloroform/isoamyl alcohol, and the aqueous top layer was transferred to a new tube. To precipitate DNA, 1/2 volume 7.5M ammonium acetate and 2 volumes 100% ethanol were added. After spinning, the supernatant was discarded, the DNA pellet washed with 70% ethanol, dried, and resuspended in water.
Library construction was performed according to Kulis et al. (2012; Nature Genetics). In short: We spiked genomic DNA (1 or 2 μg) with unmethylated λ DNA (5 ng of λ DNA per μg of genomic DNA) (Promega). We sheared DNA by sonication to 50–500 bp with a Covaris E220 and selected 150- to 300-bp fragments using AMPure XP beads (Agencourt Bioscience Corp.). We constructed genomic DNA libraries using the TruSeq Sample Preparation kit (Illumina Inc.) following the Illumina standard protocol: the ends of the fragments were repaired, an adenine was added at the 3′ extremities of the fragments and Illumina TruSeq adapters were ligated at each extremity. After adaptor ligation, we treated DNA with sodium bisulfite using the EpiTect Bisulfite kit (Qiagen) following the manufacturer’s instructions for formalin-fixed and paraffin-embedded (FFPE) tissue samples. We performed two rounds of conversion to get >99% conversion. We enriched adaptor-ligated DNA through seven cycles of PCR using the PfuTurboCx Hotstart DNA polymerase (Stratagene). We monitored library quality using the Agilent 2100 BioAnalyzer (Agilent) and determined the concentration of viable sequencing fragments (molecules carrying adapters at both extremities) by quantitative PCR using the Library Quantification Kit from KAPA Biosystem. We performed paired-end DNA sequencing (two reads of 100 bp each) using the Illumina Hi-Seq 2000.

value: WT


title: Pol2_CTD_TTE-MBD2_ChIP-seq


title: F4

source_name: Male FSHD1 biopsy F4


title: F5

source_name: Male FSHD1 biopsy F5


title: 14D Conceptus input

city: Columbia

first: Thomas

last: Spencer

address: 920 East Campus Drive

department: Animal Sciences

institute: University of Missouri

description: n=4 conceptuses, pooled

data_processing:
Sequences (50 bases, single end) were aligned to the ovine genome [U.S. National Center for Biotechnology Information (NCBI) Build 37, August 2012, Oar_v3.1] using the BWA algorithm.
Alignments were extended in silico at their 3’-ends to a length of 150 bp and assigned to 32-nt bins along the genome. The resulting histograms were stored in binary analysis results (BAR) files.
Peak locations were determined by applying a threshold of 18 (5 consecutive bins containing 18 aligns) and storing the resulting intervals in browser extensible data (BED) files. The BED files were analyzed using GenPathway software (Active Motif) that provides information on genomic annotation, peak metrics, and sample comparisons for all intervals.
The model based analysis of ChIP-Seq (MACS; ref. 18) peak-finding algorithm was used to normalize ChIP against the input control (P<107; mfold 8,30, bandwidth 150).
Genome_build: NCBI Build 37, August 2012, Oar_v3.1
Supplementary_files_format_and_content: BAM files contain reads and alignment information. Spreadsheet contains binding intervals and corresponding genes.

organism: Ovis aries

source_name: conceptus

growth_protocol: In utero conceptus development

extract_protocol:
Conceptus tissue samples (~100 mg) were submersed in 1% formaldehyde in PBS, cut into small pieces, and incubated at room temperature for 15 min. Fixation was stopped by the addition of 0.125 M glycine. Tissue pieces were then treated with a TissueTearor (BioSpecProducts, Bartlesville, OK, USA) and finally spun down and washed twice in PBS. Chromatin was isolated by disrupting the cells with a Dounce homogenizer. Lysates were sonicated using a Misonix Sonicator 3000 (Misonix, Vernon Hills, IL,USA) equipped with a microtip in order to shear the DNA to an average length of 300–500 bp. Lysates were cleared by centrifugation and stored at 80°C. Genomic DNA (input) was prepared by treating aliquots of chromatin with RNase, proteinase K, and heat for decrosslinking, followed by phenol/chloroform extraction and ethanol precipitation. Purified DNA was quantified on a NanoDrop spectrophotometer (Thermo Fisher Scientific Inc., Wilmington, DE, USA). Extrapolation to the original chromatin volume allowed quantization of the total chromatin yield. For each ChIP reaction, 30 ng of chromatin was precleared with protein A agarose beads (Invitrogen, Carlsbad, CA, USA). Immunoprecipitation was performed using 4 μg of rabbit anti PPARG polyclonal IgG (# sc-7196; Santa Cruz Biotechnology, Santa Cruz, CA, USA). Following overnight incubation at 4°C, protein A agarose was added, and incubation at 4°C continued for another 3 h. Immune complexes were washed 2 times each with a series of buffers consisting of the deoxycholate sonication buffer, high-salt buffer, LiCl buffer, and TE buffer. Immune complexes were eluted from the beads with SDS buffer and subjected to RNase treatment and proteinase K treatment. Crosslinks were reversed by incubation overnight at 65°C, and ChIP DNA was purified by phenol-chloroform extraction and ethanol precipitation.
Input and ChIP DNA were amplified using an Illumina ChIP-Seq DNA Sample Prep Kit (Illumina, San Diego, CA, USA). Briefly, DNA ends were polished and 5'-phosphorylated using T4 DNA polymerase, Klenow polymerase, and T4 polynucleotide kinase. After addition of 3’-adenine to blunt ends using Klenow (3’-5’ exo minus), Illumina genomic adapters were ligated, and the sample was size fractionated to 200–250 bp on a 2% agarose gel. After a PCR amplification(30 s at 98°C; followed by 10 s at 98°C, 30 s at 65°C, and 30 sat 72°C for 18 cycles; followed by 5 min at 72°C) with Phusion High-Fidelity DNA Polymerase (New England Biolabs, Ipswich,MA, USA), the resulting DNA libraries were tested by reverse transcription quantitative PCR (RT-qPCR) at the same specific genomic regions as the original ChIP DNA to assess quality of the amplification reactions. The DNA libraries were sent to Illumina Sequencing Services for sequencing on an Illumina HiSeq 2000.

treatment_protocol: Conceptuses were flushed from the uterine horn with 10 ml of 10 mM PBS (pH 7.2). Conceptus tissue was transferred to a 1.5ml microcentrifuge tube and frozen in liquid nitrogen.

tag: day of pregnancy

tag: chip antibody


title: 14D Conceptus PPARG

description: n=4 conceptuses, pooled

value: Santa Cruz sc-7196


title: Ty1_CTR_ChIP-seq

value: BB2 (mouse monoclonal against TY1 epitope), Diagenode


title: Input_DNA

extract_protocol:
Cells were trypsinized and re-suspended with PBS to a final concentration of 8x106 cells/ 500 μl PBS. Cell suspensions were crosslinked with 1.5mM DSG (disuccinimidyl glutarate, Thermo,  #20593) for 45’ at room temperature with gentle rotation. After two washes with 500 μl PBS, cell pellets were re-suspended in 1ml PBS and 1% formaldehyde was added for 10’ at room temperature.  Cross-linking was quenched with 125mM glycine and after two times ice-cold PBS washes, pellets were resuspended in 270 μl lysis buffer (50mM Tris pH8.0, 1%SDS, 10mMEDTA protease inhibitor) and incubated 5’ on ice.  Sonication was performed for 15’-20’ with Bioruptor sonicator (NGS, Diagenode) and lysates were centrifuged at 13000rpm 4°C, for 5min.  50 μl from TTE-MBD2 chromatin was also de-crosslinked and prepared for sequencing (referred as input chromatin).
5-10 ng DNA was subjected to end repair using Klenow DNA polymerase, T4 ligase and T4 polynucleotide kinase (T4 PNK). A 3′ protruding A base was generated using Taq polymerase and NEXTflex adapters were ligated. The DNA was subjected to 4 cycles PCR amplification using Kappa polymerase and purified DNA was loaded on E-gel for size selection (300 bp). The DNA was isolated, further amplified by PCR and used for cluster generation on the HiSeq2000 genome analyzer

value: input DNA purified from chromatin


title: F2

source_name: Female FSHD1 biopsy F2

tag: tissue


title: MBD2_ChIP-seq

extract_protocol:
Cells were trypsinized and re-suspended with PBS to a final concentration of 8x106 cells/ 500 μl PBS. Cell suspensions were crosslinked with 1.5mM DSG (disuccinimidyl glutarate, Thermo,  #20593) for 45’ at room temperature with gentle rotation. After two washes with 500 μl PBS, cell pellets were re-suspended in 1ml PBS and 1% formaldehyde was added for 10’ at room temperature.  Cross-linking was quenched with 125mM glycine and after two times ice-cold PBS washes, pellets were resuspended in 270 μl lysis buffer (50mM Tris pH8.0, 1%SDS, 10mMEDTA protease inhibitor) and incubated 5’ on ice.  Sonication was performed for 15’-20’ with Bioruptor sonicator (NGS, Diagenode) and lysates were centrifuged at 13000rpm 4°C, for 5min.
5-10 ng DNA was subjected to end repair using Klenow DNA polymerase, T4 ligase and T4 polynucleotide kinase (T4 PNK). A 3′ protruding A base was generated using Taq polymerase and NEXTflex adapters were ligated. The DNA was subjected to 4 cycles PCR amplification using Kappa polymerase and purified DNA was loaded on E-gel for size selection (300 bp). The DNA was isolated, further amplified by PCR and used for cluster generation on the HiSeq2000 genome analyzer.

tag: antibody/details


title: F15

source_name: Male FSHD2 biopsy F15

value: FSHD2 Muscle biopsy


title: Ty1_ChIP-seq


title: PC3_AR-K2R_R1881_rep1

city: Kuopio

first: Päivi

last: Sutinen

address: Yliopistonranta 1 E

department: School of Medicine

institute: University of Eastern Finland

data_processing:
The quality of raw reads was analyzed by FastQC
FASTX-toolkit was used for preprocessing (removal of artifacts, trimming (50bp), quality filtering and collapsing identical reads)
Reads were alignment Bowtie (0.12.7)(one mismatch and best alignment)
Analysis were done with HOMER software (4.2). Binding sites were determined with findpeaks (using -factor mode and input as a background).
Genome_build: hg19
Supplementary_files_format_and_content: peaks.bed files contain information of the statistically significant peaks (find in both replicates)

source_name: PC-3 cells

growth_protocol: PC-3 cells were grown in F-12 (10% FBS, 1% penicillin/streptomycin) under G418 selection (400 µg/ml).

extract_protocol:
Lysates were clarified from sonicated nuclei and AR-DNA complexes were isolated with 1 µg of antibody.
ChIP-Seq libraries were prepared for sequencing using standard Illumina protocols, as utilized at the Gene Core in EMBL, Heidelberg, Germany

treatment_protocol: PC-3 cells were growth 72h in transfection medium (5% stripped FBS in F-12) before treating 1 h with 10 nM R1881 or ethanol (vehicle).

tag: cell line

value: PC-3 prostate cancer cells

tag: antibody

value: rabbit antiserum against AR

tag: treatment

tag: genotype/variation

value: SUMOylation-deficient AR


title: F14

source_name: Female FSHD2 biopsy F14

value: Female


title: F8

source_name: Female FSHD1 biopsy F8


title: F6

source_name: Female FSHD1 biopsy F6


title: Pol2_N20_TTE-MBD2_ChIP-seq


title: F9

source_name: Male FSHD1 biopsy F9


title: F12

source_name: Male FSHD2 biopsy F12


title: PC3_AR-K2R_EtOH

value: Ethanol