title: drought-stressed DS

city: Changsha

first: Touming

last: Liu

address: NO.348, western road of xianjia lake

department: Institute of Bast Fiber Crops

institute: Chinese Academy of Agricultural Sciences

data_processing:
Tag sequencing: The 3'-tag molecules were fixed onto the Illumina Sequencing Chip (flow cell). Each molecule grew into a single-molecule cluster sequencing template through in situ amplification. Four color-labeled nucleotides were added, and sequencing was performed with the method of sequencing by synthesis. Image analysis and base calling were performed using the Illumina Pipeline, and cDNA sequence tags were revealed after purity filtering. The tags passing initial quality tests were sorted and counted. Each tunnel generates millions of raw reads with sequencing length of 49 bp (target tags plus 3’adaptor). Each molecule in the library represented a single tag derived from a single transcript.
Filtering: low quality tags  such as tags containing ‘N’ and adaptor sequences were filtered
Gene annotation :“Clean Tags” were obtained by filtering off adaptor-only tags and low-quality tags (containing ambiguous bases). Comparison of the Clean Tags sequences with ramie transcriptome sequence (published in BMC genomics, Liu et al. 14:125) by BLASTN was carried out. All clean tags were annotated based on ramie reference genes. The number of annotated clean tags for each gene was calculated and then normalized to TPM (number of transcripts per million clean tags)
Genome_build: ramie transcriptome sequence (SRA057664)
Supplementary_files_format_and_content: the text file contain the tag sequence, copy number and TPM value

organism: Boehmeria nivea

source_name: Zhongzhu 1

extract_protocol:
After severe drought stress, The CO and DS ramie tissues samples of three replicates including leaf, root, stem and shoot were individually collected. The sampled tissues were immediately frozen in liquid nitrogen and stored at -80° until use. The same tissue sample of three replicates of each group was mixed to extract RNA. Total RNAs of two treatment ramie were extracted from each tissue sample using TRIzol reagent (Transgene Company, Illkirch Graffenstaden Cedex, France) according to the manufacturer’s protocol. The RNA was equally pooled from the five tissues for cDNA library preparation
Six micrograms of total RNA for CO and DS ramie was individually purified using biotin-Oligo (dT) magnetic bead adsorption. First- and second-strand cDNA synthesis was performed after the RNA was bound to the beads. While on the beads, double strand cDNA was digested with NlaIII endonuclease to produce a bead-bound cDNA fragment containing sequence from the 3’-most CATG to the poly (A)-tail. These 3’ cDNA fragments were purified using magnetic bead precipitation and the Illumina adapter 1 (GEX adapter 1) was added to new 5’ end. The junction of Illumina adapter 1 and CATG site was recognized by MmeI, which is a Type I endonuclease (with separated recognition sites and digestion sites). The enzyme cuts 17 bp downstream of the CATG site, producing 17 bp cDNA sequence tags with adapter 1. After removing 3’ fragments with magnetic bead precipitation, the Illumina adapter 2 (GEX adapter 2) was ligated to 3’ end of the cDNA tag. These cDNA fragments represented the tag library.

treatment_protocol: the potted 30-day-old ramies were transferred to a movable rain-off shelter and were parted as two groups. One group (CO) was used as control by watering daily and the other group (DS) was treated for drought stress by withholding water. Each group was planted with three replicates. The degree of drought stress in DS ramie was determined by monitoring the relative water contents (RWC) of the ramie leaves.

tag: cultivar

value: Gaud

value: leaf, root, stem and shoot

value: control


title: No.36 Liver_SAGE

status: Public on Apr 22 2013

type: SRA

city: Kitakami-shi

first: Hidetoshi

last: Yamada

state: Iwate

country: Japan

institute: Iwate Biotechnology Reserch Center

description: Super SAGE (Serial analysis of gene expression)

library_source: transcriptomic

data_processing:
We used two original programs (kSusageA, kSusageB) witten in objective-C to analyze the Super SAGE data. The FASTAQ format fileswere split every 12,000,000 lines using Terminal.  These files were processed to create FASTA format files using kSusageA.  The kSusageA program extracts 26-base tags from reads of 35 bases, sorts the tags based on index sequence, and counts the frequency of the tags.  The tags were compared to the NCBI mouse transcript database using BLAST. The reference sequence DNA IDs were picked from the results of BLAST analysis, and the tags were annotated using kSusageB.
Supplementary_files_format_and_content: the frequency of the tags

molecule: total RNA

organism: Mus musculus

source_name: liver_High fat diet + 1% WEPAK

growth_protocol: Male C57BL/6 mice that were 4-weeks old (Charles River Laboratories, Tokyo, Japan) were housed in a temperature-controlled environment (23 ± 1°C) with a 12 h light/dark cycle.

extract_protocol: Total RNA was extracted from liver samples with an RNeasy lipid tissue kit.  Double-stranded cDNA was synthesized with biotinated oligo-dT primer and the Double-Stranded cDNA Synthesis kit (Invitrogen) from 5 µg of total RNA.  Double-stranded cDNAs were digested with NlaIII (New England Biolabs, Beverly, MA, USA) after being purified using a PCR purification kit (QIAGEN).  Double-stranded cDNAs digested with NlaIII were bound to Dynabeads M270 streptavidin (Invitrogen).  Adapter sequence 2 containing an EcoP15I restriction site was ligated to the double-stranded cDNAs that were bound to Dynabeads.  Double-stranded cDNAs were digested with EcoP15I (New England Biolabs).  Double-stranded cDNAs digested with EcoP15I and separated from Dynabeads were tag sequences.  Adaptor sequence 1 that contained index sequence was ligated to tag sequences.  Tag sequences were then amplified by PCR and gel purified.

treatment_protocol: The animals were randomly divided into four groups: normal diet (D12450B: Research Diets, Inc., New Brunswick, NJ, USA), normal diet with 1% WEPAK, high-fat diet (D12451: Research Diets, Inc.), and high-fat diet with 1% WEPAK (4 mice / group).

tag: strain

tag: gender

value: male

tag: age

value: 4 weeks

tag: feeding

value: High fat diet + 1% WEPAK for 18 wks

tag: tissue

value: liver


title: No.40 Liver_SAGE


title: No.38 Liver_SAGE


title: control CO

status: Public on Apr 23 2013

country: China

tag: developmental stage

tag: treatment

value: drought stress


title: No.28 Liver_SAGE

source_name: liver_High fat diet (45% fat)

value: High fat diet (45% fat) for 18 wks


title: No.23 Liver_SAGE


title: No.25 Liver_SAGE


title: No.20 Liver_SAGE

source_name: liver_Normal diet + 1% WEPAK

value: Normal diet + 1% WEPAK for 18 wks


title: No.8 Liver_SAGE

source_name: liver_Normal diet (10% fat)

value: Normal diet (10% fat) for 18 wks


title: No.34 Liver_SAGE


title: No.11 Liver_SAGE


title: No.7 Liver_SAGE


title: Nr37 48h H20 rep2

city: Jena

first: JenAge

last: Project

address: Beutenbergstr. 11

country: Germany

institute: Leibniz Institute for Age Research - Fritz Lipmann Institute

data_processing:
Illumina Casava software used for extraction of FASTQ files
FASTQ files were mapped using Bowtie vs. the C.elegans genome and a splice site data set created using UCSC's RefSeq annotation
Counting of uniquely mapped reads and assignment to RefSeq transcripts/genes using R Statistical Language and Bioconductor
Calculation of RPKM values for each transcript and gene from RefSeq annotation (see ce_Nr35-38_counts_rpkm.xls)
Genome_build: ce6\WBcel235
Supplementary_files_format_and_content: RPKM

organism: Caenorhabditis elegans

source_name: total RNA from whole animals

growth_protocol: Nematodes were grown and maintained on NGM agar plates according to standard protocol as described (Brenner et al. 1974)

extract_protocol:
RNA isolation was performed using a commercially available kit (Qiagen, Hilden, Germany, Rneasy Mini Kit) based on the phenol-chloroform extraction method according to the manufacturer’s instructions. Afterwards the RNA was quantified photometrically with a NanoDrop 1000 (PeqLab, Erlangen, Germany) and stored at −80°C until use.
For library preparation an amount of 5 μg of total RNA per sample was processed using Illumina’s mRNA-Seq sample prep kit (Illumina; San Diego; CA, USA) following the manufacturer’s instruction.

treatment_protocol: Treatment of C. elegans was carried out on NGM agar plates containing 100nM arsenite or the respective solvent control (H2O) and spotted with heat-inactivated OP50 E. coli. Thereby, agar plates were prepared from the same batch of NGM Agar, whereas treatment plates were supplemented with the arsenite and control plates with the corresponding solvent as described (Schmeisser et al. 2011).

value: Adult (48 hours after L4)

value: control plate

value: whole organism


title: No.29 Liver_SAGE


title: No.5 Liver_SAGE


title: No.4 Liver_SAGE


title: Larval ovary subjected to RNAi against H1

city: Bronx

first: Sandeep

middle: Nagaraj

last: Wontakal

state: NY

address: 1300 Morris Park Ave Chanin 402

department: Cell Biology

country: USA

institute: Albert Einstein College of Medicine

description: Larval ovaries were isolated from transgenic flies expressing RNAi targeting H1 and were subjected to small RNA-seq.

data_processing:
Fastq to Fasta conversion:  fastq_to_fasta -r -v -o -i INFILE -o OUTFILE  Part of FASTX Toolkit 0.0.14 by A. Gordon (gordon@cshl.edu)
Clipping the cloning adapter:  fastx_clipper -i INFILE -a ADAPTER -o OUTFILE -c -v -l 15  Part of FASTX Toolkit 0.0.14 by A. Gordon (gordon@cshl.edu)
Collapsing the fasta file:  fastx_collapser -v -i INFILE -o OUTFILE  Part of FASTX Toolkit 0.0.14 by A. Gordon (gordon@cshl.edu)
Mapping to transposon consesus with Bowtie. Iterative mapping (-v 0 -m 1) (-v 1 -m 1) (-v 2 -m 1)
sam to bam conversion with samtools view
bam to bed conversion with bedtools bamtobed. Read count (column 4 after dash) is expressed as reads per million genomic mappers.
Genome_build: Release 5; Apr. 2006 [BDGP R5/dm3]
Supplementary_files_format_and_content: UCSC bed format. Contains mapping locations with read counts over transposon consensus sequences

organism: Drosophila melanogaster

source_name: Larval Ovary

growth_protocol: Flies were crossed at 18o and knockdown was achieved by incubating at 29o from the embryo stage.

extract_protocol:
Dissected tissue was homogenized in TRIzol using a pestle and lysate was incubated for 5 minutes at RT. Following incubation, 200µl of chloroform was added to the TRIzol and sample was vortexed for 15 seconds and incubated for 3 minutes at RT before centrifugation at 4ºC at 12,000Xg for 15 minutes. The upper aqueous phase (~450µl) was transferred to a fresh tube. RNA was precipitated by adding an equal volume of isopropanol. Samples were incubated for 10 minutes at RT and the centrifuged for 10 minutes at 4ºC at a speed of 12,000Xg. RNA pellet was washed once with 70% ethanol and then air-dried for 5 minutes. RNA pellet was then resuspended in nuclease free water.
Small RNA libraries were generated as described in Malone et al, 2012 with some changes. In brief, total RNA was size selected on a denaturing polyacrylamide gel. Size selection was aided by the presence of radioactively labeled 19nt and 28nt RNAoligonucleotide cloning markers. RNA was eluted from gel slice by electro-elution using D-tube Dialyzers (Millipore). After elution, the adenylated Modban Linker-1 adaptor (IDT) was ligated to the 3’ end of the sRNA. For this ligation step, T4 RNA Ligase 2 truncated (NEB) and a buffer not containing ATP was used. Ligation reaction was examined on a polyacrylamide gel and only fragments of the expected ligated size were cut out and electro-eluted. The Solexa 5’ adapter oligo was ligated to the eluted RNA using T4 RNA Ligase. After another gel extraction of ligated products, RNA was eluted from the gel, and RNA fragments with both adaptors ligated were reverse transcribed. Resulting cDNA was the amplified by PCR followed by Pme1 digestion to eliminate all RNA oligonucleotide cloning markers. Library was single end sequenced on an Illumina Genome Analyzer II for 36 cycles.

treatment_protocol: Transgenic flies used for isolation of salivary glands and larval ovaries were described in X Lu et al. Genes Dev 2009.

value: ovary

tag: genotype/variation

value: expressing RNAi targeting histone H1


title: No.12 Liver_SAGE


title: Salivary gland subjected to RNAi against H1

description: Salivary glands were isolated from transgenic flies expressing RNAi targeting H1 and were subjected to small RNA-seq.

source_name: Salivary gland

value: salivary gland


title: Larval ovary subjected to RNAi against Nautilus

description: Larval ovaries were isolated from transgenic flies expressing RNAi targeting Nautilus and were subjected to small RNA-seq.

value: expressing RNAi targeting Nautilus


title: uninduced 2

city: Zurich

first: Ulrich

last: Omasits

address: Wolfgang Pauli-Str. 16

department: IMSB Institute of Molecular Systems Biology

country: Switzerland

institute: ETH Zurich

data_processing:
The sequenced reads were mapped using the BioScope 1.3.1 mapping pipeline (mapreads software using local alignment strategy).
SAM files were subsequently further processed to remove among all uniquely mapping reads those with an average mapping/read quality below 20, or more than two mismatches.
For the class of doubly mapped reads, we eliminated reads from duplicated 16S and 23S rRNA genes (accounting for >99%) and then filtered instances where, based on edit distance and map quality as selection criteria, one of the two mappings exhibited a better match with the reference genome sequence. This was repeated for triply mapped reads (including the 5S rRNA genes) and up to 4-7 mapped genome positions per read.
The count data summary for annotated B. henselae ORFs was generated using the HTSeq package, the quality of the reads was visually inspected with the HTSeq-qa program.
Transcript abundance was estimated via RPKM values. The number of mapped and filtered reads for each B. henselae protein-coding gene was divided by the length of the gene (in kilobases) and the number of mapped and filtered reads to all B. henselae protein-coding genes in the experiment (in million reads).
Differential transcript expression analysis was carried out with the R package DESeq (version 1.6.1).
Genome_build: B. henselae Houston-1 strain (NCBI RefSeq acc. NC_005956.1, slightly modified to include the bepD-gfpmut2 chromosomal reporter fusion, and retain the deleted batR-batS sequence)
Genome_build: ASM4670v1
Supplementary_files_format_and_content: Tab-delimited text file includes for each protein-coding gene reads and RPKM values per sample, as well as differential expression analysis for replicate 2.

organism: Bartonella henselae

source_name: bacterial colonies

growth_protocol: MQB307 was grown on Columbia agar plates containing 5% defibrinated sheep blood (CBA plates) supplemented with 30 mg/l kanamycin with (induced condition) or without (uninduced condition) 500 uM isopropyl β-D-thiogalactoside (IPTG) at 35°C and 5% CO2 for 60 h

extract_protocol:
RNA was isolated from bacterial cells using a modified hot-phenol extraction, followed by DNase I digestion, RNA cleanup (RNeasy Mini Kit, Qiagen). Integrity of total RNA was verified on an Agilent 2100 Bioanalyser using the RNA 6000 Nano LabChip kit (Agilent Technologies). The purified RNA was used directly for downstream application or stored at -70°C after precipitation with 2.5 volumes ethanol and 1/10 volume 3 M sodium acetate, pH 5.2.
The Whole Transcriptome library was produced using the Ribominus kit, Bacteria Module (Invitrogen PN 45-7014) and the SOLiD™ Total RNA-seq kit (Applied Biosystems p/n 4445374). Briefly, 5 μg of total RNA was depleted of rRNA and then fragmented using RNase III. Ligation of the adaptor mix and reverse transcription were performed following the manufacturer's protocol. cDNA libraries were size selected for fragments between 150 and 250 bp, amplified for 18 cycles of PCR using barcoded adaptor primers and purified with the PureLink PCR micro kit (Invitrogen PN K310050). Library size and concentrations were assessed on a Bioanalyzer (Agilent) and on a Qubit fluorometer (Invitrogen), respectively. The whole transcriptome library was used for emulsion-PCR based on a concentration of 0.5 pM. Sequencing beads from four barcoded libraries (2 biological replicates for the uninduced and induced condition, respectively) were pooled together and loaded on a full SOLiD™-4  slide (Applied Biosystems), according to manufacturer's instructions. SOLiD™ ToP Sequencing F3-Tag MM50 chemistry was used to produce 50 base sequencing reads.


title: T22_RNA-Seq

city: Cambridge

first: Thomas

middle: A

last: Down

address: Tennis Court Road

department: WT/CRUK Gurdon Institute

country: United Kingdom

institute: University of Cambridge

data_processing:
ChIP-seq reads were aligned to the reference sequence using bwa-0.6.6 then sorted and indexed using samtools
Read pairs with quality scores >=10 were extracted and the number of read pairs overlapping each probe on the Illumina HumanMethylation450K array were counted.
RNA-seq was aligned with TopHat 1.3.1 and FPKM values were generated using CuffLinks 1.0.3
Genome_build: GRCh37 (hg19)
Supplementary_files_format_and_content: Fragment coverage across Illumina HumanMethylation450K probes (GEO Platform GPL13534) for ChIP data.
Supplementary_files_format_and_content: Transcript FPKM numbers for nearest TSS for RNA-seq data.

organism: Homo sapiens

source_name: T22, 2nd pair, 2nd individual

extract_protocol: For RNA-seq, 200 ng of total RNA was used to prepare RNA-seq libraries using the TruSeq RNA kit from Illumina following the instructions provided in the supplier's manual, and sequenced on an Illumina GAIIx instrument.

tag: cell type

value: CD14 cells

tag: individual identifier

tag: monozygotic twin relationship

value: MZ twin of T21


title: T32_RNA-Seq

source_name: T32, 3rd pair, 2nd individual

value: MZ twin of T31


title: T11_RNA-Seq

source_name: T11, 1st pair, 1st individual


title: Salivary gland subjected to RNAi against Nautilus

description: Salivary glands were isolated from transgenic flies expressing RNAi targeting Nautilus and were subjected to small RNA-seq.


title: T32_H2AZ_ChIP-Seq

library_source: genomic

molecule: genomic DNA

extract_protocol: The chromatin immunoprecipitation (ChIP) assay was performed on 5 x 105 CD14+ cells according to previously published protocols. Chromatin was sonicated to get fragments of 100-500 bp and immunoprecipitated with 10 ul of anti-H2A.Z antibody (Active Motif, Cat no: 39113). ChIP-seq libraries were prepared following the Illumina protocol and ligated to standard PE adaptors and sequenced on an Illumina GAIIx instrument.

tag: chip antibody

value: anti-H2A.Z (Active Motif, Cat no:39113)


title: No.15 Liver_SAGE

value: male

value: 4 weeks


title: T21_RNA-Seq

source_name: T21, 2nd pair, 1st individual

value: MZ twin of T22


title: T21_H2AZ_ChIP-Seq


title: T11_H2AZ_ChIP-Seq


title: T31_H2AZ_ChIP-Seq

source_name: T31, 3rd pair, 1st individual

value: MZ twin of T32


title: T22_H2AZ_ChIP-Seq


title: Input DNA 2

status: Public on Apr 21 2013

city: Dallas

first: Joseph

middle: S

last: Takahashi

state: TX

address: 5323 Harry Hines Blvd., NA4.118

department: HHMI, Neuroscience

institute: University of Texas Southwestern Medical Center

data_processing:
Sequence reads were mapped to the mouse genome (NCBIm37/mm9) with Applied Biosystems BioScope v1.3.
PCR duplicates were removed using Picard MarkDuplicates.
The uniquely aligned reads without PCR duplicates were converted into bigWig (.bw) file to show the sequence read density across the mm9 mouse genome.  The .bw file reflects extended 120bp fragments corresponding to each ChIP-Seq read.
Genome_build: mm9
Supplementary_files_format_and_content: bigWig

source_name: Liver

growth_protocol: Male C57BL/6J at 8-12 weeks of age were housed in LD12:12 conditions.

extract_protocol: Livers from mice were immediately homogenized in 4 ml per liver of 1X PBS containing 2 mM EGS. The homogenate was incubated for 20 min at room temperature. Next, 110 µl of 36.5% formaldehyde was added followed by an 8 min incubation at room temperature. Cross-linking reactions were stopped by the addition of 250 µl of 2.5 M glycine and kept on ice. The homogenate was added to 10 ml of ice-cold 2.3 M sucrose containing 150 mM glycine, 10 mM HEPES pH 7.6, 15 mM KCl, 2 mM EDTA, 0.15 mM spermine, 0.5 mM spermidine, 0.5 mM DTT and 0.5 mM PMSF, and layered on top of a 3 ml cushion of 1.85 M sucrose (containing the same ingredients with 10% glycerol) and centrifuged for 1 hr at 24,000 rpm at 4°C in a Beckman SW32.1 rotor. The nuclei were resuspended in 1 ml of 20 mM Tris pH 7.5, 150 mM NaCl, 2 mM EDTA, transferred to a 1.5 ml microfuge tube, centrifuged for 3 min at 3000 rpm at 4°C, washed again, and stored –80°C until use. The nuclei were then resuspended in 0.8 ml per liver of lysis buffer (10 mM Tris pH 7.5, 1 mM EDTA, 0.5 mM EGTA, 0.2 M NaCl, 0.5% N-lauroylsarcosine, 0.1% sodium deoxycholate, 1 mM PMSF and EDTA-free protease inhibitor cocktail (Roche), and sonicated 5 s at 4°C 36 times with a Covaris S2. The fragmented chromatin was then diluted ten-fold with IP buffer (10 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% triton X-100, 0.1% sodium deoxycholate, 1 mM PMSF, EDTA-free protease inhibitor cocktail). Approximately 120 µg of fragmented chromatin was pre-cleared by incubating with 40 µl of protein A-agarose (Sigma) for 2 hr at 4°C with rotation. Pre-cleared chromatin was then incubated with antibody overnight at 4°C with rotation, followed by addition of 10 µl of Protein A/G Plus-agarose and incubation for 1.5 hr at 4°C. Agarose beads were then washed twice with IP buffer, twice with high salt wash buffer (20 mM Tris pH7.5, 500 mM NaCl, 2 mM EDTA, 1% Triton X-100, 1 mM PMSF), twice with LiCl wash buffer (20 mM Tris pH 7.5, 250 mM LiCl, 2 mM EDTA, 0.5% Igepal CA-630, 1% sodium deoxycholate, 1 mM PMSF), and once with TE. Co-immunoprecipitated DNA fragments were eluted with 100 µl of 20 mM Tris pH 7.5, 5 mM EDTA, 0.5% SDS, then reverse crosslinked at 65°C overnight, incubated with 10 µg of RNaseA for 30 min at 37°C, with 160 µg of proteinase K for 30 min at 55°C, and then purified using the QIAquick PCR purification Kit (Qiagen).  The immunoprecipitated DNA fragments were repaired by the End-It DNA End Repair Kit (Epicentre Biotechnology) according to the manufacturer’s instructions. The end-repaired ChIP DNA fragments were purified by MinElute Reaction Cleanup Kit (Qiagen) and eluted in 20 μl in EB buffer. The resulting DNA fragments were ligated with P1 and P2 adaptors for the Applied Biosystems SOLiD system for 20 min at room temperature using the Quick Ligase Kit (NEB), followed by purification using the MinElute Reaction Cleanup Kit (Qiagen). The purified, adaptor-ligated ChIP DNA fragments were run on 5% native polyacrylamide gel electrophoresis (PAGE) for an in-gel PCR reaction. A gel slice containing 175–200 bp adaptor-ligated ChIP DNA fragments (corresponding to 115-140 bp genomic gment sizes) was cut and shredded. PCR Platinum Supermix (100 μl, Invitrogen), 50 pmol of PCR primers with barcodes, 0.5 μl Taq DNA polymerase (NEB), and 0.15 μl p.f.u. Turbo DNA polymerase (Stratagene) were added into the shredded gel slice. The adaptor-ligated ChIP DNA fragments were amplified by 18 cycles of in-gel PCR. After the PCR reaction, gel pieces were filtered out with a 0.45 μm filter spin column, and the amplified ChIP-seq library was purified by the MinElute PCR purification kit (Qiagen). The library was purified by one more round of 5% PAGE. A gel slice containing 200-230 bp PCR products (110-130 bp fragment size) was cut and shredded, and the amplified library was extracted out of the gel by passive elution in elution buffer (2.5 M ammonium acetate in TE). Gel pieces were removed with a spin column, and the resulting ChIP-seq library was purified using the QIAquick PCR purification kit (Qiagen).

tag: genotype

value: liver


title: BMAL1 ChIP-seq 4

description: BMAL1 (Lee et al. 2001, Cell 107, 855-867)

value: Bmal1 knockout


title: Input DNA 1

value: wild-type


title: BMAL1 ChIP-seq 3

value: Clock knockout


title: T31_RNA-Seq


title: BMAL1 ChIP-seq 2


title: Egr2_IP_2

city: Chicago

first: Yan

last: Zheng

state: IL

institute: University of Chicago

data_processing:
Base calls and read alignment to mm9 were performed using the Illumina Pipeline V1.6
Peaks were called using QuEST v 2.4 using default paramters
Genome_build: mm9
Supplementary_files_format_and_content: bed file reports peak calls

source_name: CD4+ T cells_treatment with immobilized anti-CD3 mAb_Egr2_IP

extract_protocol:
Lysates were clarified from sonicated nuclei and Egr2-DNA complexes were isolated with antibody.
Libraries were prepared according to Illumina's instructions accompanying the DNA Sample Kit

treatment_protocol: overnight with immobilized anti-CD3 mAB (1ug/ml) and rested 1-2 days

value: CD4+ T cells

value: immobilized anti-CD3 mAb

value: EGR2 (PRB-236P,Covance)


title: p53-/- dox rep2_Sample 14

city: Stanford

first: Stephano

last: Spano Mello

state: CA

address: 269 Campus Drive

institute: Stanford University

data_processing:
ChIP-seq and RNA-seq reads were aligned to the mm9 genome assembly using DNAnexus.
For ChIP-seq: Peaks were called using the DNAnexus ChIP-seq analyses tool with the following specifications: peak enrichment >20, peak-to background enrichment >3, a minimum ratio between uniquely and repetitively mapped reads of 3:1, a kernel bandwith of 30.0, FDR calculation enabled. Background was combined input  reads and reads from p53 ChIP in p53-/- MEFs.
For RNA-seq: Tag counts were derived using the DNAnexus 3SEQ transcriptome-based quantification tool with the following specifications: Sense only, RefSeq genes. The DESeq library was used to normalize tag counts between samples and to call differentially expressed genes.
Genome_build: mm9
Supplementary_files_format_and_content: ChIP-seq: Processed data files are tab-delimited text files containing 1) Chromosome name, 2) Position (summit of the peak), 3) enrichment (based on background reads), 4) neg_log10_qvalue (negative log of q-value of peak) and 5) peak rank.
Supplementary_files_format_and_content: RNA-seq: Processed files are tab-delimited text files containing normalized gene expression, obtained using DESeq.

source_name: mouse embryo fibroblasts_p53-/- dox

growth_protocol: DMEM with 10% FCS, for ChIP-seq 4x106 cells were plated on 15 cm dish one day prior to treatment, for RNA-seq 1.5x 106 cells were plated on 10 cm dish

extract_protocol:
For RNA-seq, total RNA was isolated using Trizol reagent, and mRNA was purified with the Dynabeads mRNA Purification Kit from Invitrogen.
For RNA-seq, libaries were prepared according to the 3-seq protocol: RNA was heat-sheared to fragments of 100-200 bp followed by first and second strand synthesis, linker ligation, size selection on agarose gel and PCR amplification for 15 cycles.

treatment_protocol: untreated or 0.2ug/ml doxorubicin for 6h

value: mouse embryo fibroblast (MEF)

value: 0.2ug/ml doxorubicin for 6h


title: input DNA

source_name: CD4+ T cells_treatment with immobilized anti-CD3 mAb_input DNA


title: BMAL1 ChIP-seq 1


title: Egr2_IP_1


title: CLOCK ChIP-seq 1

description: CLOCK (Lee et al. 2001, Cell 107, 855-867)


title: CLOCK ChIP-seq 2


title: CLOCK ChIP-seq 3


title: USF1 ChIP-seq 4

description: USF1 (Santa Cruz Biothechnology, sc-229, Lot A1711)

tag: chip antibody manufacturer

value: Santa Cruz

tag: chip antibody catalog #

tag: chip antibody lot #


title: p53-/- dox rep1_Sample 12


title: p53-/- ut rep1_Sample 11

source_name: mouse embryo fibroblasts_p53-/- ut

value: untreated


title: USF1 ChIP-seq 3


title: Dnmt2-MEFs-Small-RNA-NBS

city: SLC

first: Vahid

last: Khoddami

state: UT

address: 4350, 2000 Circle of Hope, HCI

department: Oncological Sciences

institute: Huntsman Cancer Institute-University of Utah

data_processing:
The “Make Transcriptome” application from open source USeq package (Utah Bioinformatics Shared Resource Center) was used to build the “transcriptome index file” to include the different combinations of the splicing events for genes with multiple exons.
The commercial "Novoalign" package (Novocraft) was used for sequence alignments in normal or bisulfite modes (depending of the sample) with options to allow mismatches, reporting 18bp or larger inserts and reporting all the reads mapped to the repeats and generating SAM and Pairwise formatted alignment files.
For visualization the SAM alignment files were converted into BAM/BAI files using Samtools package.
For methylation analysis the Pairwise alignment files from bisulfite treated samples were analyzed by a set of costume Python and Perl scripts to report the base distribution (C/T) on over all sequenced cytosines.
To find the differentially methylated bases in wt vs Dnmt2-/- samples the combined (small and large) RNA datasets from each of wt and Dnmt2-/- samples were compared. To correct for mapping errors the bisulfite treated and untreated datasets were inspected using IGV to make sure the corresponding sequencing reads are present in both datasets (i.e. wt and Dnmt2-/-).
Genome_build: mm9
Supplementary_files_format_and_content: text file containing the regions which are differentially methylated  in wt and Dnmt2-/- samples (column headers and content is described  in README.txt)

growth_protocol: Wild-type (B6129PF2/J) and Dnmt2-/- (B6;129-Trdmt1tm1Bes/J) mice were obtained from Jackson Laboratory. Mouse Embryonic Fibroblasts (MEFs) were harvested from 13.5 isogenic wt or Dnmt2-/- embryos. Single embryos were dispersed and trypsinized in 10 cm dishes, and were maintained in DMEM (Invitrogen) supplemented with 10% FBS (GIBCO) and passaged several times as reported previously.

extract_protocol: Illumina’s directional mRNA-Seq sample preparation protocol was used to prepare the libraries, by ligating the adapters to the RNAs followed by reverse transcription, PCR and sample clean-up using AMPure beads (Beckman Coulter Genomics). The libraries were then subjected to 101 cycle single-end high-throughput sequencing using Illumina’s HiSeq 2000 sequencing system.

treatment_protocol: For bisulfite treated samples small and large RNA fractions of each sample (wt or Dnmt2-/-) were separately subjected to bisulfite treatment. First 5μg of RNA was dissolved in 45μl of RNase free ddH2O and added to 240μl of de-ionized formamide, mixed well and incubated at 95oC for 5min then placed on ice for 2 min. Next 3μl of the 100mM hydroquinone and 312μl of 5M sodium bisulfite (pH 5) were added and the mixture was incubated at 50oC, rotating. After 16 hours, the mixture was cleaned up using Illustra NAP-10 Columns (GE Healthcare Life Sciences) and de-sulfonation was performed for 2hrs at 37oC in 1M Tris buffer pH 9.0, followed by ethanol precipitation.

tag: tissue source

value: 13.5 isogenic embryos

value: Mouse Embryonic Fibroblasts (MEFs)

tag: bisulfite treatment of rna

value: No


title: wt ut rep3_Sample 9

source_name: mouse embryo fibroblasts_wt ut

tag: strain


title: USF1 ChIP-seq 2


title: wt dox rep3_Sample 10

source_name: mouse embryo fibroblasts_wt dox


title: wt ut rep2_Sample 7


title: wt dox rep2_Sample 8


title: wt dox rep1_Sample 6


title: Dnmt2-MEFs-Small-RNA-BS

data_processing:
Library strategy: RNA Bisulfite-Seq
The “Make Transcriptome” application from open source USeq package (Utah Bioinformatics Shared Resource Center) was used to build the “transcriptome index file” to include the different combinations of the splicing events for genes with multiple exons.
The commercial "Novoalign" package (Novocraft) was used for sequence alignments in normal or bisulfite modes (depending of the sample) with options to allow mismatches, reporting 18bp or larger inserts and reporting all the reads mapped to the repeats and generating SAM and Pairwise formatted alignment files.
For visualization the SAM alignment files were converted into BAM/BAI files using Samtools package.
For methylation analysis the Pairwise alignment files from bisulfite treated samples were analyzed by a set of costume Python and Perl scripts to report the base distribution (C/T) on over all sequenced cytosines.
To find the differentially methylated bases in wt vs Dnmt2-/- samples the combined (small and large) RNA datasets from each of wt and Dnmt2-/- samples were compared. To correct for mapping errors the bisulfite treated and untreated datasets were inspected using IGV to make sure the corresponding sequencing reads are present in both datasets (i.e. wt and Dnmt2-/-).
Genome_build: mm9
Supplementary_files_format_and_content: text file containing the regions which are differentially methylated  in wt and Dnmt2-/- samples (column headers and content is described  in README.txt)

value: Yes


title: Dnmt2-MEFs-Large-RNA-NBS


title: p53-/- ut rep2_Sample 13


title: NSUN2-Aza-IP (anti-V5-Rep1)

data_processing:
The 'Make Transcriptome' application from open source USeq package (Utah Bioinformatics Shared Resource Center) was used to build the 'transcriptome index file' to include the different combinations of the splicing events for genes with multiple exons.
The commercial "Novoalign" package (Novocraft) was used for sequence alignments with options to allow mismatches, reporting 18bp or larger inserts and reporting all the reads mapped to the repeats and generating SAM formatted alignment files.
The 'RNASeq' application of the USeq package was used for assessment of the enriched/over-expressed transcripts using SAM files as input.
The 'RNASeq' application of the USeq package was used to generate the BAM/BAI files, from SAM alignments input files, for visualization of the mapped reads.
The Integrative genomics viewer (IGV) was used to visualize the alignment files and inspect the mapped reads at base pair resolution.
The 'mpileup2cns" function of VarScan package (Washington University) was used to find the significant C>G tranversion sites to report as candidate m5C-RMT target sites.
Genome_build: hg19
Supplementary_files_format_and_content: Each processed data xls file contains detailed description in the first 'Table Legends & Header Info' spreadsheet.

extract_protocol:
After the last washing round the protein-RNA adducts were subjected to RNA fragmentation on the beads, using RNA fragmentation reagent (Ambion) at 94C for exactly 5min in a thermocycler, followed by 1min incubation on ice and then quenched by fragmentation stop solution (Ambion). The fragmented RNA was then purified by ethanol precipitation followed by Trizol (Invitrogen) RNA extraction and used for library preparation.
Illumina’s directional mRNA-Seq sample preparation protocol

treatment_protocol: 5-Azacytidine treatment at final concentration of 5μM for 24 hrs (at 32 hrs post infection by lentivral vectors)

tag: cell line

value: HeLa

tag: transgene (lentiviral infection)

value: V5-tagged hNSUN2

tag: 5-aza-c treatment

value: 5μM at 32 hrs post infection for 24 hrs

tag: cell harvest time

value: 56 hrs post infection

tag: ip antibody

value: Anti-V5 Antibody

tag: ip antibody vendor

value: Invitrogen

tag: ip antibody cat. #

tag: molecule subtype

value: Fragmented IP-RNA


title: Dnmt2-MEFs-Large-RNA-BS

tag: genotype/variation

tag: tissue


title: background_Sample 4

description: Processed data file: ChIP-seq_peaks_p53_binding_sites.txt (linked as supplementary file on Series record)

source_name: mouse embryo fibroblasts_background_p53-/- dox

extract_protocol:
For ChIP-seq, samples were prepared according to Myers lab ChIP-seq protocol: cells were cross-linked with 1% formaldehyde at room temperature for 10 min, cells were extracted with Farnham and RIPA buffers, and chromatin was fragmented by sonication.
For ChIP-Seq, libraries were prepared according to the Illumina ChIP-seq library preparation protocol: ChIP DNA was blunt ended, ligated to Solexa adaptors, amplified using adaptor primers and size-selected on agarose gel.

tag: cell type

tag: chip antibody

tag: treatment


title: NSUN2-Aza-IP (anti-V5-Rep2)


title: wt ut rep1_Sample 5

value: wild-type


title: NSUN2-Aza-IP (IgG-Control)

source_name: HeLa_NSUN2-Aza-IfG control

value: IgG


title: USF1 ChIP-seq 1

tag: genotype

tag: chip antibody manufacturer

tag: chip antibody catalog #

tag: chip antibody lot #

value: liver


title: wt-MEFs-Small-RNA-BS

source_name: wt-MEFs-Small-RNA-BS

tag: strain

value: wild type

tag: tissue source

tag: bisulfite treatment of rna


title: wt-MEFs-Small-RNA-NBS

source_name: wt-MEFs-Small-RNA-NBS


title: background_Sample 2

source_name: mouse embryo fibroblasts_background_wt dox

value: mouse embryo fibroblast (MEF)

value: 0.2ug/ml doxorubicin for 6h


title: p53 ChIP-seq_Sample 3

source_name: mouse embryo fibroblasts_p53 ChIP-seq_p53-/- dox

value: CM-5 (Vector labs, VP-P956)


title: wt-MEFs-Large-RNA-NBS

source_name: wt-MEFs-Large-RNA-NBS

value: 13.5 isogenic embryos

value: Mouse Embryonic Fibroblasts (MEFs)


title: DNMT2-Aza-Input-mRNA

description: Sample 3

data_processing:
The 'Make Transcriptome' application from open source USeq package (Utah Bioinformatics Shared Resource Center) was used to build the 'transcriptome index file' to include the different combinations of the splicing events for genes with multiple exons.
The commercial "Novoalign" package (Novocraft) was used for sequence alignments with options to allow mismatches, reporting 18bp or larger inserts and reporting all the reads mapped to the repeats and generating SAM formatted alignment files.
The 'RNASeq' application of the USeq package was used for assessment of the enriched/over-expressed transcripts using SAM files as input.
The  RNASeq  application of the USeq package was used to generate the BAM/BAI files, from SAM alignments input files, for visualization of the mapped reads.
The Integrative genomics viewer (IGV) was used to visualize the alignment files, inspect the mapped reads at base pair resolution and get the base distribution reports at desired locations.
Genome_build: hg19
Supplementary_files_format_and_content: Each processed data xls file contains detailed description in the first 'Table Legends & Header Info' spreadsheet.

source_name: HeLa cells

extract_protocol: Total RNA was purified from an aliquot of 5-Azacytidine treated cell lysate, before performing the immunoprecipation (IP). PolyA mRNA was isolated using oligo(dT) beads followed by mRNA fragmentation and library preparation according to Illumina's "directional mRNA-Seq sample preparation protocol".

treatment_protocol: 5-Azacytidine treatment at final concentration of 3μM for 12 hrs (at 18 hrs post infection by lentivral vectors)

tag: cell line

tag: transgene (lentiviral infection)

value: V5-tagged hDNMT2

tag: 5-aza-c treatment

value: 3μM at 18 hrs post infection for 12 hrs

tag: cell harvest time

value: 30 hrs post infection

value: PolyA selected mRNA


title: DsRed-Aza-Input-mRNA

description: Sample 4

value: HeLa

value: V5-tagged DsRed

tag: ip antibody

tag: molecule subtype


title: DsRed-Aza-IP

description: Sample 2

data_processing:
The “Make Transcriptome” application from open source USeq package (Utah Bioinformatics Shared Resource Center) was used to build the “transcriptome index file” to include the different combinations of the splicing events for genes with multiple exons.
The commercial "Novoalign" package (Novocraft) was used for sequence alignments with options to allow mismatches, reporting 18bp or larger inserts and reporting all the reads mapped to the repeats and generating SAM formatted alignment files.
The “RNASeq” application of the USeq package was used for assessment of the enriched/over-expressed transcripts using SAM files as input.
The “RNASeq” application of the USeq package was used to generate the BAM/BAI files, from SAM alignments input files, for visualization of the mapped reads.
The Integrative genomics viewer (IGV) was used to visualize the alignment files, inspect the mapped reads at base pair resolution and get the base distribution reports at desired locations (e.g C38 position of DNMT2 target tRNAs).
The “get_datasets.pl” script of the Biotoolbox package were used to calculate and compare the total number of mapped reads to each individual tRNA type in DNMT2- and DsRed-Aza-IP datasets.
Genome_build: hg19
Supplementary_files_format_and_content: Each processed data xls file contains detailed description in the first 'Table Legends & Header Info' spreadsheet.

extract_protocol: After the last washing round the protein-RNA adducts were subjected to RNA fragmentation on the beads, using RNA fragmentation reagent (Ambion) at 94C for exactly 5min in a thermocycler, followed by 1min incubation on ice and then quenched by fragmentation stop solution (Ambion). The fragmented RNA was then purified by ethanol precipitation followed by Trizol (Invitrogen) RNA extraction and used for library preparation. Illumina’s directional mRNA-Seq sample preparation protocol

value: Anti-V5 Antibody

tag: ip antibody vendor

value: Invitrogen

tag: ip antibody catalog #


title: wt-MEFs-Large-RNA-BS

source_name: wt-MEFs-Large-RNA-BS

tag: genotype/variation

tag: tissue

value: Yes


title: p53 ChIP-seq_Sample 1

source_name: mouse embryo fibroblasts_p53 ChIP-seq_wt dox

tag: cell type

value: wild-type

tag: chip antibody

tag: treatment


title: DNMT2-Aza-IP

description: Sample 1

value: Fragmented IP-RNA


title: S. coelicolor

status: Public on Apr 20 2013

city: Leeds

first: David

last: Romero A.

state: West Yorkshire

address: Woodhouse lane

institute: University of Leeds

description:
Enriched for mRNA
SC_gRNA-seq_data_Read_1.fastq; 5'end
SC_gRNA-seq_data_Read_2.fastq; 3'end

data_processing:
RNA sequences from the global analysis (FRT-seq) were processed in-house using Galaxy (Goecks et al. 2010. Genome Biol 11: R86). This involved trimming 3' adaptor sequence (AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCUCUUCCGAUCU) as well as 5' adaptor sequence (AGAUCGGAAGAGCGGUUCAGCAGGAATGCCGAGACCGATCTCGTATGCCGTCTTCTGCTTGC) and masking for low-quality sequence.
Reads were mapped to the reference genomes using Bowtie 2.0 (Langmead and Salzberg 2012. Nat Methods 9: 357) with custom parameter (-y -a --best --strata).
Using BEDTool, we determined or each position in the genome the number of times it was read by global RNA-seq.
The counts for each position were then used to construct BedGraphs
Genome_build: The reference genomes for E. coli K12 strain BW2113 and S. coelicolor A3(2) strain M145 were U00096.2 and AL645882, respectively.
Supplementary_files_format_and_content: Separate Bedgraphs were constucted for the forward and reverse strands of each sample.

organism: Streptomyces coelicolor A3(2)

source_name: Batch culture

growth_protocol: Escherichia coli K12 strain BW25113 (Datsenko & Wanner 2000. PNAS 97: 6640) was cultivated in 100 mL of LB broth ( Miller 1972. In: Experiments in molecular genetics. Cold Spring Harbor Laboratory, NY) in a 250 mL Erlenmeyer flask at 37°C with shaking (250 rpm) to an O.D.600nm of 0.6.  S. coelicolor A3(2) strain M145  (Kieser et al. 2000. Practical Streptomyces Genetics. The John Innes Foundation, Norwich.) was incubated in YEME broth (Kieser et al. 2000) at 30°C with shaking until the mycelia became pigmented.

extract_protocol:
RNA was isolated from the pellet of E. coli cells using a well-established, published protocol (Kime et al. 2008. In RNA Turnover in Bacteria, Archaea and Organelles, Vol 447 (ed. LE Maquat, CM Arraiano), pp. 215).  The cell pellet of S. coelicolor was resuspended in Kirby mix (Kieser et al. 2000. Practical Streptomyces Genetics. The John Innes Foundation, Norwich.), 100 µL per 1 O.D.600nm unit, and transferred to Lysing Matrix B tubes containing fine silica beads (MP Biomedical). Tubes were then placed in a high-speed benchtop homogenizer (Fastprep-24, MP Biomedical; set at 6.5 M/s). Cells were lysed by three cycles of homogenisation for 1 min with cooling between each cycle in an ice-water bath for 1 min. The lysates were extracted using an equal volume of acidic phenol: chloroform: isoamyl alcohol (50: 50: 1) and then chloroform: isoamyl alcohol (49: 1). Nucleic acid in the aqueous phase was precipitated by adding NaCl to 150 mM and 2.5 x volumes of 100% [v/v] ethanol, chilling at -20°C for 1 h, and then harvested by centrifugation (13,000 x g for 30 min at 4°C) . The nucleic acid pellet was washed twice with 700 µL of 70% [v/v] ethanol, air dried for 5 min and resuspended in RNase-free water. Contaminating DNA was removed from both E. coli and S. coelicolor samples by incubating with DNase as described by the vendor (Ambion) and extracted with phenol: chloroform as described above. The concentration and integrity of RNA samples were determined using a NanoDrop™ 1000 spectrophotometer (Thermo Fisher Scientific) and agarose gel electrophoresis (Kime et al. 2008), respectively. Samples were enriched for mRNA using MICROBExpress™-Bacteria beads, as described by the manufacturer (Ambion)
Library construction was done by Dr. Lira Mamanova of the Wellcome Trust Sanger Institute using a published protocol (Mamanovaet al. 2010. Nat Methods 7: 130)
FRT-seq: amplification-free, strand-specific transcriptome sequencing

treatment_protocol: At the appropriate phase of growth a one eighth volume of stop solution (95% [v/v] ethanol; 5% [v/v] phenol) was added to a culture to inhibit cell metabolism (Kime et al. 2008. In RNA Turnover in Bacteria, Archaea and Organelles, Vol 447 (ed. LE Maquat, CM Arraiano), pp. 215), and the cells were harvested by centrifugation. When necessary, cell pellets were stored frozen at -80ºC.

value: M145; wild-type reference strain

tag: growth stage

value: Exit from exponential growth


title: E. coli

description:
Enriched for mRNA
EC_gRNA-seq_data_Read_1.fastq; 5'end
EC_gRNA-seq_data_Read_2.fastq; 3'end

organism: Escherichia coli K-12

tag: strain

value: BW25113; wild-type reference strain

value: Mid-exponential growth


title: Nanog ChIP-Seq 1

status: Public on Apr 18 2013

city: Los Angeles

first: April

last: Kartikasari

address: 900 Veteran Avenue

department: Medical-Endocrinology

institute: UCLA

data_processing:
Basecalls performed using CASAVA version 1.4
ChIP-seq reads were aligned to the mm9 genome assembly using Bowtie
Only reads that aligned to a unique position in the genome with no more than two sequence mismatches were used for further analysis.
The number of unique reads was calculated in bins across the genome.
Bins containing statistically significant Chip-seq enrichment were identified by comparison to a Poissonian background model.
Genome_build: mm9
Supplementary_files_format_and_content: wig file contains the p-values of the bins across the genome

source_name: Mouse embryonic stem cells

growth_protocol: mouse ES cells were routinely passaged on irradiated mouse embryonic fibroblasts using medium containing Knock-out Dulbecco’s modified Eagle’s medium (KO-DMEM; GIBCO), supplemented with 10% heat-inactivated fetal bovine serum (FBS, Hyclone), 0.055 mM β-mercaptoethanol (GIBCO), 2 mM L-glutamine (GIBCO), 0.1 mM nonessential amino acid (GIBCO), 5000 U/mL penicillin-streptomycin (GIBCO), 15mM HEPES (GIBCO) and 1000 U/mL LIF (Millipore/Chemicon).

extract_protocol:
Lysates were clarified from sonicated nuclei and histone-DNA complexes were isolated with antibody.
Libraries were prepared according to Illumina's instructions accompanying the DNA Sample Kit (Part# 0801-0303). 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 15 cycles and library fragments of ~250 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 protocols.

treatment_protocol: ES cells were allowed to aggregate and differentiate to form embryoid bodies by removing Leukemia inhibitory factor (LIF)  from culture medium

value: Mouse embryonic stem cells

tag: origin

value: Vanderbilt University

tag: antibody

value: Nanog (Santa Cruz)


title: 5fC enriched DNA from Tdg fl/fl mouse EB cells, replicate 1

city: Atlanta

first: Keith

middle: E

last: Szulwach

state: GA

address: 615 Michael Street, Whitehead Suite 301

department: Human Genetics

institute: Emory University

description: processed data file: Tdg.WT.EB.5fC.regions.bed

data_processing:
library strategy: ChIP-Seq type, for Selective chemical labeling and enrichment of 5fC or 5hmC containing DNA fragments
Illumina Basecalling, CASAVA 1.8.2
FASTQ sequences were aligned to NCBIv1/mm9 with Bowtie v0.17.2 retaining unique non-duplicate matches to the genome, with no more than 3 mismatches in the first 30 bases
Enriched regions derived from MACS v1.4, --bw=200 -g 1.87e9, using genotype matched input controls
library strategy: ChIP-Methyl-Seq or ChIP-fCAB-Seq
H3K4me1-ChIP-Methyl-Seq and H3K4me1-ChIP-fCAB-Seq paired-end reads (fastq) were first pre-processed to remove adapter sequences, as well as low quality sequence on both the 3' and 5' ends using Trimmomatic 0.20, with the following parameters: LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36. This was followed by in silico conversion of each C to T (Read 1) and each G to A (Read 2). Pre-processed reads were then aligned to both C to T and G to A converted chromosomes that were computationally derived from NCBI mm9 genomic sequence using Bowtie 0.12.9 (-m  1 -l 30 -n 0 -e 90 -X 550). Reads mapping to both genomes are discarded and non-aligned pairs were re-processed as single-end data with Bowtie 0.12.9 (-m  1 -l 30 -n 0 -e 90. For both paired-end and single alignments, only uniquely mapping reads were retained and PCR duplicates were removed using MarkDuplicates (Picard Tools 1.82). To avoid counting reference positions covered by overlapping paired-end reads, overlapping regions were clipped, keeping the region of the overlap with higher quality. The original computationally converted Cs and Gs were reverted, and for each reference cytosine position the number C reads and T reads were counted using SAMTools mpileup.
Genome_build: mm9
Supplementary_files_format_and_content: BED format, containing regions of enrichment

growth_protocol: mESCs were cultured in feeder-free gelatin-coated plates in Dulbecco’s Modified Eagle Medium (DMEM) (Invitrogen Cat. No. 11995) supplemented with 15% FBS (GIBCO), 2 mM L-glutamine (GIBCO), 0.1 mM 2-mercaptoethanol (Sigma), 1×nonessential amino acids (GIBCO), 1,000 units/ml LIF (Millipore Cat. No. ESG1107), 1×pen/strep (GIBCO), 3 mM CHIR99021 (Stemgent), and 1 mM PD0325901 (Stemgent).

extract_protocol:
25 ng of 5fC-enriched DNA, 5hmC-enriched DNA, or non-enriched sonicated input genomic DNA was end-repaired, adenylated, and ligated to Illumina Genomic DNA Adapters (Genomic DNA adapter oligo mix) according to standard Illumina protocols for ChIP-Seq library construction, maintaining the proper molar ratios of adapter to insert. Adapter-ligated fragments of ~200-350 bp were gel-purified by 2% agarose gel electrophoresis and PCR-amplified for 18 PCR cycles. Libraries were checked for quality and quantified using an Agilent 2100 Bioanalyzer DNA 1000 Chip.
300 ng of H3K4me1 ChIP'd DNA (Abcam, ab8895) was subjected to fCAB treatment and subsequently bisulfite converted in parallel with untreated ChIP’d DNA, followed by end-repair, adenylation and ligation to methylated Illumina TruSeq DNA adapters according to the Illlumina Methyl-Seq Protocol

treatment_protocol: EB differentiation was induced in suspension in 10 cm bacterial dishes in medium without LIF, CHIR99021 and PD0325901. Change half of the medium every day for 5 days before harvesting mEBs by sedimentation

value: Mouse ES cells differentiated to embryoid body (EB)


title: 5hmC enriched DNA from Tdg fl/fl mouse EB cells, replicate 2

description: processed data file: Tdg.WT.EB.5hmC.regions.bed


title: Nanog ChIP-Seq 2

source_name: Mouse embryoid bodies

value: Mouse embryoid bodies


title: Tbx3 ChIP-Seq 2

value: Tbx3 (Santa Cruz)


title: 5hmC enriched DNA from Tdg fl/fl mouse EB cells, replicate 1


title: Tdg -/- H3K4me1 ChIP'd, fCAB-Seq

data_processing:
library strategy: ChIP-Seq type, for Selective chemical labeling and enrichment of 5fC or 5hmC containing DNA fragments
Illumina Basecalling, CASAVA 1.8.2
FASTQ sequences were aligned to NCBIv1/mm9 with Bowtie v0.17.2 retaining unique non-duplicate matches to the genome, with no more than 3 mismatches in the first 30 bases
Enriched regions derived from MACS v1.4, --bw=200 -g 1.87e9, using genotype matched input controls
library strategy: ChIP-Methyl-Seq or ChIP-fCAB-Seq
H3K4me1-ChIP-Methyl-Seq and H3K4me1-ChIP-fCAB-Seq paired-end reads (fastq) were first pre-processed to remove adapter sequences, as well as low quality sequence on both the 3' and 5' ends using Trimmomatic 0.20, with the following parameters: LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36. This was followed by in silico conversion of each C to T (Read 1) and each G to A (Read 2). Pre-processed reads were then aligned to both C to T and G to A converted chromosomes that were computationally derived from NCBI mm9 genomic sequence using Bowtie 0.12.9 (-m  1 -l 30 -n 0 -e 90 -X 550). Reads mapping to both genomes are discarded and non-aligned pairs were re-processed as single-end data with Bowtie 0.12.9 (-m  1 -l 30 -n 0 -e 90. For both paired-end and single alignments, only uniquely mapping reads were retained and PCR duplicates were removed using MarkDuplicates (Picard Tools 1.82). To avoid counting reference positions covered by overlapping paired-end reads, overlapping regions were clipped, keeping the region of the overlap with higher quality. The original computationally converted Cs and Gs were reverted, and for each reference cytosine position the number C reads and T reads were counted using SAMTools mpileup.
Genome_build: mm9
Supplementary_files_format_and_content: NA

source_name: Tdg -/- H3K4me1 ChIP'd, fCAB-Seq

value: Mouse ES cells

value: Anti-Histone H3 (mono methyl K4) antibody

tag: chip antibody vendor

value: Abcam

tag: chip antibody cat. #


title: Tdg -/- H3K4me1 ChIP'd, Methyl-Seq

source_name: Tdg -/- H3K4me1 ChIP'd, Methyl-Seq


title: Tdg fl/fl H3K4me1 ChIP'd, Methyl-Seq

source_name: Tdg fl/fl H3K4me1 ChIP'd, Methyl-Seq


title: A4

city: Lexington

first: Peter

last: Nelson

state: KY

address: 800 S. Limestone

department: Sanders-Brown Center on Aging

institute: University of Kentucky

description: Sample 20

data_processing:
basecall using illumina CASAVA v1.8
The 3' adaptor was removed from raw reads and reads with the length less than 10 were excluded to constitute the mappable reads.
The mappable reads were aligned against human genome and miRbase using Bowtie.
Genome_build: Homo sapiens 37.1; miRbase v17;
Supplementary_files_format_and_content: comparison.xls: raw and normalized number of miRNA reads in each sample;
statistical_analysis.xls: Statistical analysis for changed miRNAs in the various disease groups

source_name: AD VI_cerebral cortex

extract_protocol:
Trizol LS
Illumina Truseq Small RNA Preparation

tag: diagnosis

value: Braak VI Alzheimer

tag: disease group

value: AD VI

value: temporal neocortex gray matter


title: Tdg fl/fl H3K4me1 ChIP'd, fCAB-Seq

source_name: Tdg fl/fl H3K4me1 ChIP'd, fCAB-Seq


title: A1

description: Sample 17


title: A3

description: Sample 19


title: Tdg -/- polyA RNA-Seq replicate 2

description: TDG.KO.FPKM.txt

data_processing:
Illumina Basecalling, CASAVA 1.8.2
FASTQ sequences were aligned to NCBIv1/mm9 with Bowtie v0.17.2 retaining unique non-duplicate matches to the genome, with no more than 3 mismatches in the first 30 bases
Enriched regions derived from MACS v1.4, --bw=200 -g 1.87e9, using genotype matched input controls
RNA-Seq reads were aligned using tophat-1.4.1 and FPKM expression values were extracted using cufflinks-1.3.0 using RefSeq gene models.
Genome_build: mm9
Supplementary_files_format_and_content: BED format, containing regions of enrichment; FPKM expression values in text format

molecule: polyA RNA

source_name: Tdg -/- polyA RNA-Seq

extract_protocol:
25 ng of 5fC-enriched DNA, 5hmC-enriched DNA, or non-enriched sonicated input genomic DNA was end-repaired, adenylated, and ligated to Illumina Genomic DNA Adapters (Genomic DNA adapter oligo mix) according to standard Illumina protocols for ChIP-Seq library construction, maintaining the proper molar ratios of adapter to insert. Adapter-ligated fragments of ~200-350 bp were gel-purified by 2% agarose gel electrophoresis and PCR-amplified for 18 PCR cycles. Libraries were checked for quality and quantified using an Agilent 2100 Bioanalyzer DNA 1000 Chip.
RNA-Seq expression libraries were generated from duplicate samples per genotype using the Illumina TruSeq RNA Sample Preparation Kit v2. Libraries were sequenced using the Illumina HiScan platform. Cluster generation was performed with Illumina TruSeq cluster kit v2-cBot-HS. Single-read 51-bp sequencing was completed with Illumina TruSeq SBS kit v3-HS. Single-read 51-bp sequencing was completed with Illumina TruSeq SBS kit v3-HS.

tag: cell

value: ES cells


title: Tbx3 ChIP-Seq 1


title: Tdg fl/fl polyA RNA-Seq replicate 2

description: TDG.WT.FPKM.txt

source_name: Tdg fl/fl polyA RNA-Seq


title: Tdg fl/fl polyA RNA-Seq replicate 1

description: TDG.WT.FPKM.txt


title: Tdg -/- polyA RNA-Seq replicate 1

description: TDG.KO.FPKM.txt


title: Non-NaBH4 Control enrichment from Tdg fl/fl mouse ES cells b

description: processed data: NA


title: 5fC enriched DNA from Tdg fl/fl mouse EB cells, replicate 2


title: A5

description: Sample 16

source_name: AD V_cerebral cortex

value: Braak V Alzheimer

value: AD V


title: Non-NaBH4 Control enrichment from Tdg fl/fl mouse ES cells a

description: processed data: NA


title: p300 ChIP-Seq from Tdg -/- mouse ES cells, replicate b

value: p300 ChIP-Seq, sc-585 (Santa Cruz)


title: p300 ChIP-Seq from Tdg -/- mouse ES cells, replicate c


title: Non-enriched input genomic DNA from Tdg -/- mouse ES cells

description: processed data: NA


title: B2

description: Sample 14

source_name: DLB_cerebral cortex

value: Dementia with Lewy bodies (DLB)

value: DLB


title: p300 ChIP-Seq from Tdg -/- mouse ES cells, replicate a


title: A2

description: Sample 18


title: 5hmC enriched DNA from Tdg -/- mouse ES cells, replicate 2

description:
Selective chemical labeling and enrichment of 5hmC containing DNA fragments
Tdg.KO.5hmC.regions.bed


title: C1

description: Sample 11

source_name: HS aging_cerebral cortex

value: Bilateral hippocampal sclerosis

value: HS aging


title: B4

description: Sample 12


title: 5hmC enriched DNA from Tdg -/- mouse ES cells, replicate 1


title: 5fC enriched DNA from Tdg -/- mouse ES cells, replicate 2

description:
Selective chemical labeling and enrichment of 5fC containing DNA fragments
Tdg.KO.5fC.regions.bed


title: Small RNAs from adult Dirofilaria immitis_both male and female_whole body

city: Ya'an

first: Guangyou

last: Yang

address: Xinkang Road 46*

department: Department of Parasitology

institute: Sichuan Agricultural University

data_processing:
Firstly, the data is processed by the following steps: (1) Getting rid of low quality reads (The criteria for this is listed in the explanation of meaning of each row in the result table). (2) Getting rid of reads with 5' primer contaminants. (3) Getting rid of reads without 3' primer. (4) Getting rid of reads without the insert tag. (5) Getting rid of reads with poly A. (6) Getting rid of reads shorter than 18nt. (7) Summarize the length distribution of the clean reads. Secondly, map the small RNA tags to the Brugia malayi genome by SOAP to analyze their expression and distribution on the genome. Thirdly, align small RNA tags to the miRNA precursor/mature miRNA of all animals in miRBase17.0. Show the sequence and count of miRNA families(no specific species) which can be found in the samples. Align small RNA tags to repeat associated RNA to find matched tags in the sample. Then annotate the small RNA tags with sequences from Rfam and get rid of matched tags from unannotated tags. Finally, summarize all alignments and annotation before.
Di_unique counts.txt contains the length of the sequence (nt) information along with SEQUENCE, COUNT.

organism: Dirofilaria immitis

source_name: whole body

growth_protocol: Dirofilaria immitis resided in the heart of an adult dog.

extract_protocol: RNA fragments of 18-44 bases long were isolated and purified from 10 μg total RNA using a Novex 15% TBE-Urea gel. The 5’ and 3’ adaptors (Illumina) were added to the ends of fragments. Reverse transcription PCR (RT-PCR) was performed using a RT-PCR kit (Invitrogen). The fragments were purified using a 6% TBE PAGE gel and used for high-throughput sequencing with a Solexa sequencer.

tag: developmental stage

value: adult

tag: Sex

value: both male and female

value: whole body


title: B1

description: Sample 15


title: C2

description: Sample 10

value: Bilateral hippocampal sclerosis and Alzheimer changes


title: C3

description: Sample 9


title: C4

description: Sample 8


title: p300 ChIP-Seq from Tdg fl/fl mouse ES cells, replicate c


title: Non-enriched input genomic DNA from Tdg fl/fl mouse ES cells

description: processed data: NA


title: 5hmC enriched DNA from Tdg fl/fl mouse ES cells, replicate 2

description:
Selective chemical labeling and enrichment of 5hmC containing DNA fragments
Tdg.WT.5hmC.regions.bed


title: 5fC enriched DNA from Tdg -/- mouse ES cells, replicate 1


title: 5hmc-Z

city: Beijing

first: Bao-Fa

last: Sun

address: Da-Tun Road

institute: Beijing Institute of Genomics (BIG) of Chinese Academy of Sciences (CAS)

data_processing:
Pulldown reads were aligned to the hg19 genome assembly using BWA version 0.5.9-r16
Peaks were called using MACS version 1.4.2 with default parameter
Genome_build: hg19
Supplementary_files_format_and_content: MACS-generated peaks file.

source_name: sperm cells

extract_protocol:
Purified genomic DNA should be sonicated into short fragments (200~300 bp) by Covaris DNA shearing with microTUBEs according to manufacturer’s instructions. 5hmC DNA capture was performed using Hydroxymethyl Collect kit. Briefly, 5-hmC labeling reaction were performed in 75-ul solution containing 50mM HEPES buffer(PH7.9), 250 mM MgCl2, 100 µM UDP-6-N3-Glu,and 80 U βGT. The reactions were incubated for 2h at 37℃.After the reaction, The DNA substrates were purified and exchange buffer in H20 via Bio-rad micro bio spin according to the manufacturer’s instructions. The click chemistry was performed with the addition of 150µM biotin into the DNA solution and incubated 2h at 37℃.The DNA samples were then purified by Invitrogen Dynabeads MyOneTM Streptavidin C1 according to manufacturer’s instructions.
5-hmC enriched genomic DNA libraries were generated following the Illumina protocol for “Preparing Samples for CHIP sequencing of DNA”.we used 1ng 5-hmC enriched DNA to initiate the protocol. DNA fragments of 150~300bp were gel purified after the adapter ligation step. PCR-amplified DNA libraries were quantified on an Agilent 2100 Bio analyzer and using quantity PCR.We performed 100bp single end sequencing on Illumina Hiseq2000 to get 5-hmC-enriched DNA fragment sequence.

value: sperm

tag: cell phenotype

value: normal sperm cell of fertile men

tag: chemical pulldown target


title: D1

description:
Sample 7
PSP is a subset of what can be called FTLD-MAPT or FTLD-tau

source_name: PSP_cerebral cortex

value: Progressive supranuclear palsy

value: PSP

tag: tissue


title: F2

description: Sample 6

source_name: FTLD-TDP_cerebral cortex

value: FTLD (TDP43 positive)

value: FTLD-TDP

value: temporal neocortex gray matter


title: p300 ChIP-Seq from Tdg fl/fl mouse ES cells, replicate a

value: ES cells


title: B3

description: Sample 13


title: 5hmc-Y

tag: cell type

value: sperm cell of globozoospermia patient


title: Sbp1 Replicate 2

city: Boulder

first: Sarah

middle: Fitzgerald

last: Mitchell

state: CO

address: 3415 Colorado Ave

institute: University of Colorado

data_processing:
Library strategy: CLIP-Seq
raw sequences were processed using FASTX-toolkit (http://hannonlab.cshl.edu/fastx_toolkit/)
Sequences were mapped to the S288C yeast genome (SGD) using Novoalign (http://www.novocraft.com/main/page.php?s=novoalign) or Bowtie 2 (http://bowtie-bio.sourceforge.net/bowtie2/index.shtml)
Profiles of the transcriptome were generated with 50 nt extensions at both 5' and 3' ends of each mRNA. These extensions were trimmed in cases where they overlapped with annotated transcripts. Please see manuscript for full details.
Further details about analysis are available in Mitchell et al. NSMB 2013
Genome_build: S288C genome from SGD
Supplementary_files_format_and_content: .txt files, number of sequence reads across all nucleotides for all yeast mRNA

organism: Saccharomyces cerevisiae

source_name: Yeast

growth_protocol: Strains were grown in YEPD at 30° C to mid-log.

extract_protocol:
Cell lysates were partially clarified at 4,000 rpm and digested with RNAse A (Sigma). TAP-tagged proteins were pulled-down with Rabbit IgG conjugated Dynabeads (Invitrogen) and washed in lysis buffer with 1M urea to reduce non-specific binding.
The RNA for the control is extracted using glass bead lysis and phenol/chloroform extraction. mRNA is then selected by oligo(dT) pull-down. For the rest of the samples (Dhh1, Lsm1, Pat1 and Sbp1), RNA is covalently cross-linked to protein using UV-light and pulled-down with the protein via a TAP-tag purification protocol. The protein is then removed from the RNA using proteinase K. This procedure has been done according to standard CLIP-seq protocols and is described in further detail (buffers etc.) in Mitchell et al. 2013.
RNA fragments were isolated, decapped and cloned into RNA libraries as in Ule et al. Methods 2005 and Wang et al. Methods 2009

treatment_protocol: Cells were re-suspended in PBS for 10 minutes to induce P-bodies. Stressed cells were UV-crosslinked at 0.8–1.2 J/cm2.

tag: genotype/variation

value: C-terminal TAP tag

tag: rna subset

value: RNA cross-linked to Sbp1


title: p300 ChIP-Seq from Tdg fl/fl mouse ES cells, replicate b

value: p300 ChIP-Seq, sc-585 (Santa Cruz)


title: 5hmC enriched DNA from Tdg fl/fl mouse ES cells, replicate 1


title: E2

description: Sample 5


title: Pat1 Replicate 1

value: RNA cross-linked to Pat1


title: Sbp1 Replicate 1


title: 5hmc-J

value: abnormal sperm cell of fertile men


title: F1

description: Sample 4


title: Pat1 Replicate 2


title: shTDG-input-rep2

city: Boston

first: Li

last: Shen

address: 200 Longwood Ave

institute: HHMI/Boston Children's Hospital/Harvard Medical School

data_processing:
Basecalls were performed using OLB-1.8.0
Sequenced reads were de-multiplexed, trimmed and mapped to mm9 whole genome using bowtie v0.12.7
Peak calling were performed using MACS 1.4.2
Genome_build: mm9
Supplementary_files_format_and_content: wiggle files (IgG signal subtracted; scores represent tag numbers per 10 million reads)

source_name: Tdg knockdown mouse ES cells

growth_protocol: E14 mESCs are cultured on the 0.1% gelatin coated plates in the DMEM medium (Sigma) supplemented with 100U/ml penicillin/streptomycin, 15% fetal bovine serum (Sigma), 1x nonessential amino acid, 1x sodium pyruvate, 1x GlutaMax, 1x beta-mercaptoethanol (Invitrogen) and 1000 units/ml leukemia inhibitory factor (ESGRO, EMD Millipore).

extract_protocol: Libraries were prepared using NEBNext DNA Library Prep Master Mix Set for Illumina (NEB) following manufacture's instructions.

treatment_protocol: Around 1x10^5 ES cells/well in 24-well plates are infected with lentiviral vectors at 10 MOI. 48 hours after transduction, puromycin (2ug/ml) is added to the medium for selecting transduced cells.

tag: chip antibody

value: none (input)

value: embryonic stem

value: Tdg knockdown


title: Wild Type

value: Untagged

tag: strain

value: RNase A treated polyA RNA


title: Lsm1 Replicate 2

value: RNA cross-linked to Lsm1


title: 5fC enriched DNA from Tdg fl/fl mouse ES cells, replicate 2

description:
Selective chemical labeling and enrichment of 5fC containing DNA fragments
Tdg.WT.5fC.regions.bed


title: Lsm1 Replicate 1


title: shTDG-IgG-rep2

value: IgG


title: 5fC enriched DNA from Tdg fl/fl mouse ES cells, replicate 1


title: G2

description: Sample 2

source_name: control_cerebral cortex

value: Normal (no amyloid)

value: control


title: E1

description: Sample 3

source_name: FTLD_cerebral cortex

value: FTLD (TDP43 negative)

value: FTLD

value: temporal neocortex gray matter


title: Dhh1 Replicate 2

value: C-terminal TAP tag

value: RNA cross-linked to Dhh1


title: G1

description: Sample 1


title: shTDG-5mC-rep2


title: Dhh1 Replicate 1


title: shCONT-input-rep2

source_name: Control mouse ES cells


title: shTDG-5caC-rep2


title: shCONT-IgG-rep2


title: shTDG-5hmC-rep2


title: shCONT-5mC-rep2


title: shTDG-5fC-rep2


title: shCONT-5hmC-rep2


title: HIRA ChIP

city: Glasgow

middle: D.

last: Adams

address: Switchback Rd, Bearsden

institute: University of Glasgow, Beatson Institute for Cancer Research

description: ChIP-seq of histone chaperone HIRA

data_processing:
Basecalls performed using CASAVA 1.6
Sequenced reads were mapped to hg18 whole genome using bowtie v0.12.7 with parameter -p 8 -m 1 --best and remaining default settings
Aligned reads normalised by library size, extended to 150bp and converted to bigWig format
USeq 7.1.2 was used to perform peak finding with the following steps. BED files converted to Useq Point Format (Tag2Point). Satellite Repeat regions were removed from the dataset (FilterPointData). ScanSeqs used to identify enriched windows (-w 200 -p 150 -f). Enriched Regions Extracted (EnrichedRegionMaker) with 5% FDR and 2 fold change (-i 2,4 -s 13,1)
Genome_build: hg18
Supplementary_files_format_and_content: bed files represent aligned reads (one line per read) in UCSC bed format.  bigWig files represent pileups of the reads at each base.  bigWig files were normalised by library size and represent the number of reads at a given genomic location per million mapped reads and generated using UCSC wigToBigWig.  peak files were generated with USeq and thresholded at 5% FDR and 2 fold change cutoffs

source_name: HIRA ChIP

growth_protocol: HeLa cells were cultured in DMEM supplemented with 10% FBS, 2 mM L-Glutamine, 50 U Pen-Strep and incubated at 37°C in a humidified 5% CO2 atmosphere. Every 3-4 days the cells were passaged to maintain 40-90% confluence level

extract_protocol:
Cross-linked cells were solubilized by ultrasound treatment (Bioruptor Diagenode) to generate DNA fragmentation in 150-300 bp range. Protein-DNA complexes were isolated using corresponding antibody pre-immobilized on Dynabeads (Invitrogen).
Libraries were prepared according to Illumina's instructions accompanying the DNA Sample Kit (Part# 0801-0303). 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 library fragments of ~250 bp (insert plus adaptor and PCR primer sequences) were band isolated from an agarose gel and PCR-amplified using Illumina primers. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Genome Analyzer following the manufacturer's protocols.

treatment_protocol: Adherent cells were washed with PBS and cross-linked using sequential treatment with EGS and formaldehyde. Cells were incubated in 1.5mM EGS (Sigma, E3257), PBS for 45 min at room temperature, then formaldehyde (Sigma, 252549) was added to the media to 1% final concentration and cells were incubated for 15 more minutes. Cross-linking was quenched by adding 1/10 of volume of 1.25 M glycine (125mM final) and incubating for 5 min at room temperature. Media was aspirated and cells were rinsed twice with PBS, scraped into ice-cold PBS supplemented with protease inhibitors (Sigma, P8340) and collected by centrifugation (5 min at 200g, 4°C). Cell pellets were snap frozen in dry ice with ethanol and kept at -70°C before use.

value: HeLa

value: equimolar mixture of Ms mAbs WC15, WC19, WC117, WC 119 (Hall et al., 2001)


title: input DNA

description: corresponding input DNA

source_name: input DNA


title: shCONT-5fC-rep2


title: UBN1 ChIP

description: ChIP-seq of histone chaperone UBN1

source_name: UBN1 ChIP

value: Rb pAb UBN1-1358 (Banumathy et al., 2009)


title: HA.H3.3.input

description: corresponding input DNA

data_processing:
Basecalls performed using CASAVA 1.8
Sequenced reads were mapped to hg18 whole genome using bowtie v0.12.7 with parameter -p 8 -m 1 --best and remaining default settings
Aligned reads normalised by library size, extended to 150bp and converted to bigWig format
USeq 8.1.6 was used to perform peak finding with the following steps. BED files converted to Useq Point Format (Tag2Point). Satellite Repeat regions were removed from the dataset (FilterPointData). ScanSeqs used to identify enriched windows (-w 200 -p 150 -f). Enriched Regions Extracted (EnrichedRegionMaker) with 5% FDR and 2 fold change (-i 2,4 -s 13,1)
Genome_build: hg18
Supplementary_files_format_and_content: bed files represent aligned reads (one line per read) in UCSC bed format.  bigWig files represent pileups of the reads at each base.  bigWig files were normalised by library size and represent the number of reads at a given genomic location per million mapped reads and generated using UCSC wigToBigWig.  peak files were generated with USeq and thresholded at 5% FDR and 2 fold change cutoffs

extract_protocol:
Cross-linked cells were solubilized by ultrasound treatment (Bioruptor Diagenode) to generate DNA fragmentation in 150-300 bp range. Protein-DNA complexes were isolated using corresponding antibody pre-immobilized on Dynabeads (Invitrogen).
Libraries were prepared according to NEB's instructions accompanying the NEBNext® ChIP-Seq Library Prep Master Mix Set for Illumina®(E6240S). Briefly, DNA was end-repaired using a combination of T4 DNA 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 library fragments of ~250 bp (insert plus adaptor and PCR primer sequences) were isolated using Agencourt AMPure XP beads (Beckman Coulter) and PCR-amplified using Illumina primers. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Genome Analyzer following the manufacturer's protocols.

treatment_protocol: Cells were harvested by trypsinization and precipitated by centrifugation. Cells pellets were resuspended in 1% formaldehyde in PBS and incubated for 15 more minutes. Cross-linking was quenched by adding 1/10 of volume of 1.25 M glycine (125mM final) and incubating for 5 min at room temperature. Media was aspirated and cells were rinsed twice with PBS, scraped into ice-cold PBS supplemented with protease inhibitors (Sigma, P8340) and collected by centrifugation (5 min at 200g, 4°C). Cell pellets were snap frozen in dry ice with ethanol and kept at -70°C before use.


title: HA.H3.3 ChIP

description: ChIP-seq of newly synthesized H3.3 histone variant (HA-tagged at N-term)

source_name: HA.H3.3 ChIP

value: mouse mAb to HA tag (Millipore, 05-904)


title: HA.H3.3.faire

description: corresponding FAIRE-seq

data_processing:
Library strategy: FAIRE-Seq
Basecalls performed using CASAVA 1.8
Sequenced reads were mapped to hg18 whole genome using bowtie v0.12.7 with parameter -p 8 -m 1 --best and remaining default settings
Aligned reads normalised by library size, extended to 150bp and converted to bigWig format
USeq 8.1.6 was used to perform peak finding with the following steps. BED files converted to Useq Point Format (Tag2Point). Satellite Repeat regions were removed from the dataset (FilterPointData). ScanSeqs used to identify enriched windows (-w 200 -p 150 -f). Enriched Regions Extracted (EnrichedRegionMaker) with 5% FDR and 2 fold change (-i 2,4 -s 13,1)
Genome_build: hg18
Supplementary_files_format_and_content: bed files represent aligned reads (one line per read) in UCSC bed format.  bigWig files represent pileups of the reads at each base.  bigWig files were normalised by library size and represent the number of reads at a given genomic location per million mapped reads and generated using UCSC wigToBigWig.  peak files were generated with USeq and thresholded at 5% FDR and 2 fold change cutoffs


title: shTDG-input

data_processing:
Basecalls were performed using CASAVA version v.1.8.2
Sequenced reads were de-multiplexed, trimmed and mapped to mm9 whole genome using bowtie v0.12.7
Peak calling were performed using MACS 1.4.2
Genome_build: mm9
Supplementary_files_format_and_content: wiggle files (IgG signal subtracted; scores represent tag numbers per 10 million reads)

value: none (input)

value: embryonic stem


title: shTDG-5mC

value: Tdg knockdown


title: shTDG-5hmC


title: shTDG-5fC


title: Tb927.8.6650_Input

city: Montreal

first: Hamed

last: Najafabadi

state: QC

address: 740 Dr. Penfield Avenue, Room 7202

country: Canada

institute: McGill University

description: Stable transgenic procyclic form cells expressing tagged Tb927.8.6650 were induced by tetracycline and harvested after 48h. Total RNA was extracted from whole cell lysate.

data_processing:
Raw reads were mapped to annotated coding sequences (CDS) from Trypanosoma brucei TREU927 (TriTrypDB v2.4) using Bowtie v0.12.7, with the following parameters: "--best --strata -k 2 -m 1 -n 2". Mapped read counts were normalized to the length of each CDS to obtain RPKM. RPKM values were normalized in each sample by dividing by the median of the sample. Ratio of TAP vs. input for each transcript was calculated by adding a pseudocount of 0.1 to each normalized RPKM value prior to division.
Genome_build: TbruceiTreu927AnnotatedCDS_TriTrypDB-2.4;
Genome_build: ASM244v1 (Trypanosoma brucei strain 927/4 GUTat10.1)
Supplementary_files_format_and_content: Files are tab-delimited. The first column represents the gene IDs (according to TriTrypDB). The second column represents median-normalized RPKM values.

organism: Trypanosoma brucei

source_name: Tb927.8.6650 expression_tet-induced_48h_whole cell lysate

growth_protocol: Transgenic PF T. brucei cells harbouring tetracycline-inducible constructs were grown in SDM-79 medium in 27 °C. Cells were seeded at 1.3E+6 cells/ml and harvested 48h after tet-induction at a density of 3.0E+7 cells/ml.

extract_protocol: Tandem affinity purification was performed as described before (Panigrahi et al., http://www.ncbi.nlm.nih.gov/pubmed/12649499). Total RNA or RNA associated with Tb927.8.6650 was extracted using TRIzol Reagent (Invitrogen) as per manufacturers’ intsructions. RNA quality was examined using Agilent 2100 Bioanalyzer.

treatment_protocol: The cells were treated with 100ng/ml tetracycline, for a duration of 48h.

tag: cell line

value: expressing tetracycline-inducible TAP-tagged Tb927.8.6650

tag: life stage

value: Procyclic Form

tag: molecule subtype

value: total RNA


title: shTDG-5caC


title: shCONT-input


title: shCONT-IgG

value: IgG


title: shCONT-5mC

value: control


title: shCONT-5fC


title: shCONT-5caC


title: shCONT-5hmC


title: C. dubliniensis_YPS-3

status: Public on Apr 17 2013

city: Stuttgart

first: Christian

last: Grumaz

address: Nobelstr. 12

department: MBT

institute: Fraunhofer IGB

data_processing:
Basecalls were performed using standard software supplied by Illumina for HiSeq2000 sequencing.
Reads generated with HiSeq2000 were mapped with TOPHAT (version 1.4.1) using default settings.
Genome_build: C. albicans: Assembly21 at CGD (latest update 2010-06-15, http://www.candidagenome.org/download/sequence/Assembly21); C. dubliniensis: Assembly CD36 at NCBI (latest update 2010-04-09, ftp://ftp.ncbi.nih.gov/genomes/Fungi/Candida_dubliniensis_CD36)
Supplementary_files_format_and_content: TOPHAT output files were converted with BEDtools (bam-to-bed) into *.bed file format. Non-junction reads were generated from *.bed files into *.bedGraph files by a customized python-script. Scores represent coverage of raw mapped reads

organism: Candida dubliniensis

source_name: blastospore cells

growth_protocol: C. albicans_YPD-1, C. albicans_YPD-2, C. albicans_YPD-3 samples were grown in YPD at 30°C for 4 hours. C. albicans_YPS-3 and C. dubliniensis_YPS-3 samples were grown in YPD supplemented with 10% fetal calf serum at 37°C for 4 hours. C. dubliniensis_WS-3 sample was grown in water supplemented with 10% fetal calf serum at 37°C for 4 hours.

extract_protocol:
After 4h of incubation under the corresponding conditions, C. dubliniensis and C. albicans cells were harvested by centrifugation and immediately frozen with liquid nitrogen. Disruption was carried out using a Mixer Mill MM 200 (RETSCH) with a shaking frequency of 30/s. The resulting powder was resuspended in lysis buffer RLT (QIAGEN, Hilden, Germany) supplemented with 0.01% v/v of ß-mercaptoethanol. The extraction of total RNA was performed according to QIAGEN’s Mechanical Disruption Protocol for the isolation of total RNA from yeast using the RNeasy Midi Kit. After precipitation of the RNA by addition of 0.1 volume of 3M NaAc pH 5.3 and 2.5 volume of 100% EtOH, the concentration and integrity of total RNA was analyzed using the Agilent 2100 Bioanalyzer using the RNA Nano kit.
The cDNA libraries for HiSeq2000-sequencing were performed according Illumina’s TruSeq RNA Sample Prep Kit v2 protocol. The sequencing run was performed on single-read flow cell with 50bp reads and resulted in approximately 10-15 mio. reads per sample.

tag: morphology

value: blastospores

tag: growth condition

value: Cells were grown in YPD supplemented with 10% fetal calf serum at 37°C for 4 hours


title: seedling-wt

city: Berkeley

first: Toshiro

last: Nishimura

address: 211 Koshland Hall

department: Plant and Microbial Biology

institute: University of California at Berkeley

data_processing:
For Bisulfite Sequencing, we used Perl scripts to convert all the Cs in the reads (and in the scaffolds) to Ts, and aligned the converted reads to the converted  reference scaffold, allowing up to two mismatches per read. 76 base reads were divided into the first 45 and the last 31 bases, 100 base reads were divided in half and 50 base reads were processed as single reads.  Each half of the read was aligned independently using bowtie, allowing up to two mismatches. The coordinates of the two halves were subsequently correlated; the second half was discarded if it did not match the first.
Single_C: We used Perl scripts to recover the original sequence of each mapped read and, for each C (on either strand), count the number of times it was sequenced as a C or a T.
50bp_window: We used a Perl script to calculate fractional methylation (#C/(#C+#T)) within a 50 bp sliding window for each sequence context (CG, CHG, CHH).
RNA-seq: Illumina reads from cDNA libraries were aligned to genomic DNA. The file lists the log2 of the number of reads (first base) matching each 50-bp window, normalized to the total number of aligned reads in each library.
Mnase-seq: We aligned 36-base Illumina paired ends reads to genomic DNA. To measure nucleosome occupancy, we counted the number of sequenced molecules corresponding to each nucleotide of the genome. To reduce the size of the file we collapsed all adjacent positions with equal scores to one window.
Genome_build: TAIR8
Supplementary_files_format_and_content: All files are in GFF format. Files contain fractional methylation data either for individual cytosines (single-c) or in 50 bp windows.

organism: Arabidopsis thaliana

source_name: AT seedling 10 days continous light Columbia 0

growth_protocol: Arabidopsis plants were grown according to one of the following methods: 1) For shoot tissues, seeds were planted on 1x Murashige and Skoog Media with micronutrients and 1.5% Sucrose (Caisson Laboratories) and grown under 16h light/ 8h dark for 14 or 21 days in a growth chamber.  2) For roots, seeds were grown in Gamborg's B-5 liquid Media with 1.5% Sucrose (Caisson Laboratories) and grown shaking under 16h light/ 8h dark for 21 days.

extract_protocol: For bisulfite sequencing (BS-seq) About 500 ng of genomic DNA was fragmented by sonication, end repaired and ligated to custom-synthesized methylated adapters (Eurofins MWG Operon) according to the manufacturer’s (Illumina) instructions for gDNA library construction. Adaptor-ligated libraries were subjected to two successive treatments of sodium bisulfite conversion using the EpiTect Bisulfite kit (Qiagen) as outlined in the manufacturer’s instructions. The bisulfite-converted libraries were then PCR amplified using the following conditions: 2.5 U of ExTaq DNA polymerase (Takara Bio), 5 μl of 10X Extaq reaction buffer, 25 μM dNTPs, 1 μl Primer 1.1, 1 μl Primer 2.1 (50 μl final). PCR reactions were carried out as follows: 95 ̊C 3 min, then 12-14 cycles of 95 ̊C 30 sec, 65 ̊C 30 sec and 72 ̊C 60 sec. The enriched libraries were either gel-purified (~300 bp band) or purified with solid phase reversible immobilization (SPRI) method using AM-Pure beads (Beckman Coulter) prior to quantification with a Bioanalyzer (Agilent). For RNA -Sequencing, approximately 30 ug total RNA was isolated from 21 days roots using the RNEasy Plant Extraction Kit (Qiagen) with the optional on-column DNAse treatment.  mRNA was purified from total RNA by two treatments of poly-A enrichment using the Oligotex kit (Qiagen #72022), followed by a rRNA removal step using the RiboMinus Plant Kit for RNA-sequencing (Invitrogen #A1083702). Precipitated mRNA samples were eluted with 9 μl of RNase free water and fragmented with 1 μl of 10X fragmentation buffer (Ambion, #AM8740) at 70oC. Reactions were stopped after 5 minutes by adding 1 μl Stop buffer and RNA was purified by ethanol precipitation. cDNA was synthesized from 100-300 ng of mRNA using Superscript III reverse transcriptase (Invitrogen #18080-051). Double-stranded DNA was synthesized as described (3). DNA was cleaned with a QIAquick PCR spin column (Qiagen, #28106), sequencing adapters were ligated according to the Illumina protocol and library was amplified by 18 cycles of PCR using Phusion DNA polymerase (NEB, #F-530). Bands around 300 bp were gel-purified and cloned for validation. Traditional sequencing confirmed that the libraries were properly constructed, showing high percentage of mRNA over rRNA. The libraries were sequenced at the UC Berkeley Genomic Sequencing Laboratory, generating single ends (SE) 36 base reads. For MNase-sequencing, Arabidopsis roots (1 g) were ground in liquid nitrogen, resuspended in 20 ml of HBM buffer (25mM Tris, pH 7.6, 0.44M Sucrose, 10 mM MgCl2, 2 mM spermine and 0.1% Triton X-100), homogenized, filtered through miracloth, transferred to a 30 ml round bottom glass tube, centrifuged at 2000g (40C) for 10 min and resuspended in 1 ml HBB buffer (25mM Tris, pH 7.6, 0.44M Sucrose, 10 mM MgCl2 and 0.1% Triton X-100). Nuclei were further spun down at 200g, 40C for 2 min and resuspended in 1 ml of TNE buffer (10 mM Tris, pH 8.0, 100 mM NaCl and 1 mM EDTA). MNase digestion was done with 4 ul of 1M CaCl2 and 1 ul of diluted (1/20) MNase (200ul/ml; Sigma #N-3755) per 100 ul of pellet nuclei. Nuclei were then divided to several tubes and digestion was stopped at 45 sec intervals with 10mM EDTA. Digested nuclei were spun down at maximum speed for 5 min at 40C, and released soluble nucleosomes were collected from the supernatant. Following RNase A and proteinase K digestion, DNA was purified using phenol/chloroform. Purified DNA samples were run on a 2% agarose gel, digested samples with most enriched intact mononucleosomes were chosen and bands corresponding to ~150 bp were cut and purified with a Gel Purification kit (Qiagen). Illumina libraries were constructed and sequenced at the UC Berkeley Genomic Sequencing Laboratory, generating paired ends (PE) 36 base reads. For MNase sequencing, we aligned 36 bases Illumina paired reads to the TAIR8 Arabidopsis genomic scaffold, allowing up to two mismatches per read. We subsequently used a Perl script to insure that paired reads mapped to opposite strands within 300 bp of each other. To measure nucleosome occupancy, we summed the aligned reads (all bases between both paired reads got a score of 1) for every single nucleotide throughout the genome.

tag: genetic background

value: seedling

tag: treatment

value: 10 days continous light

tag: genotype

value: WT


title: shCONT-5caC-rep2


title: seedling-h1

source_name: AT seedling 10 days continous light h1


title: C. dubliniensis_WS-3

source_name: hyphal cells

value: hyphae

value: Cells were grown in water supplemented with 10% fetal calf serum at 37°C for 4 hours


title: seedling-h1ddm1-rep2

source_name: AT seedling 10 days continous light h1ddm1-rep2


title: seedling-h1ddm1-rep1

source_name: AT seedling 10 days continous light h1ddm1-rep1


title: Tb927.8.6650_TAP

description: Stable transgenic procyclic form cells expressing tagged Tb927.8.6650 were induced by tetracycline and harvested after 48h. Tb927.8.6650 was pulled down by tandem affinity purification, and co-purifying RNA was extracted.

source_name: Tb927.8.6650 expression_tet-induced_48h_tandem affinity purification

value: RNA co-purified with Tb927.8.6650


title: shTDG-IgG


title: C. albicans_YPS-3

organism: Candida albicans


title: seedling-h1.2ddm1

source_name: AT seedling 10 days continous light h1.2ddm1


title: C. albicans_YPD-3

value: Cells were grown in YPD at 30°C for 4 hours


title: C. albicans_YPD-2


title: seedling-ddm1

source_name: AT seedling 10 days continous light ddm1


title: seedling-h1.1ddm1

source_name: AT seedling 10 days continous light h1.1ddm1


title: C. albicans_YPD-1


title: cmt2-6

data_processing:
Bisulfite-Seq: We used Perl scripts to convert all the Cs in the reads (and in the scaffolds) to Ts, and aligned the converted reads to the converted reference scaffold, allowing up to two mismatches per read. 76 base reads were divided into the first 45 and the last 31 bases, 100 base reads were divided in half and 50 base reads were processed as single reads. Each half of the read was aligned independently using bowtie, allowing up to two mismatches. The coordinates of the two halves were subsequently correlated; the second half was discarded if it did not match the first.
Single_C: We used Perl scripts to recover the original sequence of each mapped read and, for each C (on either strand), count the number of times it was sequenced as a C or a T.
50bp_window: We used a Perl script to calculate fractional methylation (#C/(#C+#T)) within a 50 bp sliding window for each sequence context (CG, CHG, CHH).
RNA-seq: Illumina reads from cDNA libraries were aligned to genomic DNA. The file lists the log2 of the number of reads (first base) matching each 50-bp window, normalized to the total number of aligned reads in each library.
Mnase-seq: We aligned 36-base Illumina paired ends reads to genomic DNA. To measure nucleosome occupancy, we counted the number of sequenced molecules corresponding to each nucleotide of the genome. To reduce the size of the file we collapsed all adjacent positions with equal scores to one window.
Genome_build: TAIR8
Supplementary_files_format_and_content: All files are in GFF format. Files contain fractional methylation data either for individual cytosines (single-c) or in 50 bp windows.

source_name: AT shoots 2 weeks Columbia-0

growth_protocol: Arabidopsis plants were grown according to one of the following methods: 1) For shoot tissues, seeds were planted on 1x Murashige and Skoog Media with micronutrients and 1.5% Sucrose (Caisson Laboratories) and grown under 16h light/ 8h dark for 14 or 21 days in a growth chamber. 2) For roots, seeds were grown in Gamborg's B-5 liquid Media with 1.5% Sucrose (Caisson Laboratories) and grown shaking under 16h light/ 8h dark for 21 days.

extract_protocol:
For bisulfite sequencing (Bisulfite-Seq) About 500 ng of genomic DNA was fragmented by sonication, end repaired and ligated to custom-synthesized methylated adapters (Eurofins MWG Operon) according to the manufacturer’s (Illumina) instructions for gDNA library construction. Adaptor-ligated libraries were subjected to two successive treatments of sodium bisulfite conversion using the EpiTect Bisulfite kit (Qiagen) as outlined in the manufacturer’s instructions. The bisulfite-converted libraries were then PCR amplified using the following conditions: 2.5 U of ExTaq DNA polymerase (Takara Bio), 5 μl of 10X Extaq reaction buffer, 25 μM dNTPs, 1 μl Primer 1.1, 1 μl Primer 2.1 (50 μl final). PCR reactions were carried out as follows: 95 ̊C 3 min, then 12-14 cycles of 95 ̊C 30 sec, 65 ̊C 30 sec and 72 ̊C 60 sec. The enriched libraries were either gel-purified (~300 bp band) or purified with solid phase reversible immobilization (SPRI) method using AM-Pure beads (Beckman Coulter) prior to quantification with a Bioanalyzer (Agilent). For RNA -Sequencing, approximately 30 ug total RNA was isolated from 21 days roots using the RNEasy Plant Extraction Kit (Qiagen) with the optional on-column DNAse treatment. mRNA was purified from total RNA by two treatments of poly-A enrichment using the Oligotex kit (Qiagen #72022), followed by a rRNA removal step using the RiboMinus Plant Kit for RNA-sequencing (Invitrogen #A1083702). Precipitated mRNA samples were eluted with 9 μl of RNase free water and fragmented with 1 μl of 10X fragmentation buffer (Ambion, #AM8740) at 70oC. Reactions were stopped after 5 minutes by adding 1 μl Stop buffer and RNA was purified by ethanol precipitation. cDNA was synthesized from 100-300 ng of mRNA using Superscript III reverse transcriptase (Invitrogen #18080-051). Double-stranded DNA was synthesized as described (3). DNA was cleaned with a QIAquick PCR spin column (Qiagen, #28106), sequencing adapters were ligated according to the Illumina protocol and library was amplified by 18 cycles of PCR using Phusion DNA polymerase (NEB, #F-530). Bands around 300 bp were gel-purified and cloned for validation. Traditional sequencing confirmed that the libraries were properly constructed, showing high percentage of mRNA over rRNA. The libraries were sequenced at the UC Berkeley Genomic Sequencing Laboratory, generating single ends (SE) 36 base reads. For MNase-sequencing, Arabidopsis roots (1 g) were ground in liquid nitrogen, resuspended in 20 ml of HBM buffer (25mM Tris, pH 7.6, 0.44M Sucrose, 10 mM MgCl2, 2 mM spermine and 0.1% Triton X-100), homogenized, filtered through miracloth, transferred to a 30 ml round bottom glass tube, centrifuged at 2000g (40C) for 10 min and resuspended in 1 ml HBB buffer (25mM Tris, pH 7.6, 0.44M Sucrose, 10 mM MgCl2 and 0.1% Triton X-100). Nuclei were further spun down at 200g, 40C for 2 min and resuspended in 1 ml of TNE buffer (10 mM Tris, pH 8.0, 100 mM NaCl and 1 mM EDTA). MNase digestion was done with 4 ul of 1M CaCl2 and 1 ul of diluted (1/20) MNase (200ul/ml; Sigma #N-3755) per 100 ul of pellet nuclei. Nuclei were then divided to several tubes and digestion was stopped at 45 sec intervals with 10mM EDTA. Digested nuclei were spun down at maximum speed for 5 min at 40C, and released soluble nucleosomes were collected from the supernatant. Following RNase A and proteinase K digestion, DNA was purified using phenol/chloroform. Purified DNA samples were run on a 2% agarose gel, digested samples with most enriched intact mononucleosomes were chosen and bands corresponding to ~150 bp were cut and purified with a Gel Purification kit (Qiagen).
Illumina libraries were constructed and sequenced at the UC Berkeley Genomic Sequencing Laboratory, generating paired ends (PE) 36 base reads. For MNase sequencing, we aligned 36 bases Illumina paired reads to the TAIR8 Arabidopsis genomic scaffold, allowing up to two mismatches per read. We subsequently used a Perl script to insure that paired reads mapped to opposite strands within 300 bp of each other. To measure nucleosome occupancy, we summed the aligned reads (all bases between both paired reads got a score of 1) for every single nucleotide throughout the genome.

tag: ecotype

tag: developmental stage

value: shoots 2 weeks

tag: function

value: methylase


title: cmt2-4