title: WT1B


title: BAF155_ChIPSeq

status: Public on Jan 10 2011

type: SRA

city: Palo Alto

first: Ghia

last: Euskirchen

state: CA

address: 1501 S. California Ave.

department: Genetics

country: USA

institute: Stanford University

library_source: genomic

data_processing: Raw data from the Illumina Genome Analyzer and Illumina Genome Analyzer II were analyzed with Illumina’s Firecrest, Bustard and GERALD modules for image analysis, basecalling and run metrics, respectively. Replicates were combined for each sample. All reads were aligned against human genome build hg18. ChIP samples were scored against normal IgG (GEO Sample GSM352186) using PeakSeq (see "PeakSeq: Systematic Scoring of ChIP-Seq Experiments Relative to Controls by Rozowsky et al. 2009 Nature Biotechnology). Signal files were created using a 200 bp sliding window for all samples. The PeakSeq output was further filtered using tag count, p-value, excess and enrichment to yield a more stringent set of regions for each factor.

molecule: genomic DNA

organism: Homo sapiens

source_name: cervix; adenocarcinoma; epithelial

growth_protocol: HeLa S3 cells were grown in Minimum Essential Medium supplemented with glutamine, 10% FBS, and antibiotics (penicillin-streptomycin) at 37C in 5% CO2, to a density of 6 x10^5 cells/ml.

extract_protocol: HeLa S3 cells were fixed with 1% formaldehyde at room temperature for 10 min and the fixation was terminated by the addition of glycine to a final concentration of 125 mM. The fixed cells were washed in cold 1x Dulbecco’s PBS (Invitrogen) and swelled on ice in 10 ml hypotonic lysis buffer (20 mM Hepes, pH 7.9, 10 mM KCl, 1 mM EDTA, pH 8.0, 10% glycerol, 1 mM DTT, 0.5 mM PMSF, and protease inhibitors). Cell lysates were homogenized with 30 strokes in a Dounce homogenizer. Nuclear pellets were collected and lysed in 10 ml of 1x RIPA buffer per 3 x10^8 cells (1x RIPA buffer: 10 mM Tris-Cl, pH 8.0, 140 mM NaCl, 1% Triton X-100, 0.1% SDS, 1% deoxycholic acid, 0.5 mM PMSF, 1 mM DTT, and protease inhibitors). Chromatin was sheared with a Branson 250 Sonifier (power setting 2, 100% duty cycle for 7x 30 s intervals) to an average size of less than 500 bp as verified on a 2% agarose gel. Lysates were then clarified by centrifugation for 15 min at 20,000 xg at 4°C. Chromatin immunoprecipitations from 1x10^8 cells were conducted as previously described (Euskirchen et al. 2007). The IgG ChIP DNA samples were run though Qiagen MinElute PCR columns, eluted with 15 μl of Qiagen buffer EB and size-selected (100-350 bp) on 2% agarose E-gels (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 (3' to 5' 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.

tag: cell line

value: HeLa S3 cells (ATCC/National Cell Culture Center)

tag: antibody

value: anti-BAF155 (H-76), Santa Cruz Biotechnology, sc-10756


title: K562-TPA-GATA1

city: Rechovot

first: Ram

last: Jaschek

address: P.O Box 26

country: Israel

institute: Weizmann Institute of Science

description:
Chromatin IP against GATA1
instrument model: Illumina Genome Analyzer IIx.

data_processing: CASAVA_1.6 -  K562-TPA-Gata1.txt,K562-TPA-Gata12.txt  GAPipeline-1.4.0 -  K562-TPA-Fos2.txt, K562-TPA-FosB2.txt,  K562-TPA-Fos.txt,  K562-TPA-FosB.txt,   K562-TPA-K27.txt,  CMK-  GAPipeline-1.3.2 -  K562-TPA-NIS2.txt, K562-TPA-Runx12.txt, K562-NIS2.txt, K562-Runx12.txt, K562-NIS.txt, K562-Runx1.txt     GAPipeline-1.0 - CMK-NIS.txt, CMK-Runx1.txt, K562-TPA-NIS.txt,  K562-TPA-Runx1.txt

source_name: K562 Cells, 24 hours following induction with TPA

growth_protocol: Culture in RPMI

extract_protocol: ChIP samples were prepared using 5*10 7 cells per condition. Chromatin was prepared as described in Ainbinder et. Al Mol Cell Biol. 2002.  ChIP libraries were created following illumina sample preparation protocol from 10ng of DNA. Following end-repair and A-base addition, primers were ligated and samples were size selected on a 2% agarose gel. Fragments of 200bp were excised and purified using Qiagen gel-extraction kit with gel melting at room temperature. Enrichment was carried out using 18 cycles of amplification. Library yield was quantified using Qubit (Invitrogen) while final fragment sizes were determined using Agilent BioAnalyzer. The ChIP-seq libraries were run on an Illumina GAIIx (K562-TPA-FOS, K562-TPA-FOSB, K562-TPA-GATA1) / GAII (All other tracks) at the Department of Biological Services at WIS, using illumina cluster generation kits and sequencing reagents.

treatment_protocol: cross-linked chromatin from ~108 K562 cells, before or 24h after treatment with TPA (40nM) or from ~108 cells CMK or 107 mature FL derived megakaryocytes, was prepared and fragmented to an average size of ~200bp by 40 cycles of sonications (30sec each) in 15ml tubes using the Bioruprtor UCD-200 sonicator (Diagenode, US). For immunoprecipitation, the following antibodies were added to 12ml of diluted, fragmented chromatin: 30ul of home-made anti-RUNX12 raised against the protein C-terminal fragment; anti-monomethyl-Histone H3(Lys4) and anti trimethyl-Histone H3(Lys27) (Millipore, US); anti C-FOS (Santa Cruz) and anti GATA1 (Abcam, US). Rabbit pre-immune serum was used as control. DNA was purified using QIAquick spin columns (QIAGEN, US).     Antibodies description:  NIS – non immune serum was taken from our rabbits   RUNX1- our home made antibody, described in Aziz-Aloya RB, Levanon D, Karn H, et al. Expression of AML1-d, a short human AML1 isoform, in embryonic stem cells suppresses in vivo tumor growth and differentiation. Cell Death Differ. 1998;5(9):765-773.  GATA1 – abcam, rabbit polyclonal – ChIP grade (ab11963) - 886538  FOS – Santa cruz, rabbit polyclonal – c-fos (4):sc-52, lot - #L1609  FosB: Cell Signaling, Rabbit monoclonal. FosB (5G4) lot:2  Ref: 12/2009   H3K4me1 – Milipore, rabbit polyclonal – Cat. #07-436 , Lot. #DAM1400139  H3K4me3 - Milipore, rabbit polyclonal – Cat. #17-622, Lot. #DAM1571267

tag: treatment group

value: 24 hours following induction with TPA

tag: chip antibody


title: K562_TPA-FOS

description:
Chromatin IP against FOS
instrument model: Illumina Genome Analyzer IIx.

value: FOS


title: BAF170_ChIPSeq

value: anti-BAF170 (H-116), Santa Cruz Biotechnology, sc-10757


title: Ini1_ChIPSeq

value: anti-Ini1 (C-20), Santa Cruz Biotechnology, sc-16189


title: Brg1_ChIPSeq

value: anti-Brg1 (G-7), Santa Cruz Biotechnology, sc-17796


title: Normal IgG ChIP-Seq

city: HangZhou

first: Biaoyang

last: Lin

address: 866 Yu Hang Tang Road Zi Jin Gang Campus Zhejiang University.

department: ZCNI

country: China

institute: ZheJiang University

description: Chromatin IP against IgG

data_processing:
Alignment: Sequence reads were obtained and mapped to the human (March, 2006) genomes using the Illumina Genome Analyzer Pipeline. All reads mapping with two or fewer mismatches were retained and read starts were summed in sliding windows of 400 bp to create summary windows.
Peaks: Peak detection was performed with the Model-based Analysis of ChIP-Seq (MACS) algorithm (http://liulab.dfci.harvard.edu/MACS/)

source_name: Normal IgG ChIP

growth_protocol: LN229 cells were obtained from the American Type Culture Collection  and maintained in DMEM with 10% fetal bovine serum.

extract_protocol: LN229 cells were used for chromatin immunoprecipition (ChIP) assay, carried out according to the manufacturer’s instructions. Antibodies used for ChIP included SOX2 and IgG. For ChIP, SOX2 antibody was tested for its specificity and specific band was found. ChIP assay was performed using the ChIP kit according to the manufacturer's protocol. Briefly, cells were cross-linked by adding fresh formaldehyde to cell culture medium to a final concentration of 1%. Fixation was monitored at 37 °C for 10 min. The fixed cells were re-suspended in the lysis buffer. Nuclei were collected by centrifugation at 2000 X g, and resuspended in the nuclei lysis buffer. Samples were sonicated on ice to the length of 200–500 base pairs. 5 μg antibody and 50 μl Dynal protein G beads were incubated for 2 hours at 4 °C. Sonicated chromatin were incubated   with the protein G- antibody complex overnight at 4 °C. Precipitated immunocomplex was treated with proteinase K for 2 hours at 65 °C, and DNA was purified Qiagen Qiaquick PCR purification kit. ChIP DNA end repairing, adaptor ligation, and amplification were performed as described earlier.Fragments of about 200 bp) were isolated from agarose gel and used for sequencing using the Illumina 2 G genetic analyzer.

value: LN229 glioma cancer cells

tag: chip seq antibody

value: Normal IgG antibody


title: Sox2 ChIP-Seq

description: Chromatin IP against Sox2

source_name: Sox2 ChIP

value: Sox2 antibody (Abcam cat#ab59776)


title: colon_polyadenylated_RNA-Seq

status: Public on Jan 06 2011

city: Kiel

first: Robert

last: Haesler

address: Schittenhelmstr. 12

department: IKMB / 1st Dept. of Medicine

country: Germany

institute: University Clinic of Schleswig Holstein

description: RNA-Seq against polyadenylated RNA from colon

library_source: transcriptomic

data_processing: Sequence reads were obtained using the Applied Biosystems Modular Analysis Pipeline (MOAP). Data is unfiltered and includes all identified beads.

molecule: total RNA

organism: Mus musculus

growth_protocol: small intestine and colon from 9-10 old mice were prepared and frozen in liquid nitrogen.

extract_protocol: sample specific

tag: strain

tag: tissue

value: Colon

tag: extract protocol

value: SMART cDNA synthesis and SOLiD V2 fragment library construction

tag: library selection

value: polyadenylated RNA


title: small_intestine_polyadenylated_RNA-Seq

description: RNA-Seq against polyadenylated RNA from small intestine

value: Small Intestine


title: small-intestine_ribosomal_RNA_depleted_RNA-Seq

description: RNA-Seq against ribosomal RNA depleted RNA from small intestine

value: SOLiD V2 Whole Transcriptome Analysis Kit

value: ribosomal RNA depleted


title: colon_ribosomal_RNA_depleted_RNA-Seq

description: RNA-Seq against ribosomal RNA depleted RNA from colon


title: IgG ChIP-Seq

city: Smithville

first: Yue

last: Lu

state: TX

address: 1808 Park Road 1C

department: Epigenetics and Molecular Carcinogenesis

institute: MD Anderson Cancer Center

description: Chromatin IP against preimmun serum

data_processing:
alignment: Sequence reads were obtained and mapped to the hg18 using the Illumina Genome Analyzer Pipeline. All reads mapping with two or fewer mismatches were retained.
wig: each unique read was extended 150bp to its 3’ end. Then the number of overlapped reads was determined at each genome position and averaged over 10bp windows

source_name: MCF7 cells

growth_protocol: MCF7 cells were grown in DMEM medium supplemented with 10% FBS.

extract_protocol: Libraries were prepared using Illumina's ChIP-seq  DNA Sample Prep Kit (IP-102-1001).

tag: cell type

value: human breast cancer cell

value: Ctrl IgG (preimmun serum)


title: Heat shock complete digestion, rep1

status: Public on Jan 24 2011

city: Houston

first: Yuanxin

last: Xi

address: 1400 Pressler St

department: Bioinformatics and Computational Biology

institute: The University of Texas MD Anderson Cancer Center

description:
HS.complete_rep1
DNA fragment size selection ~150 bp.

data_processing: Alignment: Sequence reads were obtained and mapped to the yeast (October, 2003; sacCer1) genome using the Illumina Genome Analyzer Pipeline. All reads mapping with two or fewer mismatches were retained.

organism: Saccharomyces cerevisiae

source_name: yeast culture, heat shock, complete digestion

growth_protocol: 3ml pre-culture of Saccharomyces cerevisiae strain JKM139 in YE media was diluted to 500ml of YE media. For the optimal growth samples, the culture was grown to OD600= 0.7-0.8 at 28oC. For the heat-stressed samples, an ensuing step of 36oC incubation (water bath) for 20 minutes was applied.

extract_protocol: The cell pellet was subjected to zymolase treatment and MNase digestion. After MNase digestion, supernatant was collected by centrifugation and used as affinity purification material. DNA was extracted and subjected to electrophoresis. Mono-nucleosomal DNA bands (~150bp) were recovered and subjected to Illumina massive sequencing.

treatment_protocol: For crosslinking chromatin prior to MNase digestion, cells were incubated with 1% formaldehyde for 15min and quenched with 0.125M glycine prior to cell harvesting.

tag: mnase digestion time

value: 25 min

tag: growth temperature


title: Drosophila_larvae_EHMT-KO_H3K9me2_ChIP-Seq

status: Public on Jan 05 2011

city: Nijmegen

first: Hendrik

last: Marks

address: Geert Grooteplein 26/28

department: Molecular Biology

country: Netherlands

institute: Radboud University Nijmegen, RIMLS

description: Chromatin IP against H3K9me2

data_processing: Reads (36 bp) were aligned to the fly (Drosophila Melanogaster) reference genome. Sequences were aligned to the human BDGP R5/dm3 reference genome using the Illumina Analysis Pipeline allowing one mismatch. Unless specified otherwise, only the tags uniquely aligning to the genome were considered for further analysis. The 36 bp sequence reads were directionally extended to 300 bp, corresponding to the length of the original fragments used for sequencing. The output data were converted to Browser Extensible Data (BED) files for downstream analysis and Wiggle (WIG) files for viewing the data in the UCSC Genome Browser. All ChIP-Seq sequence analyses were conducted based on the Apr. 2006 Drosophila melanogaster draft assembly (BDGP Release 5) with Release 5.12 annotations (Oct. 2008)  accessed from the UCSC (assembly April 2006) Genome Browser.

organism: Drosophila melanogaster

source_name: 3rd instart Drosophila larvae

growth_protocol: Larvae were cultured on standard medium (cornmeal/sugar/agar) at 25 degrees, 45-60% humidity and 12 hour light/dark cycle

extract_protocol: Third instar larvae were homogenized in PBS followed by crosslinking with 1% formaldehyde. Crosslinked chromatin was fragmented by sonicating four times for eight minutes (high power, 30 seconds on/off) with a Bioruptor (Diagenode). Chromatin immunoprecipitation was performed with anti-H3K9me2 antibodies and Prot A/G beads (Santa Cruz) were used to capture antibody bound chromatin.

treatment_protocol: In vivo experiment of wildtype vs mutant (fly EHMT)

tag: h3k9me2 antibody

value: 07-441 Upstate antibody

tag: genotype

value: EHMT-KO


title: Drosophila_larvae_wt_H3K9me2_ChIP-Seq

description: Chromatin IP against H3K9me2

value: wild type


title: ERα sequencing in E2 (estradiol) treated ECC1 cells

city: Singapore

first: Cheng Wei

last: Chang

address: Genome Institute of Singapore Genome #02-01 60 Biopolis Street

department: Computational and Mathematical Biology 1

country: Singapore

institute: GIS

description: ERα ChIP sequencing, after E2 treatment

data_processing:
Sequence tags (26 bp reads) were obtained from Solexa sequencing of the ChIP and control samples (input DNA).  Image analysis was performed by Firecrest and base calling was performed using Bustard.  Quality assessment from Illumina’s Genome Analyzer indicated that the sequencing reads were of high quality.  The reads were aligned to the March 2006 assembly of the human genome (hg18) using ELAND.  Approximately 68~72% of the reads were mapped uniquely to the genome.   Reads that mapped to multiple locations in the genome were discarded.   Furthermore in case the same tag was sequenced repeatedly, only one copy of the tag was retained, as repetitive tags usually arise from biases during ChIP-DNA amplification and sequencing library preparation which add noise to the data. Based on the average size of the sequenced ChIP DNA fragments, the tags were extended 200 bp 3’ from the tag mapping coordinates to mimic the actual fragments.  Thus a profile of the ChIP fragments over the whole genome assembly was obtained.    File format: chromosome
Peaks: High confidence ERBS were identified with peak finding algorithm described in supplementary methods with FDR of 0.1% (De Santa et al., 2009). These high confidence peaks were used for all subsequent analyses in this report. (De Santa et al, 2009) (Reference:De Santa F., Narang V., Yap Z.H., et al. (2009) Jmjd3 contributes to the control of gene expression in LPS-activated macrophages. EMBO J.  28: 3341-52). The identified peaks were filtered. First, an estimated peak intensity threshold based on a random distribution of tags over the genome was used to remove random low-intensity peaks. The FDR for a library was determined by a Monte Carlo simulation, in which extended to 3’ direction 200 bp fragments randomly extracted from the genome were used to estimate the numbers of random peaks with different intensity values. The number of random fragments was equal to the sequencing depth of each library under analysis. The minimum intensity that satisfies the 0.001 criterion is selected as the lower cut-off for calling confident peaks.

source_name: ECC1 breast cancer cells

growth_protocol: Human mammary cancer cell line MCF-7 and uterine cancer cell line Ishikawa were obtained from European Collection of Cell Culture (ECACC, Porton Down, Salisbury, SP4 0JG, UK). Human mammary cancer cell line T47D was obtained from the American Type Culture Collection (ATCC, Manassas, VA), uterine cancer cell line ECC1.  MCF-7, ECC1 and Ishikawa cells were cultured in Dulbecco’s modified Eagle’s medium(DMEM, Invitrogen) supplemented with 5% fetal bovine serum(FBS, Invitrogen), penicillin-streptomycin(Invitrogen), and gentamycin (Invitrogen) in a 37 °C incubator at 5% CO2. T47D cells were maintained in RPM 1640 medium (Invitrogen) with same additives as the other cell lines.

extract_protocol: The cells were then cross-linked with 1% (v/v) formaldehyde for 10 mins at room temperature and stopped with 125 mM glycine for 5 min. The crude nuclei were isolated  with Triton X-100 lysis buffer ( 0.25% Triton X-100, 10 mM EDTA, 10 mM Tris-HCl (pH 8.1),  100 mM NaCl, 1x Protease Inhibitor (Roche) added freshly).  The cross-linked cells were washed with phosphate-buffered saline, resuspended in SDS lysis buffer (1% SDS (Bio-Rad), 5 mM EDTA, 50 mM Tris-HCl (pH 8.1), 1x protease inhibitor), and sonicated for 10 mins in a Biorupter (Diagenode) to generate DNA fragments with size about 500bp. The chromatin was then diluted with buffer  (1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl (pH 8.1), 150 mM NaCl, 1x protease inhibitor), precleared with 5mM BSA before immunoprecitating with ER antibody (Santa Cruz,sc-543) and rec-protein A-sephaose 4B (Invitrogen) overnight. The sephaose beads were washed with washing buffer and eluted with elution buffer (1% SDS and 0.1M NaHCO3), and then purified with QIA quick PCR purification kit (QIAGEN). For ChIP-Seq library construction, we used the same procedure as mentioned above to generate chip DNA and then processed the samples according to manufacturer’s instructions (ChIP-Seq DNA Sample Prep Kit, Illumina). Briefly, ChIP DNA was blunted and ligated to sequencing adaptors. After PCR amplification, the fragments with size between 150-300bp were purified and used for sequencing on the Illumina Genome analyzer.

treatment_protocol: For ChIP assay, the details are as described previously (Pan et al 2008). The reagents were purchased from Sigma Aldrich unless otherwise specified. Briefly, cells were cultured for 3 days in phenol red-free DMEM/F12 (or RPM1640 for T47D) (Invitrogen) containing 5% charcoal-stripped FBS before 100nM E2 treatment (or mock treatment with ethanol) for 45 mins.

value: ECC1 breast cancer cells

tag: fraction

value: ChIP DNA

tag: antibody manufacturer

value: Santa Cruz

tag: antibody catalog number

value: ERα

tag: agent


title: Control input DNA of MCF-7

description: genomic DNA sequencing

source_name: MCF-7 breast cancer cells

tag: cell line

value: MCF-7 breast cancer cells

value: input DNA


title: Control input DNA of T47D

description: genomic DNA sequencing

source_name: T47D breast cancer cells

value: T47D breast cancer cells


title: Control input DNA of ECC1

description: genomic DNA sequencing


title: ERα sequencing in ETOH (ethanol) treated ECC1 cells

description: ERα ChIP sequencing, after ETOH (ethanol) treatment

tag: antibody

value: ETOH (ethanol)


title: ERα sequencing in E2 (estradiol) treated T47D cells


title: ERα sequencing in E2 (estradiol) treated Ishikawa cells

description: ERα ChIP sequencing, after E2 treatment

source_name: Ishikawa breast cancer cells

value: Ishikawa breast cancer cells


title: ERα sequencing in ETOH (ethanol) treated Ishikawa cells

description: ERα ChIP sequencing, after E2 treatment


title: Wild type partial digestion, rep1

description:
WT.partial_rep1
DNA fragment size selection ~150 bp.

source_name: yeast culture, partial digestion

value: 5 min


title: TDRD3 ChIP-Seq

description: Chromatin IP against TDRD3

data_processing:
alignment: Sequence reads were obtained and mapped to the hg18 using the Illumina Genome Analyzer Pipeline. All reads mapping with two or fewer mismatches were retained.
wig: each unique read was extended 150bp to its 3’ end. Then the number of overlapped reads was determined at each genome position and averaged over 10bp windows
peak: Peak detection was performed with the Model-based Analysis of ChIP-Seq (MACS) algorithm (http://liulab.dfci.harvard.edu/MACS/).Pvalue cutoff is set as 1e-10.

tag: chip antibody

value: TX516 (polyclonal anti TDRD3)


title: Lin- Control IgG ChIP-Seq

status: Public on Jan 04 2011

city: RTP

first: Raja

last: Jothi

state: NC

address: 111 TW Alexander Drive; A314

department: National Institute of Environmental Health Sciences

institute: National Institutes of Health

description: Chromatin IP against IgG

data_processing: Sequence reads were mapped to the mouse genome (mm8) using the Solexa Analysis Pipeline. The mapped reads for Ldb1, Scl/Tal1, and GATA2 samples were processed using the SISSRs peak-finder (Jothi et al, Nucl Acids Res 2008; PMID: 18684996) with control IgG reads as background to identify genome-wide binding sites

source_name: Bone marrow hematopoietic progenitor cells enriched by depletion of Lin+ cells

extract_protocol: Freshly isolated hematopoietic progenitor cells were crosslinked with formaldehyde and sonicated to generate chromatin fragments, which were immunoprecipitated with Ldb1, Scl/Tal1, Gata2 or control IgG antibody. The DNA fragments were end-repaired and processed for sequencing on an Illumina Genome Analyzer using standard protocols.

treatment_protocol: Bone marrow hematopoietic progenitor cells were enriched by depletion of Lin+ cells using magnetic beads

value: Lin- bone marrow hematopoietic progenitor cells

value: IgG


title: RNA Pol II sequencing in non-treated (DMSO as vehicle) MCF-7 cells

first: Yuriy

middle: L

last: Orlov

address: 60 Biopolis Street

department: Computational Systems Biology

institute: Genome Institute of Singapore

description: Chromatin IP against RNA Pol II

data_processing:
Alignment: Sequence reads were obtained and mapped to the human (hg18) genome using the Illumina Genome Analyzer II Pipeline. ELAND program provided with the 1G analyzer software package and in-house (Genome Institute of Singapore) computational tools for mapping the sequence tags to the reference genome hg18 and clustering short sequences. In order to avoid potential PCR amplification bias, tags that shared the same mapping location on the same strand were removed. The uniquely-mapped reads with at most 2-mismatches were kept for further processing.
Peaks: The oriented 25-36 bp DNA reads (depending on library) were extended to 200 bp regions to count clusters of overlapping sequences. The enrichment peaks for corresponding libraries were identified using ChIP-seq peak calling algorithm as previously described in (Chen et al, 2008; De Santa et al, 2009) (References: Chen X., Xu H., Yuan P.,  et al. (2008) Integration of external signaling pathways with the core transcriptional network in embryonic stem cells. Cell 133:1106-1117; De Santa F., Narang V., Yap Z.H., et al. (2009) Jmjd3 contributes to the control of gene expression in LPS-activated macrophages. EMBO J.  28: 3341-52). The identified peaks were filtered in three steps. First, an estimated peak intensity threshold based on a random distribution of tags over the genome was used to remove random low-intensity peaks. The FDR for a library was determined by a Monte Carlo simulation, in which extended to 3’ direction 200 bp fragments randomly extracted from the genome were used to estimate the numbers of random peaks with different intensity values. The number of random fragments was equal to the sequencing depth of each library under analysis. The minimum intensity that satisfies the 0.001 criterion is selected as the lower cut-off for calling confident peaks. Then, we further filtered the peaks based on the 5 fold-change of peak intensity against local count of sequence reads from an input DNA control library. The peak lists provided were obtained using subsampling of the raw data, and might be not complete for larger ChIP-seq libraries. For comparison between libraries of different sequencing depth, ChIP-seq libraries were downsampled to 7 million tags each (using random tag extraction). For estimation of ER binding sites enrichment by chromatin modifications we used published data on copy number variations in MCF-7 (Shadeo and Lam, 2006: Shadeo A., Lam W.L. (2006) Comprehensive copy number profiles of breast cancer cell model genomes. Breast Cancer Res 8: R9 and GEO accession number GSE3106).

growth_protocol: MCF-7 cancer cells were grown to 70-80% confluence in D-MEM/F-12 (Invitrogen/Gibco)  supplemented with 10% FBS (Hyclone) in 150 mm dishes. In preparation for the 17 beta-estradiol (“estrogen/E2”, Sigma) treatment, cells were then split into 1:3 into serum starving medium [phenol red-free D-MEM/F-12 medium (Invitrogen/Gibco) supplemented with 5% charcoal-dextran stripped FBS (Hyclone)] and cultured for 72 hours to 70-80% confluence.

extract_protocol: All DNA samples were processed as per the Illumina Solexa ChIP-seq sample processing methods.  10 ng of ChIP DNA was end polished with T4 DNA polymerase and kinase. An A base was added to the polished DNA fragments followed by the Qiaquick column clean up. Solexa adaptors were ligated to the ChIP DNA fragments and enriched by 15 cycles of PCR amplification. 200-300 bp size fractions were selectively isolated from the 1% agarose gel and eluted by Qiagen gel extraction kit. The extracted DNA was quantified by picogreen assay and subjected to Solexa sequencing according to the manufacturer’s instruction.

treatment_protocol: Hormone-depleted cells were treated with E2 to a final concentration of 10 nM for 3 h before the ChIP/FAIRE procedure.  The control cells were treated with an equal volume and concentration of vehicle, DMSO (Merck), for 3 h. For a FAIRE experiment, we routinely used approximately 1 x 108 cells from 5-6, 150 mm diameter cell culture plates.  For isolating the open chromatin regions in the genome we performed Formaldehyde assisted isolation of regulatory elements (FAIRE) as described previously (Giresi et al, 2007).

value: breast cancer cells

value: DMSO

value: RNA Pol II

value: Abcam


title: ERα sequencing in ETOH (ethanol) treated MCF-7 cells


title: Lin- Ldb1 ChIP-Seq

description: Chromatin IP against Ldb1


title: Lin- Scl/Tal1 ChIP-Seq

description: Chromatin IP against Scl/Tal1


title: ERα sequencing in E2 (estradiol) treated MCF-7 cells

description: ERα ChIP sequencing, after E2 treatment


title: Lin- Gata2 ChIP-Seq

description: Chromatin IP against Gata2


title: SET1D_NET-seq

status: Public on Jan 19 2011

city: San Francisco

first: L. Stirling

middle: Stirling

last: Churchman

address: 1700 Fourth St. Byers Hall - Room 404

institute: University of California-San Francisco

description: IP against 3xFLAG affinity label

data_processing: Image data obtained by the Illumina Genome Analyzer 2 was analyzed using the GAPipeline to extract raw sequences. Matrix and phasing parameters were estimated from a φX control lane. Raw sequences 40 bases long were composed of the cDNA of the fragmented RNA sequence. For RNA fragments smaller than 40 bases, the sequence is followed by part of the 5’ Illumina linker sequence which was removed in silico. Alignments to the yeast genome was performed by the alignment program, Bowtie 0.12.0 (http://bowtie-bio.sourceforge.net/). Bowtie settings were chosen so that three mismatches were allowed and alignments were required to be unique. The shortest sequenced fragments were approximately 18 nucleotides due to the RNA size selection step after ligation and random fragmentation. Alignments were first performed against tRNA and rRNA sequences to remove them. The remaining sequences were aligned against a recent version of the yeast genome downloaded from the Saccharomyces Genome Database (SGD, http://www.yeastgenome.org/) on October 11, 2009

source_name: yeast BY4741, mid-exponential phase

growth_protocol: Yeast strains were grown in YEPD at 30°C with shaking from an initial OD of 0.1 to mid-log phase with an OD of 0.6-0.8.

extract_protocol: Frozen cells were cryogenically pulverized for lysis and nascent RNA was isolated via an immunoprecipitation at 4°C of Rpb3-3xFLAG and purified using the miRNeasy kit (Qiagen, 217004). An preadenylated RNA linker was ligated onto the 3’ end of the immunoprecipitated RNA. Fragmentation of the ligated samples by alkaline hydrolysis followed by a size selection after electrophoresis on a polyacrylamide gel allowed for the final DNA library to contain inserts of a narrow range. cDNA synthesis by reverse transcription (SuperScriptsIII (Invitrogen)) was performed with a long 5’ phosohorylated primer split by two 18 carbon spacers. Circularization of the RT product was performed with CircLigase (Epicentre) according to the manufacturer’s directions. The PCR was performed directly on the circularized product resulting in DNA with Illumina cluster generation sequences on each end and a sequencing primer binding site positioned so that sequencing would start at the 3’ end. DNA was purified from a PCR reaction that had not reached saturation (~10 cycles) by band extraction from a polyacrylamide gel that had not reached saturation. DNA was sequenced on the Illumina Genome Analyzer 2 according to the manufacturer’s instructions.

tag: genotype/variation

value: RPB3-3xFLAG::NAT set2D::HIS3

tag: library strategy

value: NET-Seq

tag: molecule

value: nascent RNA

tag: ip

value: IP against 3xFLAG affinity label


title: Control input DNA of Ishikawa

description: genomic DNA sequencing


title: H3K9ac sequencing in estradiol (E2) treated MCF-7 cells

description: Chromatin IP against H3K9ac

value: Upstate Biotechnology Inc.


title: RNA Pol II sequencing in estradiol (E2) treated MCF-7 cells

description: Chromatin IP against RNA Pol II


title: H3K14ac sequencing in estradiol (E2) treated MCF-7 cells

description: Chromatin IP against H3K14ac


title: H3K9ac sequencing in non-treated (DMSO as vehicle) MCF-7 cells

description: Chromatin IP against H3K9ac


title: H3K14ac sequencing in non-treated (DMSO as vehicle) MCF-7 cells

description: Chromatin IP against H3K14ac


title: ERα sequencing in ETOH (ethanol) treated T47D cells

value: ChIP DNA

value: Santa Cruz

value: ERα


title: H3K4me3 sequencing in non-treated (DMSO as vehicle) MCF-7 cells

description: Chromatin IP against H3K4me3


title: H3K4me3 sequencing in estradiol (E2) treated MCF-7 cells

description: Chromatin IP against H3K4me3


title: H3K9me3 sequencing in estradiol (E2) treated MCF-7 cells

description: Chromatin IP against H3K9me3


title: H3K9me3 sequencing in non-treated (DMSO as vehicle) MCF-7 cells

description: Chromatin IP against H3K9me3


title: H3K4me1 sequencing in estradiol (E2) treated MCF-7 cells

description: Chromatin IP against H3K4me1


title: H3K27me3 sequencing in non-treated (DMSO as vehicle) MCF-7 cells

description: Chromatin IP against H3K27me3


title: H3K4me1 sequencing in non-treated (DMSO as vehicle) MCF-7 cells

description: Chromatin IP against H3K4me1


title: FAIRE sequencing in non-treated (DMSO as vehicle) MCF-7 cells

description: FAIRE sequencing


title: H3K27me3 sequencing in estradiol (E2) treated MCF-7 cells

description: Chromatin IP against H3K27me3


title: FAIRE sequencing in estradiol (E2) treated MCF-7 cells

description: FAIRE sequencing, after E2 treatment


title: MEFControl_ChIP-Seq_WCE

status: Public on Jan 03 2011

city: Boston

first: Richard

last: Koche

state: MA

address: 185 Cambridge St.

institute: Broad Institute of Harvard and MIT

description: input DNA

data_processing: Primary sequence data were filtered and aligned to mm8 as described in Mikkelsen et al. Nature 2007, PMID 17603471. The WIG files were similarly created by extending mapped reads 300bp and calculating the overlapping density in 25 bp bins.

source_name: Uninduced MEFs

growth_protocol: Standard 10% Fetal Bovine Serum containing growth medium

extract_protocol: Library construction protocol: Single read - Illumina

treatment_protocol: Either 15% FBS containing medium supplemented with 2 ug/ul Doxycyline or 0.5% FBS containing Serum Starvation medium

tag: cell division cohort

value: MEFControl

value: control

value: secondary, inducible MEFs

tag: strain

value: 129/SvJ x C57/B6 hybrid


title: >3Div_WCE_ChIP-Seq_WCE

description: input DNA

source_name: Doxycycline-induced MEFs

value: Doxycycline

tag: cell type


title: >3Div_ChIP-Seq_H3K36me3

description: Chromatin IP against H3K36me3

tag: antibody vendor

tag: antibody catalog #

tag: antibody lot #


title: Locusta_nerve

city: Chongqing

first: Zhengyi

last: Zhang

address: Shapinba

institute: Chongqing University

data_processing: Transcriptome de novo assembly is carried out with short reads assembling program SOAPdenovo (Li, Zhu et al . 2009). SOAPdenovo firstly combines reads with certain length of overlap to form longer fragments without N, which are called contigs. Then the reads are mapped back to contigs; with paired-end reads it is able to detect contigs from the same transcript as well as the distances between these contigs. Next, SOAPdenovo connects the contigs using N to represent unknown sequences between each two contigs, and then Scaffolds are made. Paired-end reads are used again for gap filling of scaffolds to get sequences with least Ns and cannot be extended on either end. Such sequences are defined as Unigenes.

organism: Locusta migratoria manilensis

source_name: Central nervous system cell

extract_protocol: Beads with Oligo(dT) are used to isolate poly(A) mRNA after total RNA is collected from the tissue. Fragmentation buffer is added for interrupting mRNA to short fragments. Taking these short fragments as templates, random hexamer-primer is used to synthesize the first strand cDNA. The second-strand cDNA is synthesized using buffer, dNTPs, RNaseH and DNA polymerase I, respectively. Short fragments are purified with QiaQuick PCR extraction kit and resolved with EB buffer for end reparation and adding poly(A). After that, the short fragments are connected with sequencing adapters. And, after the agarose gel electrophoresis, the suitable fragments are selected for the PCR amplification as templates. At last, the library could be sequenced using Illumina HiSeq™ 2000.

tag: tissue

value: Brain, thoracic ganglion chain and the ventral nerve cord

value: Central nervous system cell


title: >3Div_ChIP-Seq_H3K4me3

description: Chromatin IP against H3K4me3


title: 1Div_ChIP-Seq_H3K36me3

description: Chromatin IP against H3K36me3


title: >3Div_ChIP-Seq_H3K4me1

description: Chromatin IP against H3K4me1


title: MEFControl_ChIP-Seq_H3K4me1

description: Chromatin IP against H3K4me1


title: 1Div_ChIP-Seq_H3K4me3

description: Chromatin IP against H3K4me3


title: >3Div_ChIP-Seq_H3K27me3

description: Chromatin IP against H3K27me3

value: Millipore


title: 1Div_ChIP-Seq_H3K27me3

description: Chromatin IP against H3K27me3


title: 1Div_ChIP-Seq_H3K4me1

description: Chromatin IP against H3K4me1

value: Abcam


title: MEFControl_ChIP-Seq_H3K36me3

description: Chromatin IP against H3K36me3


title: 2Div_ChIP-Seq_H3K27me3

description: Chromatin IP against H3K27me3


title: 0Div_ChIP-Seq_H3K27me3

description: Chromatin IP against H3K27me3


title: 2Div_ChIP-Seq_H3K4me2

description: Chromatin IP against H3K4me2


title: MEFControl_ChIP-Seq_H3K27me3

description: Chromatin IP against H3K27me3


title: >3Div_ChIP-Seq_H3K4me2

description: Chromatin IP against H3K4me2


title: 1Div_ChIP-Seq_H3K4me2

description: Chromatin IP against H3K4me2


title: >3Div_RRBS

description: Reprogramming factors were induced with doxycycline for 96 hours in MEFs undergoing an average of three or more cell divisions.

data_processing:
Primary sequence data were filtered and aligned to mm9 as described in Meissner et al. Nature 2008, PMID 18600261.
Each *cpgs.txt.gz file contains information in the same format as Meissner et al. Nature 2008, PMID 18600261, here aligned to mm9:
<chromosome>  <CpG position mm9>  <X/Y>
where X is the number of CGs observed and Y the number of TGs.
Also, note that each sample is run on two lanes (high and low molecular weight), resulting in two srf files per sample. However,
the processed data file combines each of these lanes to capture all data for a given sample, hence one processed file per two lanes.

extract_protocol: Libraries were constructed as described in Meissner et al. 2008, PMID 18600261. Bisulfite treated, MspI digested. Size selection, 40-220bp range.

value: doxycycline


title: 1Div_WCE_ChIP-Seq_WCE

description: input DNA


title: 0Div_RRBS

description: Reprogramming factors were induced with doxycycline for 96 hours in MEFs undergoing an average of zero cell divisions.


title: 1Div_RRBS

description: Reprogramming factors were induced with doxycycline for 96 hours in MEFs undergoing an average of one cell division.


title: MEFControl_RRBS


title: MEFControl_ChIP-Seq_H3K4me2

description: Chromatin IP against H3K4me2


title: 0Div_ChIP-Seq_H3K4me2

description: Chromatin IP against H3K4me2


title: MEFControl_ChIP-Seq_H3K4me3

description: Chromatin IP against H3K4me3


title: HL1_siNon_smallRNAseq

city: Berlin

first: Silke

last: Sperling

state: Berlin

address: Lindenberger Weg 80

department: Cardiovascular Genetics

institute: Experimental and Clinical Research Center (ECRC), Charité / MDC for Molecular Medicine

description: Total RNA was isolated using TRIzol reagent.

data_processing: Image analysis and base calling was done using the open source Firecrest and Bustard applications (Solexa Pipeline 1.4.0). For each of these samples, we determined the abundance value for each sequence in the library. The numbers in the VALUE column represent the expression level of the corresponding sequence for the library.

source_name: mouse cardiomyocytes (HL1-cells)

growth_protocol: HL-1 cells were provided by Prof. William C. Claycomb and cultured as described (Claycomb et al. (1998) HL1 cells: a cardiac muscle cell line that contracts and retains phenozypic characteristics of the adult cardiomyocytes. Proc Natl Acad Sci USA 95(6):2979-2984.). The cells were harvested for experiments at their maximum concentration.

extract_protocol: Small RNAs were isolated from total RNA of HL-1 cells and prepared for miRNA sequencing using Illumina Kit FG-102-1009 according to manufacturer’s protocol. Small RNA Libraries were sequenced on the Genome Analyzer following the manufacturer's protocols.

treatment_protocol: For Srf knockdown HL-1 cells were transfected with two different siRNAs. Therefore cells were grown for at least two days without addition of antibiotics to 70-80% confluence. 3 × 105 cells were seeded into 6-well plates with 2ml media resulting in 70-80% confluence after 4h. 9μl of 20μM siRNA was mixed with 270μl of DMEM media and 16μl of Lipofectamin™ 2000 was mixed with 470μl DMEM media. Both mixtures were combined, incubated for 20min at room temperature and added drop wise to the cells. Cells were harvested for total RNA preparation 48h after transfection.

value: cardiomyocytes

tag: sirna

value: Srf si1


title: HL1_siSrf2_smallRNAseq

value: siNon


title: HL1_Srf_ChIPseq

description: Chromatin IP against Srf

data_processing: Image analysis and base calling was done using the open source Firecrest and Bustard applications (Solexa Pipeline 1.4.0). The resulting sequence reads were mapped to the mouse reference genome (NCBI v37, mm9) using the read mapper RazerS. Only unique mapped 36nt reads with at most two mismatches were retained. To identify transcription factor Srf binding sites as well as histone 3 acetylated regions we used the CisGenome software. The final processed peak list contains peak identifier, chromosome number, start and end position.

extract_protocol: Libraries were prepared for sequencing according to Illumina's library preparation instructions. 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.

value: Srf


title: HL1_H3ac_ChIPseq

description: Chromatin IP against H3ac


title: Sin3B MT 5

status: Public on Jan 01 2011

city: New York

first: Brian

last: Dynlacht

state: NY

department: Cancer Institute

institute: NYU Medical Center

description: Chromatin IP against Sin3B

data_processing:
Alignment: Sequence reads were obtained and mapped to the mouse (July, 2007) genomes using the Illumina Genome Analyzer Pipeline. Only prefectly matched single unique reads were retained for further analysis
Peaks: Peak detection was performed with the Qeseq algorithm (Parisi et al., manuscript in preparation)
Parameter settings  Qeseq: sigma 100, tag cutoff 12

source_name: C2C12 myotubes

growth_protocol: C2C12 cells were grown in DMEM supplemented with 10% fetal bovine serum.

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 End-it DNA repair kit (Epicentre). The blunted ends were treated with Klenow exo (3' to 5' exo minus, NEB)  and dATP to yield a protruding 3- 'A' base for ligation (Liga fast, Promega) 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 18 cycles and library fragments of ~350 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: Differentiated cells were obtained by culturing confluent C2C12 cells in DMEM supplemented with 2% horse serum. Differentiated cells were separated from undifferentiated cells using diluted trypsin

tag: origin

value: C2C12 cell line

tag: genetic background

value: myotube

tag: chip antibody manufacturer

value: Santa Cruz Biotechnology

tag: chip antibody catalog #

tag: chip antibody lot #


title: Sin3B MT 3

description: Chromatin IP against Sin3B

tag: chip antibody


title: Sin3B GM 1

description: Chromatin IP against Sin3B

source_name: C2C12 myoblasts

value: myoblast


title: Blattella germanica whole body 6th instar nymph

city: Brisbane

first: Alexandre

middle: Santos

last: Cristino

state: Queensland

address: 79 QBI Building

department: The Queensland Brain Institute

country: Australia

institute: The University of Queensland

data_processing: The adaptors sequences were trimmed and all identical sequences were clustered. Refer to methods of the related manuscript for further details.

organism: Blattella germanica

source_name: whole body

extract_protocol: Freshly ecdysed sixth (last) instar nymphs and adult females of the cockroach B. germanica were obtained from a colony reared in the dark at 30 ± 1°C and 60–70% relative humidity. For the WB-6 samples, the entire animal except the head (to avoid interferences with the eye pigments) and the digestive tube (to avoid contamination with parasites) was used. Stage specific samples of 2-3 individuals were collected for each of the 8 days of the last instar nymph. Then a pool composed by day 0 to 7 aliquots was built in order to cover the entire last instar nymph. For the Ov-A samples, we dissected the ovary pair from adult virgin cockroaches in each day of the first gonadotrophic cycle, which lasts 7 days. The pooling procedure to get an extract covering in this case the whole first gonadotrophic cycle was equivalent to that followed in the WB-6 extract. All dissections and tissue sampling were carried out on carbon dioxide-anaesthetized individuals. RNA isolation from WB-6 and Ov-A samples was carried out with mirVana miRNA Isolated kit (Ambion), which increases the yield of small RNAs. After ligation of 5' and 3' RNA adapters, the first strand was synthesized by reverse transcription and PCR amplified with Illumina primers for 16 cycles.

value: whole body

tag: developmental stage

value: 6th instar nymph


title: Sin3A MT 1

description: Chromatin IP against Sin3A


title: Blattella germanica ovaries adult

source_name: ovaries

value: ovary

value: adult


title: Sin3B MT 4

description: Chromatin IP against Sin3B


title: Sin3B MT 1

description: Chromatin IP against Sin3B


title: Sin3B MT 2

description: Chromatin IP against Sin3B


title: Sin3A GM 2

description: Chromatin IP against Sin3A


title: HL1_siSrf1_smallRNAseq

tag: cell line

value: Srf si2


title: Input MT 2

description: input DNA


title: Input MT 1

description: input DNA


title: Sin3B GM 2

description: Chromatin IP against Sin3B


title: Sin3A GM 1

description: Chromatin IP against Sin3A


title: Input GM

description: input DNA


title: ThioMac-GroSeq-RNA-Seq

city: La Jolla

first: Christopher

last: Benner

state: California

address: 9500 Gilman Dr. MC 0640

department: Medicine

institute: University of California, San Diego (UCSD)

description:
strand-specific RNA-Seq of Nuclear Run-on
Nuclear run-on RNA from ThioMac

data_processing:
Alignment: Reads were truncated to 25 bp (ChIP-Seq) or 32 bp (RNA, GRO-Seq) and aligned to the mouse mm8 genome (NCBI Build 36) using eland. Only reads that mapped to a single, unique position were used for downstream analysis.
Data analysis was performed using HOMER, a software suite for ChIP-Seq analysis and created in part to support this study (http://biowhat.ucsd.edu/homer/). Each ChIP-Seq experiment was normalized to a total of 10^7 uniquely mapped tags by adjusting the number of tags at each position in the genome to the correct fractional amount given the total tags mapped. UCSC Genome Browser bedGraph files were created by calculating ChIP-Fragment pileups given a fragment size of ~150 bp.
bedGraph files were replaced Sept 01, 2010 and have been re-formatted for use with the updated UCSC Genome Browser.

growth_protocol: Peritoneal macrophages were harvested by peritoneal lavage with 10 ml ice-cold PBS 3 days after peritoneal injection of 3 ml thioglycollate broth. Peritoneal cells were washed once with PBS, and seeded in 10% fetal calf serum (FCS)/DMEM containing 100 U/ml penicillin/streptomycin in tissue culture-treated petri dishes overnight. Non-adherent cells were washed off with room temperature PBS.

extract_protocol: For GRO-Seq, twenty million peritoneal macrophages were washed with ice cold PBS. The nuclei was allowed to swell on ice for 5 min in buffer containing 10mMTris-cl pH7.5, 2mM MgCl2 and 3mM CaCl2. Cells were scraped and pelleted for 10 min at 400 g. Supernatant was removed and cells suspended to 500 µl of Swelling buffer + 10 % glycerol + 2U/ml Superase In (Ambion). The cells were vortexed slowly while adding 500 µl of Swelling buffer + 10 % glycerol + 1 % Igepal drop by drop. Nuclei were washed twice with 10 ml of lysis buffer (swelling buffer, 0.5% Igepal, 10% glycerol, 2units/ml SUPERase In) and once with 1 ml of freezing buffer (50mM Tris-CL pH 8.3, 40% glycerol, 5mM MgCl2, 0.1 mM EDTA) before counting. Nuclei were centrifuged for 6 min at 900 gand suspended to a concentration of 5 x 106 nuclei per 100 µl of freezing buffer. Nuclei were mixed with an equal volume of reaction buffer (10mM Tris-Cl pH 8.0, 5mM MgCl2, 1mM DTT, 300mM KCL, 20 units of SUPERase In, 1% sarkosyl, 500uM ATP, GTP, and Br-UTP, 2M CTP) and the reaction was allowed to proceed for 5 min at 30C. The reaction was stopped and RNA isolated using Trizol LS (Invitrogen). RNA was treated with TURBO DNase (Ambion). Base hydrolysis was performed using RNA fragmentation Reagents (Ambion) and the reaction was purified through p-30 RNAse-free spin column (BioRad Hercules, CA, USA). Samples were dephosphorylated with Antarctic phophatase and immunopurified using Anti-deoxyBrU beads (Santa Cruz). Samples were then loaded on a Novex 15% polyacrylamide TBE-urea gel (Invitrogen) and run according to the manufacturer’s instructions. Fragments of 36-55 nucleotides were cut and RNA was eluted from the gel debris and recovered by ethanol precipitation. E. coli Poly(A) Polymerase (NEB) was used to create a priming site for cDNA synthesis. Reverse transcription was carried out using Superscript III reagents (Invitrogen) and oligonucleotide 5’-pGATCGTCGGACTGTAGAACTCTxCAAGCAGAAGACGGCATACGATTTTTTTTTTTTTTTTTTTTVN (p indicates 5’ phosphorylation, x indicates the abasic dSpacer furan and V, N indicates degenerate nucleotides). The RNA was hydrolyzed and cDNA extracted from 10% TBE-Urea Gel (Invitrogen) followed by ethanol precipitation. cDNA was circularized using CircLigase (Epicentre) and linearized using APE1 (New England Biolabs). The relinearized product band was recovered from Novex 10% polyacrylamide TBE-urea gel. cDNA was amplified by 8 cycles of PCR using the Phusion High-Fidelity enzyme (NEB) and Small RNA PCR primers (Illumina). Reactions were separated on a non-denaturing 8% polyacrylamide gel and the recovered libraries were sequenced for 36 cycles on an Illumina Genome Analyzer according to the manufacturer’s instructions.

value: macrophage

tag: treatment

tag: transgenes


title: ThioMac-GroSeq-Kdo2-1h-RNA-Seq

description:
strand-specific RNA-Seq of Nuclear Run-on
Nuclear run-on RNA from ThioMac treated with Kdo2 for 1h

value: Kdo2 Lipid A for 1 hour


title: RAW-Tbp-ChIP-Seq

description: Chromatin IP against TBP

source_name: RAW264.7 cells

growth_protocol: RAW264.7 Macrophage cell line was cultured in RPMI 1640 containing 10% FCS and 100 U/ml penicillin/streptomycin

extract_protocol: For ChIP-Seq, lysates were clarified from sonicated nuclei and protein-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, ChIP DNA was PCR-amplified with Illumina primers for 12-16 cycles and library fragments of 200-300 bp (ChIP) (insert plus adaptor and PCR primer sequences) were isolated from a 2% agarose gel. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Genome Analyzer or Genome Analyzer II following the manufacturer's protocols.

value: RAW264.7 Macrophage cell line

value: TBP (Santa Cruz sc-204, LOT H0508)

value: wild-type


title: ThioMac-totalRNA-RNA-Seq

description:
strand-specific RNA-Seq
Total RNA from ThioMac

extract_protocol: For total RNA-Seq, cells were scraped and RNA purified using TRIZOL reagent (Invitrogen). Ribosomal RNA was depleted from DNase-treated samples using Ribominus Eukaryote Kit (Invitrogen) followed by ethanol precipitation. RNA was fragmented using RNA Fragmentation Reagents (Ambion) followed by ethanol precipitation. Samples were dephosphorylated using Antarctic phosphatase (New England Biolabs) and then loaded on a Novex 15% polyacrylamide TBE-urea gel (Invitrogen) and run according to the manufacturer’s instructions. Fragments of 36-55 nucleotides were cut and RNA was eluted from the gel debris and recovered by ethanol precipitation. E. coli Poly(A) Polymerase (NEB) was used to create a priming site for cDNA synthesis. Reverse transcription was carried out using Superscript III reagents (Invitrogen) and oligonucleotide 5’-pGATCGTCGGACTGTAGAACTCTxCAAGCAGAAGACGGCATACGATTTTTTTTTTTTTTTTTTTTVN (p indicates 5’ phosphorylation, x indicates the abasic dSpacer furan and V, N indicates degenerate nucleotides). The RNA was hydrolyzed and cDNA extracted from 10% TBE-Urea Gel (Invitrogen) followed by ethanol precipitation. cDNA was circularized using CircLigase (Epicentre) and linearized using APE1 (New England Biolabs). The relinearized product band was recovered from Novex 10% polyacrylamide TBE-urea gel. cDNA was amplified by 8 cycles of PCR using the Phusion High-Fidelity enzyme (NEB) and Small RNA PCR primers (Illumina). Reactions were separated on a non-denaturing 8% polyacrylamide gel and the recovered libraries were sequenced for 36 cycles on an Illumina Genome Analyzer according to the manufacturer’s instructions.


title: ThioMac-H4K8ac-Kdo2-1h-ChIP-Seq

description: Chromatin IP against H4K8ac in ThioMac treated with Kdo2 for 1h


title: ThioMac-H4K12ac-Kdo2-1h-ChIP-Seq

description: Chromatin IP against H4K12ac in ThioMac treated with Kdo2 for 1h


title: ThioMac-H4K8ac-ChIP-Seq

description: Chromatin IP against H4K8ac


title: ThioMac-H4K5ac-Kdo2-1h-ChIP-Seq

description: Chromatin IP against H4K5ac in ThioMac treated with Kdo2 for 1h


title: Sin3A MT 2

description: Chromatin IP against Sin3A


title: Benign neurofibroma MeDIP-seq

city: London

first: Gareth

last: Wilson

address: 23 Huntley Street

country: United Kingdom

institute: UCL Cancer Institute

description: DNA IP against 5-Methylcytosine

data_processing:
Alignment: Sequence reads were obtained and mapped to the human (March, 2006) genomes using the software 'maq' (http://maq.sf.net/). PA\aired reads with a maq score of <10 were removed for downstream analysis.
DNA Methylation:Absolute DNAm states were inferred from raw reads using the Batman algorithm (Down et al., Nat Biotech 2008)

source_name: Benign neurofibroma

growth_protocol: Primary human Schwann cells (SC) were isolated from 10-15 cm length of human sciatic/brachial nerve from amputation amputations specimens from patient without NF1 or nerve sheath tumours. The protocol was adapted from Muir et al., (1994). Briefly, after removal of the perineurium, the nerve was cut into ~ 2mm transverse sections with a scalpel and placed in cell culture plates with hSC medium, containing DMEM/F12 supplemented with 2uM of Forskolin (Calbiochem), recombinant GGF (20 ng/ml, R&D Systems), 3% FCS (Invitrogen), Kanamycin Monosulfate (80ug/ml)  (Sigma) and Gentamicin (40ug/ml) (Gibco). The plates were incubated at 37°C and 5% CO2 and fed every 2-3 days for 2-3 weeks. Then the nerve fragments were dissociated overnight at 37°C degrees with a mixture of 300U/ml collagenase Type XI (Sigma) and 1.25U/ml of Dispase (Roche) containing antibiotics kanamycin/gentamicin. The single cell suspension was then plated into 1xPoly-L-Lysin/Laminin-coated tissue culture plates (Sigma) and incubated at 37°C 5% CO2 for another few days before CD271 (low-affinity nerve growth factor receptor, LNGFR) –positive cells were selected  by MACS according to the manufacturer’s instructions (Miltenyi Biotec Ltd., Surrey, UK).

extract_protocol: 5ug of DNA was sonicated giving fragment sizes <500bp.Fragments were end repaired, phosphorylated and A-tailed the fragmented DNA and ligated Illumina paired end adapters to fragments in accordance with the Illumina Paired End Sample Preparation protocol. These samples were then subjected to MeDIP as described previously (Weber et al. 2005; PMID: 16007088). The immunoprecipitated (IP) sample was purified using Zymo-5 kit according to the manufacturers instructions.The sample isolated by meDIP then underwent gel electrophoresis and library size selection, prior to PCR amplification using Illumina paired-end PCR primers for 18 cycles.  Bands were excised to produce libraries with insert sizes of 100-150 bp and 150-200bp, libraries were quantified using an Agilent Bioanalyzer 2100.

tag: sample source

value: DNA pooled from 10 benign neurofibroma patients

tag: medip antibody

tag: sample type

value: Benign neurofibroma

tag: antibody manufacturer

value: Eurogentec

tag: lot #

tag: antibody product name

value: 5-methylcytidine, Monoclonal Antibody, Clone-33D3


title: Schwann cell MeDIP-seq

description: DNA IP against 5-Methylcytosine

source_name: Cultured normal schwann cells

value: DNA pooled from 6 normal schwann cell culuters

value: Cultured normal schwann cells

tag: antibody catalog #


title: MPNST MeDIP-seq

description: DNA IP against 5-Methylcytosine

source_name: Malignant peripheral nerve sheath tumour

value: DNA pooled from 10 MPNST patients


title: ThioMac-totalRNA-Kdo2-1h-RNA-Seq

description:
strand-specific RNA-Seq
Total RNA from ThioMac treated with Kdo2 for 1h

tag: cell type


title: mouse blastomere in 4-cell embyro #2_4_cell_B6

status: Public on Dec 31 2010

city: Foster City

first: Catalin

last: Barbacioru

address: 850 Lincoln Centre Dr

institute: Applied Biosystems

data_processing:
Raw data is aligned to Mouse genome (Jul. 2007 assembly of the mouse genome: mm9, NCBI Build 37, with dbSNP128 positions replaced with IUB codes) using SOLiD whole transcriptome pipeline: http://solidsoftwaretools.com/gf/project/transcriptome/. Reads that aligned to a unique location are used for downstream analysis and saved in *_max.srf.tar files.
srf conversion is generated by SOLiD srt tool: http://solidsoftwaretools.com/gf/project/srf/
Algorithm description: Tang F, Barbacioru C, Wang Y, Nordman E, Lee C, Xu N, Wang X, Bodeau J, Tuch BB, Siddiqui A, Lao K, Surani MA., mRNA-Seq whole-transcriptome analysis of a single cell, Nat Methods. 2009 May;6(5):377-82.

molecule: polyA RNA

source_name: mouse blastomere in a 4-cell embyro

extract_protocol: Oocyte, Two-cell, Four-cell, and 8-cell stage embryos were recovered from MF1 females mated with MF1 male mice (Nagy et al. 2003). The zona pellucida was removed by treatment with acidic tyrode solution. The individual blastomeres were separated by gentle pipeting using a glass capillary. Whole transcriptome Amplification by RT-PCR from a single cell.

value: mouse blastomere in 4-cell embyro #2

tag: gender

value: female

tag: strain

value: MF1 wild type


title: mouse blastomere in 2-cell embyro #3_2cell_B8

source_name: mouse blastomere in a 2-cell embyro

value: mouse blastomere in 2-cell embyro #3


title: mouse blastomere in 8-cell embyro #3_8_cell_A15

source_name: mouse blastomere in a 8-cell embyro

value: mouse blastomere in 8-cell embyro #3


title: mouse blastomere in 4-cell embyro #3_4_cell_B10

value: mouse blastomere in 4-cell embyro #3


title: mouse blastomere in 4-cell embyro #2_4_cell_B5


title: mouse blastomere in 2-cell embyro #3_2cell_B7


title: mouse blastomere in 2-cell embyro #2_2cell_B6

value: mouse blastomere in 2-cell embyro #2


title: mouse blastomere in 2-cell embyro #4_2cell_B9

value: mouse blastomere in 2-cell embyro #4


title: mouse blastomere in 4-cell embyro #3_4_cell_B9


title: ThioMac-H4K12ac-ChIP-Seq

description: Chromatin IP against H4K12ac

value: macrophage

tag: genetic background


title: mouse blastomere in 8-cell embyro #3_8_cell_A9


title: ThioMac-H4K5ac-ChIP-Seq

description: Chromatin IP against H4K5ac


title: mouse blastomere in 2-cell embyro #2_2cell_B5


title: mouse blastomere in 8-cell embyro #2_8_cell_A5

value: mouse blastomere in 8-cell embyro #2


title: mouse blastomere in 4-cell embyro #1_4_cell_B1

value: mouse blastomere in 4-cell embyro #1


title: mouse Dicer knockout oocyte #4_Dicer_5

source_name: mouse oocyte

value: mouse Dicer knockout oocyte #4

value: mixed genetic background of C57BL/6J, 129/sv, MF1


title: mouse blastomere in 4-cell embyro #1_4_cell_B2


title: mouse Dicer knockout oocyte #3_Dicer_2A

value: mouse Dicer knockout oocyte #3


title: mouse blastomere in 8-cell embyro #2_8_cell_A6


title: wild type mouse oocyte #2_WT_10

value: wild type mouse oocyte #2


title: mouse blastomere in 8-cell embyro #1_8_cell_A1

value: mouse blastomere in 8-cell embyro #1


title: mouse blastomere in 2-cell embyro #4_2cell_B10


title: mouse blastomere in 2-cell embyro #1_2cell_A12

value: mouse blastomere in 2-cell embyro #1


title: wild type mouse oocyte #1_WT_A7

value: wild type mouse oocyte #1


title: mouse blastomere in 8-cell embyro #1_8_cell_A4

value: female


title: mRNASeq_hy5-215

city: New Haven

first: Hang

last: He

state: CT

address: 165 Prospect St

department: MCDB

institute: Yale University

description: mRNA-Seq

data_processing: Bowtie (Langmead et al., 2009) was used to map the sequence reads to the Arabidopsis genome (TAIR 7), and only uniquely mapped reads were used in subsequent analyses. Reads mapped to exonic regions of annotated gene models were normalized against the length of the transcript. To compare gene expression between wild type and the hy5-215 mutant, length-normalized read density was quantile-normalized. A two-fold difference was used as the threshold for identification of differentially expressed genes.

organism: Arabidopsis thaliana

source_name: whole seedling, hy5-215

growth_protocol: Arabidopsis ecotype Columbia (Col-0) was the genetic background used for this study. Wild type and hy5-215 mutant seeds were stratified for four days at 4°C, then germinated and grown for six days at 22°C in continuous white light. The whole seedlings were harvested and used for mRNA isolation.

extract_protocol: Total RNA was isolated using TRIzol reagent (Invitrogen) according to the manufacturer's instructions, and was treated with RNase-free DNase I (New England Biolabs) to remove any possible contaminating genomic DNA. mRNA was extracted from total RNA using Dynabeads Oligo(dT) (Invitrogen Dynal) following the manufacturer's directions. First-and second strand cDNA was generated using SuperScript II reverse transcriptase (Invitrogen) and random hexamer primers. Double-stranded cDNA was fragmented by nebulization and used for mRNA library construction following the standard Illumina protocol.

tag: ecotype

value: Columbia (Col-0)

tag: genotype

value: hy5-215 mutation

tag: tissue

value: whole seedling

tag: growth condition

value: wild type light (WL)


title: mouse blastomere in 2-cell embyro #1_2cell_A11

value: MF1 wild type


title: 193_hom_enrich

city: Utrecht

first: Michal

last: Mokry

address: Lundlaan 6

institute: Wilhelmina Children's Hospital, University Medical Center Utrecht

data_processing: SOLiD sequence tags were mapped to the full genome (Arabidopsis tair8 and rgsc3.4) with the MAQ package 40 (V0.7.1, options: -c, n3, e180, C10)

source_name: seedling

extract_protocol:
One microgram of each individual genomic DNA sample was fragmented to ~ 100 nt double-stranded DNA fragments by sonication in 100 µl of 10 mM Tris buffer pH 8 using Covaris S2 sonicator (6 x 16 mm AFA fiber Tube, duty cycle: 20%, intensity: 5, cycles/burst: 200, frequency sweeping, 6 minutes). After fragmentation, fragments were blunt-ended and phosophorylated at 5'-prime end using End-it Kit (Epicentre) according to the manufacturer’s instructions. Double stranded adapters compatible with SOLiD sequencing (adapter 1: pre-annealed duplex of 5'-CTA TGG GCA GTC GGT GAT-3' and 5'-ATC ACC GAC TGC CCA TAG TTT-3' and adapter 2 (sequence depends on multiplexing); adapter 2 for non- multiplex libraries: pre-annealed duplex of 5'- CCG GGT TCC TCA TTC TCT-3' and 5'- AGA GAA TGA GGA ACC CGG TTT-3', adapter 2 for multiplexed libraries: pre-annealed duplex of 5'-CGC CTT GGC CGT ACA GCA G-3' and 5'-GCT GTA CGG CCA AGG CG-3'; all oligo’s were acquired through Integrated DNA Technologies (Coralville, IA) and pre-annealing was done by mixing complementary oligonucleotides at 500 µM concentration and running on thermocycler with the following program: 95˚C for 3 min, 80˚C for 3 min, 70˚C for 3 min, 60˚C for 3 min, 50˚C for 3 min, 40˚C for 3 min and 4˚C hold), were ligated to the fragmented DNA (~ 1 µg) using Quick ligation kit (New England Biolabs) with 750 mM adaptor 1 and adaptor 2, 150 µl of 2x Quick ligation buffer, 5 µl Quick Ligase in a total volume of 300 µl. Samples were purified using Ampure beads (Agencourt) and amplified in 400 µl of Platinum PCR Supermix with 750 mM of both amplification PCR primers (P1_short: 5'-CTA TGG GCA GTC GGT GAT-3' with P2_short: 5’-CCG GGT TCC TCA TTC TCT -3’ for non-barcoded samples, with Bar_primer: 5’-GCT GTA CGG CCA AGG CG-3’ for multiplexed samples and with BC-primer: 5’-CTG CCC CGG GTT CCT CAT TCT CTN NNN NNN NNN CTG CTG TAC GGC CAA GGC G-3’ for barcoded samples multiplexed before enrichment, where N represent unique barcode sequence for each library), 2.5 U of Pfu DNA polymerase (Stratagene) and 5 U Taq DNA polymerase (Bioline). Before ligation-mediated amplification, the PCR sample was incubated at 72˚C for 5 minutes in PCR mix to perform nick translation on non-ligated 3'-ends. After 6 to 8 cycles of amplification, the library DNA was purified on Ampure beads and the quality was checked on a gel for the proper size range and the absence of adapter dimers and heterodimers. Finally, single samples or equimolar pools of multiplexed barcoded samples were size selected on 4% agarose gel for 125-175bp fraction.
microarray hybridization: DNA was mixed with 31.7 μl of Nimblegen aCGH hybridization solution and denatured at 95˚C for 5 minutes. After denaturing the sample was hybridized for 72 hours at 42˚C on MAUI hybridization station. After hybridization, the array was washed using Nimblegen Wash Buffer Kit according the user’s guide for aCGH hybridization. Elution was performed using 800 μl of elution buffer (10 mM Tris pH 8.0) in an Agilent Microarray Hybridization Chamber at 95˚C for 30 minutes. After 30 minutes the chamber was quickly dissembled and elution buffer was collected. Eluted library DNA was concentrated by speedvac to a volume of 50 μl

value: MUTANT for unknown causal mutation

tag: phenotype

value: Mutant 193 (qc19-3), displays both a root and shoot meristem phenotype, where stem cell identity is reduced, lost or re-organized

tag: enrichment

value: yes

value: seedling


title: rbr_hom_enrich

value: supressor of RBR over expression


title: 136_hom_enrich

value: The 136 mutant has a shorter root meristem and a reduction in root length of about 75% compared to wild type.


title: 136_het

value: WILDTYPE for unknown causal mutation

value: no


title: 193_hom


title: KSO_H3K9me3_ChIP_Seq

city: Cambridge

first: Steve

last: Bilodeau

address: 9 Cambridge Center

institute: Whitehead Institute

data_processing: Images acquired from the Illumina/Solexa sequencer were processed through the bundled Solexa image extraction pipeline which identified polony positions, performed base-calling and generated QC statistics. Sequences were aligned using ELAND software to NCBI Build 36 (UCSC mm8) of the mouse genome. Only sequences that mapped uniquely to the genome with zero or one mismatch were used for further analysis. When multiple reads mapped to the same genomic position, a maximum of two reads mapping to the same position were used.    Analysis methods were derived from previously published methods (PMID:19884255). Sequence reads from multiple flow cells for each IP target were combined.  Each read was extended 100bp, towards the interior of the sequenced fragment, based on the strand of the alignment.  Across the genome, in 25 bp bins, the number of ChIP-Seq reads within a 1kb window surrounding each bin (+/- 500bp) was tabulated.  The 25bp genomic bins that contained statistically significant ChIP-Seq enrichment were identified by comparison to a Poissonian background model.  Assuming background reads are spread randomly throughout the genome, the probability of observing a given number of reads in a 1kb window can be modeled as a Poisson process in which the expectation can be estimated as the number of mapped reads multiplied by the number of bins (40) into which each read maps, divided by the total number of bins available (we estimated 70%).  Enriched bins within 1kb of one another were combined into regions.    The Poissonian background model assumes a random distribution of background reads, however we have observed significant deviations from this expectation.  Some of these non-random events can be detected as sites of apparent enrichment in negative control DNA samples and can create many false positives in ChIP-Seq experiments. To remove these regions, we compared genomic bins and regions that meet the statistical threshold for enrichment to a set of reads obtained from Solexa sequencing of DNA from whole cell extract (WCE) in matched cell samples.  We required that enriched bins and enriched regions have five-fold greater ChIP-Seq density in the specific IP sample, compared with the control sample, normalized to the total number of reads in each dataset.  This served to filter out genomic regions that are biased to having a greater than expected background density of ChIP-Seq reads

source_name: Chromatin IP against H3K9me3

growth_protocol: Mouse Embryonic Fibroblasts were derived from day e13 embryos.  MEF and KSO were cultured in Dulbecco’s modified Eagle medium supplemented with 10% cosmic calf serum, β-mercaptoethanol, nonessential amino acids, L glutamine and penicillin/streptomycin.

extract_protocol: ChIP was done following the Agilent Mammalian ChIP-on-chip protocol (version 9.1, Nov 2006).  In summary, MEF and mES cells were grown as described above and cross-linked for 10 minutes at room temperature by the addition of one-tenth of the volume of 11% formaldehyde solution (11% formaldehyde, 50mM Hepes pH7.3, 100mM NaCl, 1mM EDTA pH8.0, 0.5mM EGTA pH8.0) to the growth media followed by two washes with PBS.  Cells were scraped and frozen in liquid nitrogen.  100ul of Dynal magnetic beads (Sigma) were blocked with 0.5% BSA (w/v) in PBS.  Magnetic beads were bound with 10ug of the indicated antibody.  Antibodies used are as follows: H3K4me3 (AbCam, Ab8580), H3K9me3 (AbCam, Ab8898)  and H3K27me3 (Ab6002). Cells were resuspended, lysed in lysis buffers and sonicated to solubilize and shear crosslinked DNA. Sonication conditions vary depending on cells, culture conditions, crosslinking and equipment. For H3K9me3, the sonication buffer was Tris-HCl pH8 20mM, 150mM NaCl, 2mM EDTA, 0.1% SDS, Triton X-100 1%. We used a Misonix Sonicator 3000 and sonicated at approximately 24 watts for 10 x 30 second pulses (60 second pause between pulses). Samples were kept on ice at all times.  The resulting whole cell extract was incubated overnight at 4°C with 100 μl of Dynal Protein G magnetic beads that had been pre-incubated with approximately 10 μg of the appropriate antibody. Beads were washed 4 times with: 1 time with the sonication buffer, 1 time with 20mM Tris-HCl pH8, 500nM NaCl, 2mM EDTA, 0.1% SDS, 1%Triton X-100, 1 time with 10mM Tris-HCl pH8, 250nM LiCl, 2mM EDTA, 1% NP40 and 1 time with TE containing 50 mM NaCl. Bound complexes were eluted from the beads by heating at 65°C for 1 hour with occasional vortexing and crosslinking was reversed by overnight incubation at 65°C. Whole cell extract DNA reserved from the sonication step was also treated for crosslink reversal. RNA and protein were digested using RNAse A and Proteinase K and DNA was purified with phenol chloroform extraction and ethanol precipitation.      DNA was prepared for sequencing according to a modified version of the Illumina/Solexa Genomic DNA protocol. Fragmented DNA was prepared for ligation of Solexa linkers by repairing the ends and adding a single adenine nucleotide overhang to allow for directional ligation. A 1:100 dilution of the Adaptor Oligo Mix (Illumina) was used in the ligation step. A subsequent PCR step with limited (18) amplification cycles added additional linker sequence to the fragments to prepare them for annealing to the Genome Analyzer flow-cell. After amplification, a narrow range of fragment sizes was selected by separation on a 2% agarose gel and excision of a band between 150-300 bp (representing shear fragments between 50 and 200nt in length and ~100bp of primer sequence). The DNA was purified from the agarose and diluted to 10 nM for loading on the flow cell. The DNA library (2-4 pM) was applied to the flow-cell (8 samples per flow-cell) using the Cluster Station device from Illumina. The concentration of library applied to the flow-cell was calibrated such that polonies generated in the bridge amplification step originate from single strands of DNA. Multiple rounds of amplification reagents were flowed across the cell in the bridge amplification step to generate polonies of approximately 1,000 strands in 1μm diameter spots. Double stranded polonies were visually checked for density and morphology by staining with a 1:5000 dilution of SYBR Green I (Invitrogen) and visualizing with a microscope under fluorescent illumination. Validated flow-cells were stored at 4 degrees C until sequencing. Flow-cells were removed from storage and subjected to linearization and annealing of sequencing primer on the Cluster Station. Primed flow-cells were loaded into the Illumina Genome Analyzer 1G. After the first base was incorporated in the Sequencing-by-Synthesis reaction the process was paused for a key quality control checkpoint. A small section of each lane was imaged and the average intensity value for all four bases was compared to minimum thresholds. Flow-cells with low first base intensities were re-primed and if signal was not recovered the flow-cell was aborted. Flow-cells with signal intensities meeting the minimum thresholds were resumed and sequenced for 26 or 32 cycles.

value: mouse embryonic fibroblasts (MEF)

tag: transfection

value: Dox-inducible vectors for Klf4, Sox2 and Oct4 (KSO)

tag: passages

tag: antibody


title: MEF_H3K4me3_ChIP_Seq

source_name: Chromatin IP against H3K4me3


title: MEF_WCE_Seq

source_name: WCE


title: soybean_degradome-library

status: Public on Dec 30 2010

city: beijing

first: Jinsong

address: No.1 West Beichen Road,Chaoyang District

institute: IGDB

description: construction of degradome library from mRNA in soybean seed

data_processing: Only 20-21 nt sequences with high quality were collected for subsequent analysis. The raw sequences were first normalized to “reads per 10 million” (RP10M). The distinct reads that perfectly matched soybean cDNA sequences remained. The 15 nt of sequence upstream and downstream of the 5’ end of matched reads were extracted to constitute 30-nt sequence tags for searching corresponding miRNA. The CleaveLand pipeline was used to align the 30 nt sequence to soybean known miRNAs from miRBase and our newly identified miRNAs. All alignments with scores up to 7 and no mismatches at the cleavage site (between the 10th and 11th nucleotides) were considered candidate targets.

organism: Glycine max

source_name: soybean seed

growth_protocol: Soybean seeds of (Glycine max) cultivar Heinong44 were directly planted in the Experimental Station of Institute of Genetics and Developmental Biology, Chinese Academy of Sciences in Beijing on May. Soybean seeds of 15 days after flowering (DAF) were collected

extract_protocol: In brief, poly(A) RNA was extracted from 200 μg of total RNA using the Oligotex kit (Qiagen). A 5’ RNA adapter containing a MmeI recognition site was ligated to the poly(A) RNA possessing a 5’-phosphate, by T4 RNA ligase (Ambion), and the ligated products were repurified using the Oligotex kit. Five PCR cycles were then performed to amplify the reverse transcription products. The PCR products were digested with MmeI and ligated to a 3’ double DNA adapter. The ligation products were amplified by 20 PCR cycles and gel-purified for SBS sequencing.

tag: cultivar

value: seed

tag: developmental stage

value: 15 days after flowering


title: MEF_H3K9me3_ChIP_Seq


title: 193_het


title: TSS-seq_oocyte

first: Gert Jan

last: Veenstra

address: Geert Grooteplein 28

department: Molecular Developmental Biology

institute: Radboud University

description: 5' ends of capped transcripts

data_processing:
Reads were mapped to the X.tropicalis genome (version JGI 4.1) using ELAND. All unmapped reads, or reads mapping to multiple positions were discarded. To call transcription start sites, all positions with at least 20 overlapping reads were ﬁltered to overlap with either a TBP peak or a H3K4me3 peak (Akkers et al., 2009).

The included bed-files contain xenTro2/JGI4.1 coordinates. These tab-delimited files contain up to 6 columns according to the BED specification.
1) chromosome
2) start
3) end
4) score (see description below)
5) not used
6) strand

The file TSS-seq.bed contains the start position of every TSS-seq read that uniquely mapped to the xenTro2 genome.

The file TSS.bed contains all called TSS based on the TSS-seq reads. To call transcription start sites, all positions with at least 20 overlapping reads were filtered to overlap with either a TBP peak or a H3K4me3 peak (Akkers et al., 2009). The score column (4) contains the number of overlapping TSS-seq reads.

organism: Xenopus tropicalis

source_name: whole embryo

growth_protocol: Xenopus tropicalis embryos were obtained from a natural mating procedure after human chorion gonadotropin injection, dejellied in 3% cysteine and collected at the indicated stage

extract_protocol: Total RNA was isolated using Trizol and the QIAGEN RNeasy Kit. 50 μg of puriﬁed total RNA was dephosphorylated with Bac- 14 terial Alkaline Phosphatase, ligated with oligo-RNA (5’-AAUGAUACGGCGACCACCGAGAUCUACACUCUUUCCCUACACGACGCUCUUCCGAUCUGG- 3’) using T4 RNA ligase, and cDNA was then synthesized from a random hexamer primer (5’-CAAGCAGAAGACGGCATACGANNNNNNC-3’) with Super Script II (Invitrogen). The cDNA was ampliﬁed by 20 cycles of PCR.

value: :oocyte


title: KSO_H3K27me3_ChIP_Seq

source_name: Chromatin IP against H3K27me3


title: TBP_ChIPSeq

description: Chromatin IP against TBP

data_processing:
Reads were mapped to the X.tropicalis genome (version JGI4.1) with ELAND, reads in identical positions were removed and peaks were called using MACS with p-value 1e-7

The included bed-files contain xenTro2/JGI4.1 coordinates. These tab-delimited files contain up to 6 columns according to the BED specification.
1) chromosome
2) start
3) end
4) score (see description below)
5) not used
6) strand

The file TBP.bed contains the coordinates of the filtered unique 35-basepair reads, mapped strand-specific to the xenTro2 genome.
The TBP_peaks.bed file contain the peaks as determined by MACS using a p-value threshold of 1e-7. The score column contains the number of reads in the peak as determined by MACS.

growth_protocol: Xenopus tropicalis embryos were obtained from a natural mating procedure after human chorion gonadotropin injection, dejellied in 3% cysteine and collected at Nieuwkoop-Faber stage 12

extract_protocol: Chromatin (400 embryos) was fixed in 1% formaldehyde, sheared using a Branson sonifier, immunoprecipitated using 2 µl of antibody (SL33 against TBP, Ruppert et al. 1996,PMID: 9064287), washed, decrosslinked, DNA was purified and sequenced using a Illumina Genome Analyzer according to the manufacturer.

tag: development stage

value: SL33 against TBP


title: rbr_het


title: MEF_H3K27me3_ChIP_Seq


title: TSS-seq_stage12

description: 5' ends of capped transcripts


title: mRNASeq_WT

description: mRNA-Seq

source_name: whole seedling, WT

value: Columbia (Col-0)

value: wild type

value: whole seedling


title: Stam_K562

status: Public on Dec 29 2010

city: Stanford

first: ENCODE

last: DCC

address: 300 Pasteur Dr

institute: ENCODE DCC

data_processing: For data processing details see: http://genome.ucsc.edu/cgi-bin/hgTrackUi?db=hg19&g=wgEncodeUwDgf

growth_protocol: Cells were grown according to ENCODE cell culture protocols: http://genome.ucsc.edu/ENCODE/protocols/cells/human/K562_protocol.pdf

extract_protocol: For extraction protocol details see: http://genome.ucsc.edu/cgi-bin/hgTrackUi?db=hg19&g=wgEncodeUwDgf

tag: cell

tag: cell organism

value: Human

tag: cell description

value: leukemia

tag: cell karyotype

value: cancer

tag: cell lineage

value: "The continuous cell line K-562 was established by Lozzio and Lozzio from the pleural effusion of a 53-year-old female with chronic myelogenous leukemia in terminal blast crises." - ATCC

tag: cell sex

value: F


title: 136_hom

tag: genotype

value: MUTANT for unknown causal mutation

value: The 136 mutant has a shorter root meristem and a reduction in root length of about 75% compared to wild type.

value: seedling


title: Stam_SK-N-SH_RA

growth_protocol: Cells were grown according to ENCODE cell culture protocols: http://genome.ucsc.edu/ENCODE/protocols/cells/human/UW_Stam:Stam_15_protocols.pdf

value: neuroblastoma cell line differentiated w/ retinoic acid

value: "SK-N-MC was isolated in September of l971 and was found to have moderate dopamine - beta - hydroxylase activity as well as formaldehyde induced fluorescence indicative of intracellular catecholamines." - ATCC


title: UW_DnaseDgf_HMVEC-dBl-Ad

source_name: HMVEC-dBl-Ad

extract_protocol: Instrument model unknown. ("Illumina Genome Analyzer" specified by default). For more information, see http://genome.ucsc.edu/cgi-bin/hgTrackUi?db=hg19&g=wgEncodeUwDgf

tag: lab

value: UW

tag: lab description

value: Stamatoyannopoulous - University of Washington

tag: datatype

value: DnaseDgf

tag: datatype description

value: DNase Digital Footprinting

value: HMVEC-dBl-Ad

value: human

value: adult blood microvascular endothelial cells, dermal-derived

value: normal

value: mesoderm

tag: labversion


title: UW_DnaseDgf_Th1

value: Human

value: primary human Th1 T cells

value: U


title: rbr_het_enrich

value: WILDTYPE for unknown causal mutation

tag: phenotype

value: supressor of RBR over expression

tag: enrichment

value: yes

tag: tissue


title: KSO_WCE_Seq

tag: cell type

value: mouse embryonic fibroblasts (MEF)

tag: transfection

value: Dox-inducible vectors for Klf4, Sox2 and Oct4 (KSO)

tag: passages

tag: antibody


title: UW_DnaseDgf_HCPEpiC

source_name: HCPEpiC

value: UW

tag: cell

value: HCPEpiC

tag: cell organism

tag: cell description

value: choroid plexus epithelial cells

value: ectoderm


title: UW_DnaseDgf_NHDF-Ad

source_name: NHDF-Ad

value: Stamatoyannopoulous - University of Washington

value: DnaseDgf

value: DNase Digital Footprinting

value: NHDF-Ad

value: adult dermal fibroblasts

tag: cell karyotype

tag: cell lineage

tag: cell sex

value: F


title: rbr_hom

tag: genotype

value: MUTANT for unknown causal mutation

value: no

value: seedling


title: UW_DnaseDgf_H7-hESC

tag: lab

tag: lab description

tag: datatype

value: human

value: undifferentiated embryonic stem cells

value: inner cell mass

value: U


title: UW_DnaseDgf_HAEpiC

source_name: HAEpiC

value: UW

tag: datatype description

value: HAEpiC

value: amniotic epithelial cells

tag: labversion


title: Stam_GM06990

value: Human

value: lymphoblastoid (CEPH)

value: relatively normal

value: CEPH/Utah - European Caucasion; Epstein-Barr Virus


title: Stam_HepG2

growth_protocol: Cells were grown according to ENCODE cell culture protocols: http://genome.ucsc.edu/ENCODE/protocols/cells/human/HepG2_protocol.pdf

value: liver carcinoma

value: cancer

value: endoderm

value: M


title: Gingeras_K562_cytosol_longPolyA

status: Public on Dec 28 2010

data_processing: For data processing details see: http://genome.ucsc.edu/cgi-bin/hgTrackUi?db=hg18&g=wgEncodeCshlLongRnaSeq

extract_protocol: For extraction protocol details see: http://genome.ucsc.edu/cgi-bin/hgTrackUi?db=hg18&g=wgEncodeCshlLongRnaSeq

value: leukemia

value: "The continuous cell line K-562 was established by Lozzio and Lozzio from the pleural effusion of a 53-year-old female with chronic myelogenous leukemia in terminal blast crises." - ATCC

value: F

tag: localization

value: cytosol

tag: rnaextract

value: longPolyA


title: UW_DnaseDgf_HMF

source_name: HMF

value: Stamatoyannopoulous - University of Washington

value: DnaseDgf

value: DNase Digital Footprinting

value: HMF

value: human

value: mammary fibroblasts


title: Gingeras_K562_cell_total

value: Human

value: cell

value: total


title: Gingeras_GM12878_cell_total

growth_protocol: Cells were grown according to ENCODE cell culture protocols: http://genome.ucsc.edu/ENCODE/protocols/cells/human/GM12878_protocol.pdf

value: lymphoblastoid

value: relatively normal

value: International HapMap Project - CEPH/Utah - European Caucasion; Epstein-Barr Virus


title: mixed_stage_wild-type_run2

status: Public on Dec 24 2010

first: calvin

last: jan

address: 9 Cambridge center

institute: WIBR/MIT/HHMI

data_processing: Reads were reverse complemented and aligned to the C. elegans genome (ce6/ws190) using Bowtie.  Reads that aligned best to one genomic locus and had one or more mismatches at their 3' end within a terminal adenylate-run were carried forward as 3P tags.  Reads mapping to the same locus with the same number of terminal adenylates were consolidated in the processed data file.

organism: Caenorhabditis elegans

source_name: whole animal

growth_protocol: Nematodes were grown under standard conditions

tag: strain

value: Bristol N2

tag: developmental stage

value: mixed stage


title: adult_glp-4_run1

value: adult


title: mixed_stage_wild-type_run1


title: adult_wild-type_run1


title: L4_wild-type_run1


title: L3_wild-type_run1


title: L1_wild-type_run1


title: L2_wild-type_run1


title: Prdm14 Knockdown_RNAseq

status: Public on Dec 23 2010

first: Ziyang

last: Ma

address: 269 Campus Dr, Room 3130

institute: Stanford University

description: Oligo-dT selected, double stranded cDNA

data_processing: Alignment: Reads were aligned to the mm9 genome and a splice junction database was used to identify uniquely mapping reads. The data were mapped to RefSeq gene model, using a professional website DNAnexus (https://dnanexus.com/).

source_name: mouse ES cells

growth_protocol: LF2 mouse ES cells were cultured under feeder free conditions according to standard procotols.

extract_protocol: mRNA-Seq was performed as recommended by the manufacturer (Illumina). Briefly, poly(A) mRNA was fragmented and first strand cDNA generated using random hexamers. Following second strand cDNA synthesis, end repair, addition of a single A base, adaptor ligation, agarose gel isolation of ~200 bp cDNA and PCR amplification of the ~200 bp cDNA.

tag: cell type

value: mESCs

tag: genotype/variation

value: Prdm14 Knockdown


title: control Knockdown_RNAseq

description: Oligo-dT selected, double stranded cDNA

value: control


title: Degenerative

city: Madison

first: Wen

last: Huang

state: WI

address: 1675 Observatory Dr. Room 634

department: Department of Dairy Science

institute: University of Wisconsin - Madison

description: Bovine IVF degenerative embryos

data_processing: For RNA-Seq read counts, we first aligned reads to the genome using Tophat v1.0.13. We then extracted uniquely mapped reads and count the number of reads mapped to non-overlapping exons of Ensembl Genes (release 57) using BEDtools. For paired end reads, we counted fragments while for single end reads, we counted reads. The read counts were then quantile normalized using the 'limma' package in R.

organism: Bos taurus

source_name: IVF degenerative embryos (d8)

extract_protocol: Total RNA was extracted from pools of 20 embryos using Ambion's RNaqueous Micro kit according to the manufacturer's instructions. After extraction, total RNA was linearly amplified using Ambion's MessageAmp II. 50ng of the amplified RNA (aRNA) was subjected to Illumina's library preparation protocol for RNA-Seq. For Degenerative: library insert size (excluding primer) = 120 and standard deviation = 20.

treatment_protocol: Morphological evaluation of bovine embryos

tag: tissue

value: IVF embryos


title: Blastocyst

description: Bovine IVF blastocysts

source_name: IVF blastocysts (d8)

extract_protocol: Total RNA was extracted from pools of 20 embryos using Ambion's RNaqueous Micro kit according to the manufacturer's instructions. After extraction, total RNA was linearly amplified using Ambion's MessageAmp II. 50ng of the amplified RNA (aRNA) was subjected to Illumina's library preparation protocol for RNA-Seq. For Blastocyst: library insert size (excluding primer) = 120 and standard deviation = 20.


title: H3K4me1-ChIPseq

description: Chromatin IP against H3K4me1

data_processing:
Alignment: Sequence reads were obtained and mapped to the mm9 genome using the Illumina Genome Analyzer II Pipeline. All uniquely mapped reads with two or fewer mismatches were retained.
Peaks: Peak detection was performed using QuEST software

extract_protocol: Lysates were clarified from sonicated nuclei and chromatin fragments were enriched with antibodies.  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 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 18 cycles and library fragments of ~280 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 II following the manufacturer's protocols.

value: wildtype

tag: chip antibody(ies)

tag: chip antibody details

value: Rabbit polyclonal to Histone H3K4me1, Abcam, Catalog ab8895 (lot 721955)


title: Input_DNA_sequencing_ChIPseq (control)

description: input DNA


title: Prdm14_ChIPseq

description: Chromatin IP against FLAG and HA

value: FLAG and HA

value: FLAG:mouse monoclonal anti-FLAG (clone M2), Sigma, Catalog A2220 (lot N/A)

value: HA:mouse monoclonal anti-HA (clone HA-7), Sigma, Catalog A2095 (lot N/A)


title: UW_DnaseDgf_SKMC

source_name: SKMC

value: UW

value: Stamatoyannopoulous - University of Washington

value: DnaseDgf

value: DNase Digital Footprinting

tag: cell

value: SKMC

tag: cell organism

value: human

tag: cell description

value: skeletal muscle cells

tag: cell lineage

value: mesoderm

value: U


title: HNF4g_ChIPSeq

status: Public on Dec 22 2010

city: Meguro

first: Kenji

last: Daigo

state: Tokyo

department: Research Center for Advanced Science and Technology

country: Japan

institute: University of Tokyo

description: Chromatin IP against HNF4g

data_processing:
Alignment: Sequence reads were obtained and mapped to the human (Feb, 2009) genomes using the Illumina Genome Analyzer Pipeline. All reads mapping with two or fewer mismatches were retained and read starts were summed in sliding windows of 400 bp to create summary windows.
Peaks: Peak detection was performed with the Model-based Analysis of ChIP-Seq (MACS) algorithm (http://liulab.dfci.harvard.edu/MACS/)

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.

tag: cell line

value: hepatocellular carcinoma

tag: chip antibody


title: HNF4a_ABdomain_ChIPSeq

description: Chromatin IP against HNF4a AB domain

value: HNF4a AB domain


title: dauer_wild-type_run1

value: Bristol N2

value: dauer


title: HNF4a_Fdomain_ChIPSeq

description: Chromatin IP against HNF4a F domain

value: HNF4a F domain


title: untreated_repII_lane01

first: Peter

middle: J

last: Park

address: 10 Shattuck St

department: Center for Biomedical Informatics

institute: Harvard Medical School

description:
RNA-Seq of nascent RNA transcripts.
GRO-Seq Untreated, Replicate II, lane1

data_processing: Reads were mapped to the D. melanogaster genome (dm3) using Bowtie alignment software (bowtie-bio.sourceforge.net), allowing at most one base mismatch and discarding all non-uniquely mapping reads

source_name: SL2 Cells

growth_protocol: Drosophila SL2 cells were grown in Schneider's medium with 10% FBS

extract_protocol: GRO-Seq Nuclei extraction and nascent RNA purification protocol is detailed in Larschan et al., Submitted

tag: biological replicate

value: Replicate II


title: untreated_repI_lane02

description:
RNA-Seq of nascent RNA transcripts.
GRO-Seq Untreated, Replicate I, lane2

value: Replicate I


title: untreated_repI_lane01

description:
RNA-Seq of nascent RNA transcripts.
GRO-Seq Untreated, Replicate I, lane1


title: egg_wild-type_run1

source_name: egg

value: egg


title: Stage HH16_Heart_tube_smallRNA_multilpexRun

status: Public on Dec 21 2010

city: Leiden

first: Henk

last: Buermans

state: ZH

address: Albinusdreef 2

department: Human Genetics

institute: Leiden University Medical Center

data_processing:
The data were processed using the E-miR analysis pipeline. This pipeline is a custom made tool for smallRNA expression profiling specific for next gen sequencing data.
1: 3' adapters introduced during the library sample preparation were identified and removed from the reads via regular expression matching. the first 8 nt of the adapter sequence was used, allowing for one mismatch. Sequences that were shoerter than 15 nt after truncation were discarded. Sequences longer than 32 nt after truncation were truncated at 32 nt.
2: Sequence reads were aligned to the unmasked Gallus gallus-2.1, May 2006 genome index using the Eland aligner allowing for max 1 mismatch
3: Individual reads were scaled to transcripts per million relative to the total numner of aligned sequence tags. Wig files were generated from the Eland alignments.

organism: Gallus gallus

source_name: stage HH16 heart tube -> smallRNA extraction

growth_protocol: Fertilized chicken eggs were obtained from a local hatchery (Drost BV, Nieuw Loosdrecht, The Netherlands), incubated at 39°C in a moist atmosphere, and automatically turned every hour. After the appropriate incubation time, embryos were isolated in Earl's balanced salt solution (EBBS, Life Technologies) and staged according to Hamburger and Hamilton Hamburger and Hamilton (1951).

extract_protocol: Total RNA and miRNA enriched RNA fractions were isolated with the mirVana miRNA Isolation Kit (Ambion) according to the manufacturers protocol. RNA integrity was confirmed with Agilent 2100 Bioanalyzer pico-RNA and small-RNA chips. Sequencing libraries were generated using a modification on the SOLiD Small RNA Expression Kit (SREK) to make it compatible with Illumina Genome Analyzer technology. MirVana-enriched miRNA fractions from HT or EM and EM totalRNA were hybridized and ligated to the A adapter mix to prepare 5' to 3' sequencing libraries. Reverse transcription and RNaseH treatment were as described in the SREK protocol. Library pre-amplification PCR was performed with Phusion® Hot Start High-Fidelity DNA Polymerase (Finnzymes) with a set of alternative primers (Figure 1) (Integrated DNA Technologies) that contain sequence features compatible with both the adapters that were introduced during the ligation step as well as the oligonucleotides deposited on the Illumina Genome Analyzer flow cell. DNA was denatured for 30'' at 98°C followed by 18 cycles with 30'' at 98°C, 30'' at 65°C, 30'' at 72°C and final extension for 5' at 72°C. During amplification, the RNaseH treated cDNA input did not exceed 1% of total PCR volumes. Final primer concentration was 100 nM. All libraries were amplified using a different 6-nucleotide multiplex sequence tag in the 5'-primer. Library fragments were separated on a native 6% gradient pre-cast PAGE gel (Novex, Invitrogen). The 140-155 bp size fractions containing the miRNA inserts were excised, DNA was eluted from the gel, precipitated and dissolved in 15 uL nuclease free water. Library yield was quantified on a Agilent Bioanalyzer DNA1000 chip. Single read cluster generation and single read sequencing were performed according to the standard Illumina v2 and v3 kits, respectively. An alternative set of sequencing primers was used during the sequence-by-synthesis steps to determine the RNA insert sequence and multiplex tag. Multiplexed and single read samples were 35 and 32 nt long, respectively.

treatment_protocol: The complete heart tube (HT) was dissected from stage HH16 embryos (EM). Tissue samples were stored at -80°C prior to use.

tag: Stage

tag: rna input

value: heart tube

tag: srek ligation


title: Stage HH16_whole_Embryo_totRNA_singleRun

source_name: stage HH16 whole chicken embryo without the heart tube -> totalRNA extraction

tag: stage

value: whole embryo


title: Stage HH16_whole_Embryo_smallRNA_multilpexRun

source_name: stage HH16 whole chicken embryo without the heart tube -> smallRNA extraction


title: Stage HH16_whole_Embryo_smallRNA_singleRun


title: Stage HH16_Heart_tube_smallRNA_singleRun