title: pol-sRNA Seq

city: Shanghai

first: Yuanshuai

last: Fu

address: College of Fisheries and Life Science, Shanghai Ocean University

department: College of Fisheries and Life Science

institute: Shanghai Ocean University

description: total RNA of nine stages during metamorphosis

data_processing:
Sequences were processed using the Pipeline software from the Genome Analyzer GA-1 instrument (Illumina Inc.) at first, then were subject to a series of data filtration steps to obtain "mappable sequences", which are sequences satisfy the following criteria from the statistics of miRBbase 14.0: (1) not sequencing adapters; (2) containing no more than 80% A, C, G, or T; (3) containing no more than two N (undermined bases); (4) containing no stretches of A7, C8, G6, or T7; (5) been observed more than two times; (6) being longer than 14 nts and shorter than 27 nts; and (7) not originating from mRNA, rRNA, tRNA, snRNA, snoRNA and repetitive sequence elements.
Further data analysis of the "mappable sequences" mainly used ACGT101-miR, our own development software for discovering miRNAs from next-generation sequencing (NGS) data (to be published separately), based on the databases containing genome & EST sequences and Metazoa konwn miRNA genes in miRBase 14.0

organism: Paralichthys olivaceus

source_name: Japanese flounder larvae

growth_protocol: To breed Larva of the metamorphosis phase which is from 14dph (day post hatching) to 42dph in the Japanese flounder postembryonic development

extract_protocol: A total RNA pool of the metamorphosis by collecting nine-stage(17, 19, 21, 24, 29, 31, 33, 36 and 42 dph) total RNA and pooled them together, Small RNA fragments 14-30 bases length were isolated from total RNA by Novex 15% TBE-Urea gel (Invitrogen). The purified small RNAs were then ligated with 5’ adapter and 3' adapter (Illumina). After gel purification on Novex 10% TBE-Urea gel (Invitrogen), RNA fragments with the adapters at both ends (70-90 bases in length) were reversely transcribed and the resulted cDNA was subjected to 15 PCR cycles. The amplification products were excised from 6% TBE-Urea gel (Invitrogen). The purified DNA was used directly for cluster generation and sequencing analysis with the Illumina/Solexa G1 sequencer

treatment_protocol: To extract total RNA by Trizol reagent

value: metamorphosis stages

value: whole fish


title: FLAG-HA_ChIPseq

status: Public on Dec 23 2010

type: SRA

city: Stanford

first: Ziyang

last: Ma

address: 269 Campus Dr, Room 3130

country: USA

institute: Stanford University

description: Chromatin IP against FLAG and HA

library_source: genomic

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

molecule: genomic DNA

organism: Mus musculus

source_name: mouse ES cells

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

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.

tag: strain

tag: cell type

value: mESCs

tag: genotype/variation

value: wildtype

tag: chip antibody(ies)

value: FLAG and HA

tag: chip antibody details

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: BFU-E_CD71-24_4h_Negative_RNA-Seq

status: Public on Dec 21 2010

city: Cambridge

first: Whitehead

last: Institute

state: MA

address: 9 Cambridge Center

department: Genome Technology Core

institute: Whitehead Institute

description:
Hematopoietic
Paired End

library_source: transcriptomic

data_processing: Images acquired from the Solexa sequencer were processed through the bundled Solexa image extraction pipeline version 1.4. mRNA-Seq reads were aligned to mouse NCBI build 37 (mm9) using ELAND, Briefly, the first 32 bases of a read were used as a seed. Each matched seed was then extended up to 36 bases and scored to break any ties between multi-matches. All reads mapping to unique positions in the known transcriptome were counted to asign a count for each gene which where further normalized to gene length to get Reads Per Kb per Million (RPKM)

molecule: total RNA

source_name: Fetal liver E13.5-14.5

growth_protocol: Fresh BFU-E, CFU-E and Ter119+ erythroblasts were purified from E 13.5-14.5 fetal liver by flow cytometry cell sorting. "Ter119+ erythroblasts" are positive for Ter119 and negative for kit. "CFU-E" cells are the 20% with highest expression of CD71 of cells that are negative for Ter119, B220, Mac-1, CD3, Gr-1, CD16/32 (FcgR), CD41 and CD34; and positive for kit. "BFU-E" cells are the 10% with lowest expression of CD71 of cells that are negative for Ter119, B220, Mac-1, CD3, Gr-1, CD16/32 (FcgR), CD41 and CD34; and positive for kit. In the experimetns labelled "BFU-E (CD71,CD24a)" BFU-E cells were purified from E 13.5-14.5 fetal liver by flow cytometry cell sorting."BFU-E" cells are the 10% with lowest expression of CD71 AND CD24a of cells that are negative for Ter119, B220, Mac-1, CD3, Gr-1, CD16/32 (FcgR), CD41 and CD34; and positive for kit.

extract_protocol: Samples for Paired-End mRNA-Seq were prepared using the Illumina kit according to the manufacturer’s instructions with the exception that we extracted 300bp bands, before and after the PCR step (Illumina).

treatment_protocol: In experiments labelled " BFU-E (CD71) 4 h..." BFU-E cells were cultured 4 hours in serum free erythroid liquid expansion medium (SFELE), which consists of 100ng/mL rmSCF, 40ng/mL rmIGF-1 and 2U /mL rhEPO, in StemSpan® SFEM. After 4 hours 100nM Dexamethasone was added to half the cells and water to the other half. Total RNA was isolated after 4 additional hours of culture. In experimetns labelled " BFU-E (CD71, CD24a) 4 h..." BFU-E cells were cultured 4 hours in serum free erythroid liquid expansion medium (SFELE), which consists of 100ng/mL rmSCF, 40ng/mL rmIGF-1 and 2U /mL rhEPO, in StemSpan® SFEM. After 4 hours 100nM Dexamethasone and 333uM DMOG was added to 25% of the cells,  100nM Dexamethasone  was added to 25% of the cells,  333uM DMOG was added to 25% of the cells and water to the other 25%. Total RNA was isolated after 4 additional hours of culture.

tag: developmental stage

tag: cell population

value: BFU-E

tag: agent

tag: tissue

value: Fetal liver


title: BFU-E_CD71_4h_100nMDex_RNA-Seq

description:
Hematopoietic
Paired End


title: BFU-E_CD71-24_4h_100nMDex_333uMDMOG_RNA-Seq

description:
Hematopoietic
Paired End


title: BFU-E_CD71-24_4h_333uMDMOG_RNA-Seq

description:
Hematopoietic
Paired End


title: BFU-E_CD71_4h_Dexnegative_RNA-Seq

description:
Hematopoietic
Paired End


title: CFU-E_RNA-Seq

description:
Hematopoietic
Paired End

value: CFU-E


title: BFU-E_RNA-Seq

description:
Hematopoietic
Paired End


title: Ter119+_RNA-Seq

description:
Hematopoietic
Paired End


title: BFU-E_CD71-24_4h_100nMDex_RNA-Seq

description:
Hematopoietic
Paired End


title: S2-DRSC-ChIP-Input replicate 1

city: 4058 Basel

first: Daniel

last: Enderle

state: Basel Stadt

address: Mattenstrasse 26

department: D-BSSE

country: Switzerland

institute: ETH Zürich

data_processing: The Apr. 2006 D. melanogaster genome assembly (dm3, BDGP Release 5) provided by the Berkeley Drosophila Genome Project (BDGP, http://www.fruitfly.org/) was used as a basis for all analyses. Low-complexity reads were filtered out based on their dinucleotide entropy (removing <1% of the reads). Alignments to the D. melanogaster genome were performed by the software bowtie (version 0.9.9.1) (Langmead et al. 2009) with parameters -v 2 -a -m 100, tracking up to 100 best alignment positions per query and allowing at most two mismatches.

organism: Drosophila melanogaster

source_name: DRSC-S2 cell line

growth_protocol: Drosophila S2-DRSC cells were grown in Schneider’s Drosophila Medium (Invitrogen) supplemented with 10 % FBS (Hyclone) at 25°C.

extract_protocol: Chromatin fixation and immunoprecipitation were performed essentially as described in (Orlando V et al. (1997) Methods 11:205–214.). Cells (1x109) were fixed in 200-ml medium with 1% formaldehyde for 10 min at room temperature. Cross-linked cells were sonicated to produce chromatin fragments of an average size of 200–400 bp. Soluble chromatin was separated from insoluble material by centrifugation. The supernatant containing chromatin of 5x107 cells was taken for immunoprecipitation. Psc and Ph antibodies were described in (Strutt H and Paro R (1997) Mol Cell Biol 17:6773–6783.). Pc and Trx-C antibodies were described in (Beisel et al. (2007) PNAS 104:16615-16620). Anti-H3K4me3 purchased from Millipore (Anti-trimethyl-Histone H3 (Lys4), clone MC315, Cat# 05-745). Libraries for sequencing were prepared with the ChIP-Seq DNA Sample Prep Kit (Cat# IP-102-1001) according to Illumina's instructions. After adapter ligation library fragments of ~250 bp were band isolated from an agarose gel. The DNA was PCR amplified with Illumina primers for 18 (ChIP-seq) cycles, purified and loaded on an Illumina flow cell for cluster generation. Libraries were sequenced on the Genome Analyzer II and Genome Analyzer IIx following the manufacturer's protocols.

tag: cell line

tag: chip antibody


title: S2-DRSC-ChIP-Input replicate 2


title: S2-DRSC-5'MACE

extract_protocol: 10 µg of Trizol extracted, TurboDNAse digested total RNA was dephosphorylated with CIAP (20 U, Invitrogen), decapped with TAP (1 U, Epicentre) and ligated to 0.42 µg RNAoligo (35.5 pMol) with T4RNA ligase (10 U, Epicentre). The RNAoligo sequence corresponds to the sequence of Illumina’s Genomic DNA PCR Primer 1.1 with addition three adenines at the 3’-end (5’-acacu cuuuc ccuac acgac gcucu uccga ucuaaa-3’). Column based purifications (RNeasy MinElute, Quiagen) were used between each step to clean reactions from enzymes and excess RNAoligo. To create a pool of 1st strand cDNAs with a suitable size distribution half of ligation product was reverse-transcribed with Supercript III (Invitrogen) and 1 µl of tagged random-hexamers (2 µM, 5’-caagc agaag acggc atacg agctc ttccg atctn nnnnn-3’). To create 2nd strand cDNA for size-selection on a 1.5% agarose gel half of the 1st strand cDNA was subjected to a limited PCR amplification (8 cycles) using Phusion Polymerase (NEB) and Genomic DNA PCR Primer 1.1 / 2.1. cDNAs running at 350±50 bp were gel-extracted (Quiaquick, Quiagen) and half of the eluate was used in PCR enrichment (18 cycles) for cluster generation according to Illumina’s standard protocols. The final cDNA library corresponds to capped transcripts present in ~1.25 µg total RNA and sequencing reads after the initial three adenines match the first nucleotides transcribed.


title: Embryo-0-16h-5'MACE

source_name: 0-16h Embryos

value: mixed-stage embryos


title: S2-DRSC-ChIP-Trx-C replicate 1

value: Trx-C


title: S2-DRSC-ChIP-H3K4me3


title: S2-DRSC-ChIP-Trx-C replicate 2


title: S2-DRSC-ChIP-Psc replicate 2

value: Psc


title: S2-DRSC-ChIP-Ph replicate 1

value: Ph


title: S2-DRSC-RNA

molecule: polyA RNA

extract_protocol: RNA was isolated using TRIzol reagent (Invitrogen) following the manufacturer’s instructions. Libraries for sequencing were prepared with the Illumina mRNA-Seq 8-Sample Prep Kit (Cat# RS-100-0801) according to Illumina's instructions. After adapter ligation library fragments of ~250 bp were band isolated from an agarose gel. The DNA was PCR amplified with Illumina primers for 15 cycles, purified and loaded on an Illumina flow cell for cluster generation. Libraries were sequenced on the Genome Analyzer II following the manufacturer's protocols.


title: S2-DRSC-ChIP-Ph replicate 2


title: S2-DRSC-ChIP-Psc replicate 1


title: ER E2 treat

status: Public on Dec 20 2010

city: Columbus

first: Fei

last: Gu

state: Ohio

department: Molecular Virology, Immunology & Medical Genetics

institute: The Ohio State University

description: Chromatin IP against ER

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.

organism: Homo sapiens

source_name: Tamoxifen resistance MCF7 cell line

growth_protocol: MCF7-T cells were maintained in a hormone-free medium (phenol red–free MEM with 2 mmol/L L-glutamine, 0.1 mmol/L nonessential amino acids, 50 units/mL penicillin, 50 Ag/mL streptomycin, 6 ng/mL insulin, and 10% charcoal-stripped FBS) supplemented with 10-7 mol/L 4-hydroxytamoxifen for 1 week to deplete any residual OHT, and then MCF7-T cells were cultured in hormone-free basal medium (phenol-red free MEM with 2 mM L-glutamine, 0.1 mM non-essential amino acids, 50 units/ml penicillin, 50 μg/ml streptomycin, and 3% charcoal-dextran stripped FBS) for three days.

extract_protocol: cells were crosslinked with 1% formaldehyde for 10 min, at which point 0.125 M glycine was used to stop the crosslinking. In brief, after crosslinking, cells were treated by lysis buffers and sonicated to fragment the chromatin to a size range of 200bp-1kb.

treatment_protocol: MCF7-T cells were treated with E2 (108 mol/L) for 3 hours

value: Breast cancer cell

value: MCF7, tamoxifen resistance

value: E2 treated

value: ER

tag: vendor

value: Santa Cruz Biotechnology

tag: catalog number


title: ER control

description: Chromatin IP against ER

value: untreated


title: S2-DRSC-ChIP-Pc replicate 1

value: Pc


title: Ezh2 RIP-seq Ezh2 null control

status: Public on Dec 22 2010

city: Boston

first: Mark

middle: L

last: Borowsky

address: 185 Cambridge Street

department: Molecular Biology

institute: MGH

description:
RIP against Ezh2
library strategy: RIP-seq
library source: RNA
library selection: RIP

data_processing:
Image processing and base calling were performed using the Illumina RTA pipeline.
Preprocessing: 3' adapter sequences were identified using cross_match and trimmed if a match of at least 5 bp was found. Reads matching mtDNA or rRNA sequences, reads < 12 bp long, reads containing N's, and homopolymer reads were removed. Alignment: reads were aligned to the mouse genome (mm9) using shortQueryLookup with a maximum of 1 error and no indels.  Frequencies of unique reads were then tabulated.

source_name: embryonic stem cells

growth_protocol: Mouse embryonic stem cells were routinely growed and maintained in T25 or T75 flasks with LIF supplemented in the culture medium.

extract_protocol: Cell nuclei were isolated, nuclear lysate was prepared and incubated with an anti-Ezh2 antibody. Afterwards, the RNA-protein complexes were immunoprecipitated with protein A agarose beads and the RNA fraction was extracted using Trizol reagent. Template switching method was used for the library construction per manufacturer's recommendation (Clontech's SMART cDNA Library Construction Manual).

value: embryonic stem cell

tag: antibody

tag: genome/variation

value: Ezh2 null


title: 2270 AF, small RNA

city: Nanchang

first: Lusheng

last: Huang

state: Jiangxi Province

address: No. 1101, Zhimin Avenue

country: China

institute: Jiangxi Agricultural University

description: expression profile of miRNAs

data_processing:
Sequence reads were obtained and filtered using the Illumina Genome Analyzer Pipeline.
small RNA tags were mapped to genome by SOAP2 (Li et al. 2009) to analyze their expression and distribution on the genome

organism: Sus scrofa

source_name: abdominal fat

growth_protocol: All animals were housed in the same environmental conditions and had a good body condition. The room temperatures were uncontrolled with natural lighting. Animals were floor fed three times a day.

extract_protocol: Total RNA was isolated with TRIzol (invitrogen) from each sample according to the manufacture’s instructions. Remaining DNA was removed with RNase-free DNase I (New England Biolabs) for 30 min at 37 ℃. The quality of total RNA was assessed by the 2100 Bioanalyzer (Agilent) and agarose gel electrophoresis.Small RNA library preparation was performed according to the illumina alternative v1.5 protocol for small RNA Sequencing. Briefly, small RNA sized at 18-30 nt was purified from total RNA through PAGE gel for each of six samples. 3’ and 5’ Illumina RNA adapters were ligated to the small RNA molecules by T4 RNA ligase. The ligated small RNA was subsequently transcribed into cDNA and amplified for 15 cycles with PCR using primers that anneal to the ends of the adapters. After purified with gel, the amplified cDNA constructs were sequenced according to the Illumina GA platform sequencing protocols.

treatment_protocol: All samples were put into the liquid nitrogen within 30 min after slaughtering, and then conserved in -80oC ultra freezer until RNA extraction.

value: F2 female from White Duroc × Erhualian

tag: sib

value: abdominal fat

value: Age of 240 days


title: 2268 LD, small RNA

description: expression profile of miRNAs

source_name: longissimus dorsi muscle

value: longissimus dorsi muscle


title: 2270 LI, small RNA

description: expression profile of miRNAs

source_name: Liver

value: Liver


title: 2268 LI, small RNA

description: expression profile of miRNAs


title: 2270 LD, small RNA

description: expression profile of miRNAs


title: 2268 AF, small RNA

description: expression profile of miRNAs


title: Ezh2 RIP-seq technical replicate


title: PolII control

description: Chromatin IP against PolII

value: PolII


title: Ezh2 RIP-seq

data_processing:
Image processing and base calling were performed using the Illumina pipeline.
Preprocessing: 3' adapter sequences were identified using cross_match and trimmed if a match of at least 5 bp was found.  Reads matching mtDNA or rRNA sequences, reads < 12 bp long, reads containing N's, and homopolymer reads were removed.
Alignment: reads were aligned to the mouse genome (mm9) using shortQueryLookup with a maximum of 1 error and no indels.

growth_protocol: mouse embryonic stem cells were routinely growed and maintained in T25 or T75 flasks with LIF supplemented in the culture medium.

treatment_protocol: Untreated


title: S2-DRSC-ChIP-Pc replicate 2


title: NEC_input

status: Public on Dec 15 2010

first: Alvaro

last: Rada

state: CA

address: 269 Campus Drive, CCSR Building

department: Chemical and Systems Biology

description: Total genomic DNA (input from ChIPs)

data_processing: All sequences were mapped by ELAND software (Illumina Inc) using the hg18 human genome assembly and analyzed by QuEST 2.4 software (Valouev et al (2008)). ChIP-seq enrichment regions were determined using  settings according to QuEST recommendations.  Valouev, A. et al. Genome-wide analysis of transcription factor binding sites based on ChIP-Seq data. Nat Methods 5, 829-834 (2008).

source_name: Neuroectodermal sphere cells (NEC) derived from H9 ESC

growth_protocol: hESCs (H9 line, Wi-Cell) were expanded in feeder free, serum free medium, mTESR-1 from StemCell technologies. Cells were passaged 1:7 every 5-6 days by incubation with accutase (Invitrogen) long and resultant small cell clusters were subsequently re-plated on tissue culture dishes coated overnight with growth factor reduced matrigel (BD biosciences). hESCs quality was regularly tested by evaluating the expression of a panel of hESC markers (e.g. Alkaline phosphatase, OCT4) and the capacity to differentiate into cell types derived from the three germ layers.

extract_protocol:
Lysates were clarified from sonicated nuclei and ChIP-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 size selected on agarose gel from 200-700 bp. Then isolated DNA was PCR amplified with Illumina primers for 18 cycles. 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.
Antibodies used: p300: Santa Crux Biotechnologies (sc-585). Lot#K1609BRG1: clone JA1, a kind gift from Dr. G. CrabtreeH3K4me1: Abcam (ab8895), lot#730178H3K27ac: Abcam (ab4729), lot#733245H3K27me3: Active Motif (39536) lot#09508002H3K4me3: Active Motif (39159) lot#01609004

treatment_protocol: hESCs were differentiated into hNECs using a previously described differentiation protocol 21. Briefly, hESC were incubated with 2mg/ml collagenase. Once detached, cells were plated in NEC differentiation media: 1:1 Neurobasal medium/D-MEM F-12 medium (Invitrogen), 0.5x B-27 supplement minus Vitamin A (50x stock, Invitrogen),  0.5x N-2 supplement (100x stock, Invitrogen),  20 ng/ml bFGF (Peprotech),  20 ng/ml EGF (Sigma-Aldrich), 5 µg/ml bovine insulin (Sigma-Aldrich),  0.1 µg/ml recombinant human NOGGIN (Peprotech), 1x Glutamax-I supplement (100x stock, Invitrogen). Cells were differentiated for seven days, changing media every other day.

value: NEC

value: input


title: NEC_H3K27ac

description: ChIP against H3K27ac


title: ESC_FAIRE

description: FAIRE (formaldehyde assisted isolation of regulatory elements) in crosslinked H9 ESC

source_name: Human embryonic stem cells

value: H9 ESC

value: FAIRE


title: NEC_H3K4me1

description: ChIP against H3K4me1


title: NEC_p300

description: ChIP against p300


title: ESC_H3K4me3

description: ChIP against H3K4me3


title: NEC_H3K27me3

description: ChIP against H3K27me3


title: ESC_H3K4me1

description: ChIP against H3K4me1


title: PolII E2 treat

description: Chromatin IP against PolII

value: Breast cancer cell

value: MCF7, tamoxifen resistance

value: E2 treated


title: ESC_H3K27ac

description: ChIP against H3K27ac


title: ESC_BRG1

description: ChIP against BRG1


title: ESC_input

description: Total genomic DNA (input from ChIPs)


title: NEC_H3K4me3

description: ChIP against H3K4me3

extract_protocol: Lysates were clarified from sonicated nuclei and ChIP-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 size selected on agarose gel from 200-700 bp. Then isolated DNA was PCR amplified with Illumina primers for 18 cycles. 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.


title: Ezh2 RIP-seq biological replicate


title: NEC_RNAseq

description: polyA RNA

data_processing: RNA-seq libraries were sequenced with Illumina Genome Analyzer and both mapping and analysis of resulting reads were perfomed with DNAnexus software tools (https://dnanexus.com)

extract_protocol: RNAs from hESC and NEC were extracted with Trizol (invitrogen), following manufacturer’s recommendations. 10 µg of total RNA were subjected to two rounds of oligo-dT purification using Dynal oligo (dT) beads (Invitrogen). 100 ng of the purified RNA were fragmented with 10x Fragmentation Buffer (Ambion). Fragmented RNA was used for first strand cDNA synthesis, using random hexamer primers (Invitrogen) and SuperScript II enzyme (Invitrogen). Second strand cDNA was obtained by adding RNAseH (Invitrogen) and DNA Pol I (New England Biolabs) to the first strand cDNA mix. The resulting double-stranded cDNA was used for Illumina library preparation 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 size selected on agarose gel from 200-700 bp. Then isolated DNA was PCR amplified with Illumina primers for 18 cycles. 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: hESCs were differentiated into hNECs using a previously described differentiation protocol 21. Briefly, hESC were incubated with 2mg/ml collagenase. Once detached, cells were plated in NEC differentiation media: 1:1 Neurobasal medium/D-MEM F-12 medium (Invitrogen), 0.5x B-27 supplement minus Vitamin A (50x stock, Invitrogen), 0.5x N-2 supplement (100x stock, Invitrogen), 20 ng/ml bFGF (Peprotech), 20 ng/ml EGF (Sigma-Aldrich), 5 µg/ml bovine insulin (Sigma-Aldrich), 0.1 µg/ml recombinant human NOGGIN (Peprotech), 1x Glutamax-I supplement (100x stock, Invitrogen). Cells were differentiated for seven days, changing media every other day.


title: ESC_H3K27me3

description: ChIP against H3K27me3


title: ESC_p300

description: ChIP against p300


title: ESC_RNAseq

description: polyA RNA


title: Adult_F_Cb_MeDIP

status: Public on Dec 12 2010

city: Atlanta

first: Keith

middle: E

last: Szulwach

state: GA

address: 615 Michael Street, Whitehead Suite 301

department: Human Genetics

institute: Emory University

description: 5hmC Capture and Sequencing

data_processing: Processed data not provided.

source_name: C57/BL6 Cerebellum

extract_protocol: Genomic DNA was purified from tissue using Wizard genomic DNA purification kit (Promega) with additional Proteinase K treatment and rehydrated in 10 mM Tris (pH 7.9). Genomic DNA samples were sonicated to 100-500 bp by Misonix sonicator 3000 (using microtip, 3 pulses of 30 seconds each with 2 min of rest and a power output level of two) or Bioruptor UCD-200 sonicator (Diagenode, Sparta). The output selector switch was set on High (H), and sonication interval was 30 seconds with 30 cycles of sonication performed. 5-hmC labelling reactions were performed in a 100-µL solution containing 50 mM HEPES buffer (pH 7.9), 25 mM MgCl2, 300 ng/μL sonicated genomic DNA (100-500 bp), 250 μM UDP-6-N3-Glu or UDP-Glu, and 2.25 μM wild-type BGT. The reactions were incubated for 1 hr at 37 °C. After the reaction, the DNA substrates were purified by Qiagen DNA purification kit or by phenol-chloroform precipitation and reconstituted in H2O. Biotin addition by click chemistry was performed by adding 150 μM dibenzocyclooctyne modified biotin into the DNA solution, and the reaction mixture was incubated for 2 hr at 37 °C. The DNA samples were then purified by Qiagen DNA purification kit. Biotin Labeled 5-hmC DNAs were purified/enriched by Pierce Monomeric Avidin Kit (Thermo) following manufacturer’s recommendations. After elution, the biotin-5-N3-gmC containing DNA was concentrated by 10 K Amicon Ultra-0.5 mL Centrifugal Filters (Millipore) and purified by Qiagen DNA purification kit. 25 ng genomic DNA, 5-hmC-captured DNA, or control captured DNA (in the absence of biotin) were used to initiate library preparation following the Illumina protocol for “Preparing Samples for ChIP Sequencing of DNA” (Part# 111257047 Rev. A). DNA fragments ~150-300 bp were gel purified after the adapter ligation step and amplified by 18 cycles PCR. MeDIP was performed using 4 μg of the same DNA used for 5hmC enrichment diluted into 450 μL 1X TE. DNA was denatured for 10 min at 100°C in a dry heat block, and then immediately placed on ice for 5-10 min. 51 μL of 10X IP Buffer (100 mM Na-phosphate pH7.0 made from a 1M stock solution (2M monobasic sodium phosphate, 2M dibasic sodium phosphate at 1:1.564 ratio, and equal volume H2O), 1.4M NaCl, and 0.5% Triton-X100 was added along with 10 μg 5-methylcytidine antibody (Eurogentec #BI-MECY-0500). IP was done at 4°C with rotation for 2 h. Antibody-DNA complexes were pulled down by adding 40 μL Dynabeads (M-280) Sheep anti-Mouse IgG (Dynal Biotech, Invitrogen, #112.01) directly to the IP reaction at 4°C for 2 h with rotation. Beads were collected with a 1.5-mL microfuge tube holder magnet and washed 3 times in 1X IP Buffer at room temperature, 10 min per wash with rotation. Washed beads were collected by magnet and resuspended in 250 μL Proteinase K Digestion Buffer (50 mM Tris, pH8.0, 10 mM EDTA, 0.5% SDS). 3.5 μL of 20 mg/mL Proteinase K was added and digestion was performed at 50°C for 3 h in an Eppendorf Thermomixer set at 800 rpm. Beads were collected by magnet, and DNA was extracted from the supernatant 1X with phenol and 1X with chloroform. DNA was precipitated with 400 mM NaCl, 15 μg linear acrylamide, and 2 volumes 100% ethanol at -35°C overnight. 25ng 5mC enriched DNA was used to initiate library preparation following the Illumina protocol for “Preparing Samples for ChIP Sequencing of DNA” (Part# 111257047 Rev. A). DNA fragments ~150-300 bp were gel purified after the adapter ligation step and amplified by 18 cycles PCR.

tag: age

tag: Sex

value: Female

tag: treatment

value: 5mC enriched DNA (MeDIP)


title: Adult_M_Cb_5hmC_2

description: 5hmC Capture and Sequencing

tag: strain

value: Male

value: 5hmC enriched DNA


title: Adult_F_Cb_5hmC_4

description: 5hmC Capture and Sequencing


title: Adult_M_Cb_5hmC_1

description: 5hmC Capture and Sequencing


title: Adult_F_Cb_5hmC_3

description: 5hmC Capture and Sequencing


title: ATRX_Mouse_ES_Input

status: Public on Dec 13 2010

city: Oxford

first: Richard

middle: J

last: Gibbons

state: Oxon

address: John Radcliffe Hospital,

department: MRC Molecular Haematology Unit

country: United Kingdom

institute: Weatherall Institute of Molecular Medicine

description: Input DNA

data_processing: For the mouse ES cell ATRX ChIP-seq 51bp reads were used. A total of 42,550,172 ATRX ChIP reads and 61,646,974 input reads were generated. Reads were mapped to the mm9 (NCBI37) mouse genome build without repeatmasking using Bowtie (Langmead, B., Trapnell, C., Pop, M., and Salzberg, S.L. (2009). Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol 10, R25.) with –best, -strata and m -5 parameters. 22,426,377 ChIP reads and 4,251,942 input reads aligned to the genome. Peaks were called using Seqmonk v0.9 with a contig probe generator and the input sequence as a reference. For both mouse and human data, peaks within 1 kb of an adjacent peak were merged into one.

source_name: Mouse Embryonic Stem Cells

growth_protocol: Mouse ES cells were cultured according to standard procedures.

extract_protocol: Chromatin Immunoprecipitation coupled with Microarray Analysis (ChIP-chIP): I. Dynabead Preparation -Make PBS/BSA -For each ChIP reaction. Wash 100ul of beads 3-4 times with PBS BSA (5mg/ml) -Transfer beads to a 500ul PCR tube. -For ATRX antibody binding, Add 200ul ATRX H300, 22ul 50mg/ml BSA, 4ul ssDNA (Invitrogen 10mg/ml) -For NoAb Rxn. Add 222ul PBS BSA 4ul ssDNA -Incubate 3-4 hrs on roller at 4 oC -Wash 4 times in PBS BSA -Resuspend in 100ul PBSBSA and add 4ul (40ug) ssDNA For each preclear reaction -Take 100ul of Dynabeads -Wash 3-4 times with 1ml PBS BSA (5mg/ml freshly prepared) -Add 100ul of PBS BSA(5ug/ul) -Add 4ul ssDNA (10ug/ul) -Incubate at 4oC on roller with antibody binding reactions -Use 100ul for each preclearing reaction II. Cell cross-linking: Need 4oC PBS, Formaldehyde, EGS 1M glycine, Trypan blue, gilsons, timer 1.5ml tubes, calculator, A. Suspension Cells: 1. Make up EGS (Pierce 21565) stock fresh: 0.046g EGS in 200ul DMSO for 500mM stock 2. Chill PBS 3. Wash twice in PBS and resuspend in 20mls PBS (Important as FCS inhibits EGS) 4. Count cells 5. Use 3x107 – 5x107 cells for each ChIP reaction 6. Add EGS (it will form a white PPT which disperses afer a second) to 2mM and incubate at room temperature of 45 mins with occasional mixing. (80ul per 20mls of PBS). 7. Add formaldehyde solution to 1% for 20mins (526ul /20mls. 4 x 131.5ul) 8. Add 1/8 volume of 1M glycine to tubes to quench formaldehyde.(2.5ml glycine / 20ml PBS). 9. Spin cells at 1k for 5 min at 4oC. Make up protease inhibitors and add to lysis buffers. 10.Wash twice with 20ml 4oC PBS III. Cell sonication (keep all steps on ice): Note: Add protease inhibitors to all lysis buffers before use. If using a protease inhibitor cocktail tablet from Complete, dissolve one tablet in 2 ml water for 25X solution. Store aliquots at -20oC. 1. Resuspend each tube of cells in 10 ml of lysis buffer I. Rock at 4oC for 10 min, and then spin at 2k in a table-top centrifuge for 2 min at 4oC. 2. Resuspend each tube of cells in 5-10 ml of lysis buffer 2. Pellet nuclei in table-top centrifuge by spinning at 2.5k for 2 min at 4oC. 3. Resuspend the pellet in each tube in 3 ml lysis buffer 3. (Retain the buffer for step 6) 4. Sonicate the suspension in 15ml corning blue top tubes. 2mls per tube. Use the Bioruptor at high power for 20mins (4x5mins). After each 5 minute pulse, replenish the ice. 5. Add 1/10 volume of 10% Triton X-100. Transfer to 1.5 ml centrifuge tubes. Spin out debris at 14k for 10 min at 4oC. The lysate contains the fragmented DNA. 6. Save pellet for western analysis and take 50ul lysate for western analysis 7. Transfer lysate to 15 ml conical tube, adjusting the volume to 3 ml using lysis buffer 3. 8. Save 50µl of cell lysate from each sample as input. Save 50ul to assess sonication efficiency. IV. Chromatin immunoprecipitation: -Add 100ul of the preclearing beads from step I to the 3mls of chromatin lysate -Preclear on roller at 4 oC for 1 hour -Precipitate beads from chromatin using magnet and remove chromatin to new 3ml tubes -Add the washed antibody bound beads from step 1 -Incubate overnight on roller at 4 oC V. Washing, eluting, and reverse cross-linking: Note: Carry steps 1-3 in the cold room and all wash solution must be ice cold. 1. Transfer lysates from the 3 ml tubes to a 1.5ml tube. 2. Wash the IPs 5 times with 1 ml wash buffer (be sure to add protease inhibitors to the wash buffer). Use the magnetic stand to precipitate the beads. 3. Wash once with 1 ml cold PBS. Aspirate residual PBS. 4. Add 250µl of elution buffer. Elute on a roller at room temperature for 15 minutes. 5. Add 250ul more of elution buffer to beads, vortex, precipitate and combine with previous round of elution. 6. Take 50ul of eluate for western blot analysis 7. PPT beads and remove eluate to a new tube. Add 20ul 5M NaCl and incubate at 65oC for 4 hours. Thaw input, make up to 500ul with elution buffer, add 20ul 5M NaCl and incubate for 4hrs at 65oC. VI. RNase, Proteinase K, Phenol/Chloroform, EtOH PPT: Remove beads. Then add 20ul of 1M Tris pH6.5, 10ul 0.5M EDTA, 2ul 10mg/mg Proteinase K. Incubate for 1hrs at 45°C. Phenol chloroform and EtOH PPT with 20ug of carrier glycogen. Resuspend in 21ul sigma water. Use low volume to give the option of labelling for ChIP on Chip. For real time PCR, use 1/12 ul per reaction Solutions PBS BSA 5mg/ml For 50mls: 5mls 10XPBS, 250mg BSA Fraction V, Filter sterilise. Make up fresh every time. Lysis Buffer 1 (100 ml): 100mM HEPES 140mM NaCl 1mM EDTA 10% Glycerol 0.5% NP40 0.25% Triton X Lysis Buffer 2 (100 ml): 200mM NaCl 1mM EDTA 0.5mM EGTA 10mM Tris pH 8 Lysis Buffer 3 (100 ml): 1mM EDTA 0.5mM EGTA 1mMTris-HCl pH8 100mM NaCl 0.1% Na DOC 0.5% N-lauroyl Sarcosine Wash buffer (RIPA Buffer, 100 ml): 50mM Hepes, pH 7.6 1mM EDTA 0.7% Na DOC 1% NP40 500mM LiCl

tag: passage no.


title: Pol2-S2P_Day2+8h_ChIP-seq

city: University Park

first: Shaun

last: Mahony

state: PA

address: 404 South Frear Bldg

department: Biochemistry & Molecular Biology

institute: Penn State University

description: Chromatin IP against Pol2-S2P

data_processing:
Alignment: Sequence reads were aligned to the mouse genome (version mm8) using Bowtie (Langmead et al Genome Biology 2009) version 0.9.9.2 with options -k 2 --best. Only uniquely mapping reads were analyzed further. Multiple hits aligning to the same nucleotide position are discarded above the level expected at a 10^-7 probability from a per-base Poisson model of the uniquely mappable portion of the mouse genome. In practice, this caps the number of hits that start at the same nucleotide to 3 in the RAR ChIP-seq experiments.
Peaks: Binding event detection is carried out using a customized methodology named SEEDS (Sequencing Experiment Event Detection Statistics) that uses statistical significance testing to find regions producing an over-abundance of sequenced reads in the signal experiments compared with the control. The algorithm is run twice across the data. The first pass estimates a scaling factor for control sequencing read depth and a model of the distribution of sequencing read alignment hits around binding events. The second pass applies these parameters to predict a final set of significant events. Before the first pass, the scaling factor is initialized to be the ratio of total hit counts between the signal and control channels. The binding distribution model is initialized to be an empirical distribution estimated around predicted binding events in Oct4 ChIP-seq data (Marson, et al. Cell 2008). All alignment hits are extended in both 3’ and 5’ directions, mirroring the observed distribution of hits around binding events. The extension magnitudes are set equal to the positions where the binding model distribution intersects a uniform distribution over the same area. Control channel hit counts are scaled using the signal-control scaling factor. A sliding window of bin width 50bp and offset 25bp is run over the genome. Overlapping extended hit counts are calculated for both the signal and (scaled) control channels. The background distribution of ChIP-seq hits is modeled as a non-homogenous Poisson process with parameters estimated from the scaled control hit counts. Specifically, the Poisson parameter is chosen as the maximum mean overlapping hit count in 50bp windows of those observed from i) the entire genome, ii) a 5Kbp window centered on the current location, and iii) a 10Kbp window centered on the current location. The use of this dynamic background model is motivated by the desire to correct local ChIP-seq enrichment biases that appear in the signal and control channels, and is similar to the model employed by MACS (Zhang, et al. Genome Biol 2008). A given bin is denoted as potentially enriched if the overlapping hit count exceeds that expected from the background model at a p-value of 10^-9. P-values for each potentially enriched bin’s over-representation in the signal channel over the control are calculated using the binomial distribution CDF (see Rozowsky, et al. Nature Biotech 2009). Neighboring regions in the set of potentially enriched regions are merged, and the maximal p-value observed for the constituent bins is attached to the resulting merged region. The p-values are corrected for multiple hypothesis testing using Benjamini & Hochberg’s method, and all regions with corrected p-values above 0.001 are discarded. False-discovery rates are estimated by repeating the event discovery procedures after swapping the scaled control channel and the signal channel. After the first pass, the scaling factor is estimated by carrying out linear regression on the hit counts observed in 10,000 bp windows that are devoid of potentially significant events in both the signal and control channels. The binding model is estimated from enriched regions with p-values < 10^-7 and signal/control hit count enrichment greater than 10. These regions are aligned around the 'peak' location, defined as the position of maximum probability when scanning the current binding model over the region's hit landscape. To capture broader domains of enrichment for Pol2-S2P ChIP-seq, the bin width was set to 500bp and the bin offset to 250bp. The background model was also set to a homogeneous Poisson threshold estimated from the entire genome. The significance threshold was raised to p<0.01 for domain calling.

source_name: Day2+8hrs post-RA exposure

growth_protocol: ES cells were differentiated as previously described (Wichterle, et al. Cell 2002).  Briefly, ES cells were trypsinized and seeded at 5x10e5 cells/ml in ANDFK medium (Advanced DMEM/F12:Neurobasal (1:1) Medium, 10% Knockout-SR, Pen/Strep, 2 mM L-Glutamine, and 0.1 mM 2-mercaptoethanol) to initiate formation of embryoid bodies (Day 0). Medium was exchanged on Day 1, Day 2 and Day 5 of differentiation.  Patterning of embryoid bodies was induced by supplementing media on Day 2 with 1 µM all-trans-Retinoic acid (RA, Sigma) and 0.5 µM agonist of hedgehog signaling (SAG, Calbiochem). For ChIP experiments, the same conditions were used but scaled to seed 1x10e7 cells on Day 0

extract_protocol: ChIP protocols were adapted from http://jura.wi.mit.edu/young_public/hESregulation/ChIP.html.  Descriptions of these protocol modifications have been previously published [51]. Briefly, approximately 6x10e7 cells taken from each developmental time point were cross-linked using formaldehyde and snap-frozen in liquid nitrogen.  Cells were thawed on ice, resuspended in 5ml lysis buffer 1 (50 mM Hepes-KOH, pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100) and mixed on a rotating platform at 4°C for 5 minutes.  Samples were spun down for 3 minutes at 3000rpm, resuspended in 5ml lysis buffer 2 (10 mM Tris-HCl, pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA), and mixed on a rotating platform for 5 minutes at room temperature.  Samples were spun down once more, resuspended in lysis buffer 3 (10 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% Na-Deoxycholate, 0.5% N-lauroylsarcosine) and sonicated using a Misonix 3000 model sonicator to sheer cross-linked DNA to an average fragment size of approximately 500bp.  Triton X-100 was added to the lysate after sonication to final concentrations of 1% and the lysate spun down to pellet cell debris.  The resulting whole-cell extract supernatant was incubated on a rotating mixer overnight at 4°C with 100 µL of Dynal Protein G magnetic beads that had been preincubated for 24 hours with 10 µg of the appropriate antibody in a PBS/BSA solution. Pan-RAR (Santa Cruz Biotechnology, sc-773), Pol2-S5P (Abcam, ab5131), and Pol2-S2P (Abcam H5 clone ab24758) antibodies were used for ChIP experiments. After approximately 16 hours of bead-lysate incubation, beads were collected with a Dynal magnet.  ChIP samples probing for transcription factor binding were washed with the following regimen, mixing on a rotating mixer at 4°C for 5 minutes per buffer: low-salt buffer (20 mM Tris at pH 8.1, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% SDS), high-salt buffer (20 mM Tris at pH 8.1, 500 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% SDS), LiCl buffer (10 mM Tris at pH 8.1, 250 mM LiCl, 1 mM EDTA, 1% deoxycholate, 1% NP-40), and TE containing 50 mM NaCl.  ChIP samples probing for histone and chromatin marks were washed 4 times with RIPA buffer (50 mM Hepes-KOH, pH 7.6, 500 mM LiCl, 1 mM EDTA, 1% NP-40, 0.7% Na-Deoxycholate) and then once with TE containing 50 mM NaCl, again mixing on a rotating mixer at 4°C for 5 minutes per buffer.  After the final bead wash, samples were spun down to collect and discard excess wash solution, and bound antibody-protein-DNA fragment complexes were eluted from the beads by incubation in elution buffer at 65°C with occasional vortexing.  Cross-links were reversed by overnight incubation at 65°C.  Samples were digested with RNase A and Proteinase K to remove proteins and contaminating nucleic acids, and the DNA fragments precipitated with cold EtOH. Purified DNA fragments were processed according to a modified version of the Illumina/Solexa sequencing protocol (Illumina, http://www.illumina.com/pages.ilmn?ID=252).

treatment_protocol: Ectopic exposure to retinoic acid and hedgehog agonist at day 2 during the differentiation protocol.

tag: cell-line

tag: cell-stage

tag: chip-antibody

value: Pol2-S2P (Abcam H5 clone ab24758)


title: P7_Cb_5hmC

description: 5hmC Capture and Sequencing


title: Adult_F_Cb_5hmC_2

description: 5hmC Capture and Sequencing


title: Adult_F_Cb_5hmC_1

description: 5hmC Capture and Sequencing


title: Adult_F_Cb_Input_1

description: 5hmC Capture and Sequencing

value: Non-enriched genomic DNA


title: ASSAGE_breast_epithelium_cancer_T1

status: Public on Dec 10 2010

first: Kornelia

last: Polyak

address: 450 Brookline Ave

department: Medical Oncology

institute: Dana-Farber Cancer Institute

data_processing: We generated a bisulfite-converted genome for both plus and minus strands (6.2 Gb) as a reference for ASSAGE reads. We used SeqMap (http://biogibbs.stanford.edu/~jiangh/SeqMap/)to align reads to the bisulfite-converted genome under default conditions, allowing up to two mismatches in 32-bp reads. We used reads that uniquely mapped to either plus or minus strand-derived sequences.

source_name: biopsy and surgery

extract_protocol: ASSAGE libraries were generated essentially as described (PMID: 19056939), except that we used polyA RNA. Briefly,we treated polyA RNA with bisulfite, which changes all cytidine residues to uridine residues. We generated cDNA from bisulfite-treated RNA with reverse transcriptase, the double-stranded cDNA was used to construct libraries for sequencing following Illumina's standard genomic DNA sample preparation instructions. The sequence of a bisulfite-treated RNA molecule can only be matched to one of the two possible DNA template strands.

value: breast

tag: cell type

value: epithelial cell

tag: disease state

value: cancer

tag: cell marker


title: Adult_F_Cb_Input_2

description: 5hmC Capture and Sequencing


title: ASSAGE_breast_epithelium_cancer_T2

tag: tissue


title: Adult_F_Cb_Control

description: 5hmC Capture and Sequencing

value: UDP-Glucose, no biotin control


title: breast_epithelium_normal_CD44_P3

description: Sage-Seq

data_processing: Sequencing was carried out at the Wistar Institute. The first 17bps of each read is used as a tag. The number of each unique tag is counted.   Processed data file includes tag sequences (17bps) in the first column and counts (frequencies) in the second column. Illumina software pipeline OLB version 1.6, 1.7, 1.8 with CASAVA 1.5 was used for generation  of sequence: frequency processed files.

source_name: reduction mammoplasty

extract_protocol: cDNA made from polyA RNA isolated from purified cells using antibody attached to beads were fragmented with NlaIII/MmeI restriction enzyme with subsequent adapter ligation.

value: normal

tag: antibody vender

value: BD-Pharmingen

tag: antibody catalog #


title: breast_epithelium_cancer_SSEA4_IDC38

description: Sage-Seq

value: R&D Systems


title: breast_epithelium_normal_CD44_P2

description: Sage-Seq


title: breast_epithelium_normal_CD24_P2

description: Sage-Seq


title: Adult_M_Cb_Input

description: 5hmC Capture and Sequencing


title: breast_epithelium_normal_CD44_P1

description: Sage-Seq


title: ASSAGE_breast_epithelium_normal_N1


title: Adult_M_Cb_Control

description: 5hmC Capture and Sequencing


title: E16-24h_anti-Bonus-[GP37]_INPUT_1

status: Public on Dec 09 2010

city: Chicago

first: Kevin

middle: P.

last: White

state: IL

address: 900 E. 57th STR. 10th FL.

department: Institute for Genomics and Systems Biology

institute: University of Chicago

data_processing: Reads were aligned to the flybase BDGPv5 reference genome using Bowtie 0.12.5. Signal files were generated with the R 2.7.1 package SPP 2.7.1. Peaks were called with MACS 1.3.7.

growth_protocol: 1. the iso1 (y; bw cn sp) flies or the transgenic flies are cultivated in cages with apple juice agar plates covered with yeast powder. and the egg laying is performed for the desired amount of time to correspond to the proper stage. The biological material is collected using a filter mesh and a brush and rinsed extensively with Embryonic Wash Buffer (EWB). The material is then used freshly for cross-linking.

extract_protocol: After chromatin immuno-precipitation, the DNA is purified in 30ul of water.  Libraries were prepared according to Illumina's instructions accompanying the DNA Sample Kit (Part# 0801-0303). Briefly, DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (32 to 52 exo minus) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3' end. After adapter ligation DNA was PCR amplified with Illumina primers for 15 cycles and library fragments of ~250 bp (insert plus adaptor and PCR primer sequences) were band isolated from an agarose gel. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Genome Analyzer following the manufacturer's protocols.

treatment_protocol: No Treatment

tag: developmental stage

tag: fly

value: iso1 (y; bw cn sp)


title: breast_epithelium_cancer_CD24_IDC36

description: Sage-Seq


title: WCE_Day3_seq

description: WCE control

data_processing:
Alignment: Sequence reads were aligned to the mouse genome (version mm8) using Bowtie (Langmead et al Genome Biology 2009) version 0.9.9.2 with options -k 2 --best. Only uniquely mapping reads were analyzed further. Multiple hits aligning to the same nucleotide position are discarded above the level expected at a 10^-7 probability from a per-base Poisson model of the uniquely mappable portion of the mouse genome. In practice, this caps the number of hits that start at the same nucleotide to 3 in the RAR ChIP-seq experiments.
Peaks: Binding event detection is carried out using a customized methodology named SEEDS (Sequencing Experiment Event Detection Statistics) that uses statistical significance testing to find regions producing an over-abundance of sequenced reads in the signal experiments compared with the control. The algorithm is run twice across the data. The first pass estimates a scaling factor for control sequencing read depth and a model of the distribution of sequencing read alignment hits around binding events. The second pass applies these parameters to predict a final set of significant events. Before the first pass, the scaling factor is initialized to be the ratio of total hit counts between the signal and control channels. The binding distribution model is initialized to be an empirical distribution estimated around predicted binding events in Oct4 ChIP-seq data (Marson, et al. Cell 2008). All alignment hits are extended in both 3’ and 5’ directions, mirroring the observed distribution of hits around binding events. The extension magnitudes are set equal to the positions where the binding model distribution intersects a uniform distribution over the same area. Control channel hit counts are scaled using the signal-control scaling factor. A sliding window of bin width 50bp and offset 25bp is run over the genome. Overlapping extended hit counts are calculated for both the signal and (scaled) control channels. The background distribution of ChIP-seq hits is modeled as a non-homogenous Poisson process with parameters estimated from the scaled control hit counts. Specifically, the Poisson parameter is chosen as the maximum mean overlapping hit count in 50bp windows of those observed from i) the entire genome, ii) a 5Kbp window centered on the current location, and iii) a 10Kbp window centered on the current location. The use of this dynamic background model is motivated by the desire to correct local ChIP-seq enrichment biases that appear in the signal and control channels, and is similar to the model employed by MACS (Zhang, et al. Genome Biol 2008). A given bin is denoted as potentially enriched if the overlapping hit count exceeds that expected from the background model at a p-value of 10^-9. P-values for each potentially enriched bin’s over-representation in the signal channel over the control are calculated using the binomial distribution CDF (see Rozowsky, et al. Nature Biotech 2009). Neighboring regions in the set of potentially enriched regions are merged, and the maximal p-value observed for the constituent bins is attached to the resulting merged region. The p-values are corrected for multiple hypothesis testing using Benjamini & Hochberg’s method, and all regions with corrected p-values above 0.001 are discarded. False-discovery rates are estimated by repeating the event discovery procedures after swapping the scaled control channel and the signal channel. After the first pass, the scaling factor is estimated by carrying out linear regression on the hit counts observed in 10,000 bp windows that are devoid of potentially significant events in both the signal and control channels. The binding model is estimated from enriched regions with p-values < 10^-7 and signal/control hit count enrichment greater than 10. These regions are aligned around the 'peak' location, defined as the position of maximum probability when scanning the current binding model over the region's hit landscape.

source_name: Day3, seq

growth_protocol: ES cells were grown on ES Medium (DMEM, 15% FBS, Non-essential amino acids, Glutamine, Pen/Strept, 2-mercaptoethanol and LIF) on feeder cells for amplification or on Gelatin for differentiation and experiments.  Motor neuron differentiation was done as described (Wichterle & Peljto, Curr Protoc Stem Cell Biol, 2009). Briefly, ES cells were tripsinized  and allowed to form aggregates in ANDFK medium (Advanced DMEM/F12,  Neurobasal Medium, Knockout-SR, of Pen/Strep, L-Glutamine, and 2-mercaptoethanol. Day 0 is the day of tripsinization. At Day 2, RA is added.

extract_protocol: ChIP protocols were adapted from http://jura.wi.mit.edu/young_public/hESregulation/ChIP.html.  Briefly, approximately 3x10e7 cells taken from each developmental time point were cross-linked using formaldehyde and snap-frozen with liquid nitrogen.  Cells were thawed on ice, resuspended in 5ml lysis buffer 1 (50 mM Hepes-KOH, pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100) and mixed on a rotating platform at 4°C for 5 minutes.  Samples were spun down for 3 minutes at 3000rpm, resuspended in 5ml lysis buffer 2 (10 mM Tris-HCl, pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA), and mixed on a rotating platform for 5 minutes at room temperature.  Samples were spun down once more, resuspended in lysis buffer 3 (10 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% Na-Deoxycholate, 0.5% N-lauroylsarcosine) and sonicated using a Misonix 3000 model sonicator to sheer cross-linked DNA to an average fragment size of approximately 500bp.  Triton X-100 was then added to the lysate after sonication to final concentrations of 1% and the lysate spun down to pellet cell debris.  The resulting whole-cell extract supernatant was incubated on a rotating mixer overnight at 4°C with 100 µL of Dynal Protein G magnetic beads that had been preincubated the previous 24 hours with 10 µg of the appropriate antibody in a PBS/BSA solution.  After approximately 16 hours of bead-lysate incubation, beads were collected with a Dynal magnet.  ChIP samples probing for transcription factor binding were washed with the following regimen, mixing on a rotating mixer at 4°C for 5 minutes per buffer: low-salt buffer (20 mM Tris at pH 8.1, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% SDS), high-salt buffer (20 mM Tris at pH 8.1, 500 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% SDS), LiCl buffer (10 mM Tris at pH 8.1, 250 mM LiCl, 1 mM EDTA, 1% deoxycholate, 1% NP-40), and TE containing 50 mM NaCl.  ChIP samples probing for histone and chromatin marks were washed 4 times with RIPA buffer (50 mM Hepes-KOH, pH 7.6, 500 mM LiCl, 1 mM EDTA, 1% NP-40, 0.7% Na-Deoxycholate) and then once with TE containing 50 mM NaCl, again mixing on a rotating mixer at 4°C for 5 minutes per buffer.  After the final bead wash, samples were spun down to collect and discard excess wash solution, and bound antibody-protein-DNA fragment complexes were eluted from the beads by incubation in elution buffer at 65°C with occasional vortexing.  Cross-links were reversed by overnight incubation at 65°C.  Samples were digested with RNase A and Proteinase K to remove proteins and contaminating nucleic acids, and the DNA fragments precipitated with cold EtOH.  Resulting purified DNA fragments were processed according to a modified version of the Illumina/Solexa sequencing protocol (Illumina, http://www.illumina.com/pages.ilmn?ID=252).  Descriptions of these protocol modifications and data handling have been previously published (Guenther, et al. Genes Dev 2008).

treatment_protocol: Ectopic exposure to retinoic acid

tag: fraction

value: WCE

tag: agent

value: RA


title: breast_epithelium_normal_CD24_P3

description: Sage-Seq


title: RAR_Day2_ChIP-seq

description: Chromatin IP against RAR

source_name: Day2 pre-RA, ChIP-seq

value: RAR sc-773

value: IP

value: none (pre-RA)


title: WCE_Day2_seq

description: WCE control

source_name: Day2 pre-RA, seq


title: RAR_Day2+8hrsRA_ChIP-seq

description: Chromatin IP against RAR

source_name: Day2+8hrs post-RA exposure, ChIP-seq


title: breast_epithelium_normal_CD24_P1

description: Sage-Seq


title: HPC GABPa ChIP-Seq

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 GABP-alpha

data_processing: Sequence reads were mapped to the human genome (hg18) using the Solexa Analysis Pipeline. The mapped GABP-alpha reads 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 GABP-alpha binding sites.

source_name: CD34+CD133+ hematopoietic progenitor cells (HPCs)

growth_protocol: CD34+CD133+ HPCs were isolated from healthy blood donors.

extract_protocol: Freshly isolated HPCs were crosslinked with formaldehyde and sonicated to generate chromatin fragments, which were immunoprecipitated with GABP-alpha or control IgG antibody. The DNA fragments were end-repaired and processed for sequencing on an Illumina Genome Analyzer using standard protocols.

tag: antibody

value: anti-GABP-alpha

tag: antibody manufacturer

value: Santa Cruz Biotechnology


title: HPC control IgG ChIP-Seq

description: Chromatin IP against IgG

value: CD34+CD133+ hematopoietic progenitor cells (HPCs)

value: anti-IgG


title: E16-24h_anti-Bonus-[GP37]_ChIPSeq_1 and 2

tag: antibody name

tag: manufacturer

value: Bellen Lab

tag: lot/batch#

tag: catalog


title: E0-12h_Su[var]3-9_INPUT_1


title: E16-24h_KW3-Kr-D2_INPUT_1


title: E0-12h_Su[var]3-9_ChIPSeq_1 and 2

value: Abcam


title: R13-E1-6h-Set2_GFP_INPUT_1

value: transgenic


title: E16-24h_KW3-Kr-D2_INPUT_2


title: E16-24h_KW3-Kr-D2_ChIPSeq_1 and 2

value: White Lab


title: 5T-8-16h_GFP_INPUT_1


title: 5T-8-16h_GFP_ChIPSeq_1 and 2

value: GFP


title: R13-E1-6h-Set2_GFP_INPUT_2


title: [E-MTAB-407] polyA+_kidney_7110096

status: Public on Dec 04 2010

city: Hinxton

first: ArrayExpress

last: EBI

state: Cambridgeshire

address: Wellcome Trust Genome Campus

institute: European Bioinformatics Institute

description: Performer: LGTF

data_processing: processed data not provided

extract_protocol:
nucleic_acid_extraction | RNA from tissues was isolated by a modified guanidium thiocynate methods followed by polyA+ RNA selection with two rounds of oligo(dT)-cellulose columns. Selected targets were then amplified by RT-PCR.
sequencing | Standard Illumina protocol, performed at the University of Lausanne, Lausanne, Switzerland

tag: material type

tag: organismpart

value: kidney

tag: Sex

tag: biosourcetype

tag: biosourceprovider

value: Clontech


title: Th2_IgG_control

status: Public on Dec 07 2010

city: Coventry

last: Hebenstreit

address: Gibbet Hill Rd

institute: University of Warwick

description: Chromatin IP with non-specific IgG antibody.

data_processing: Reads were mapped to genome build mm9 with Bowtie. Only uniquely mapping reads were kept.

source_name: Th2 cells, IgG

growth_protocol: Murine CD4+CD62L+ splenocytes were activated for 5 days with anti-CD3/CD28 in the presence 10 ng/ml IL-4 and 2 ug/ml anti-IFN-g, followed by 3 days of resting.

extract_protocol: Preparation of the samples was based on Wilson NK et al. Blood, 2009 (PMID 19346495). Briefly, 3 x 10^7 cells were crosslinked in 0.4% formaldehyde for 10 min at room temperature. After lysing cells and nuclei, samples were sonicated with a Diagenode Bioruptor at the maximum power setting for 15 min with 30 sec intervals. Immunoprecipitation was then performed overnight with 7 microgram of antibody. After crosslink reversal and PCI extraction, samples were isopropanol-precipitated and subjected to Illumina ChIP-seq sample preparation using a single-end kit. The manufacturer's protocol was followed except for an extended adapter ligation step (30 min) and carrying out the PCR step before gel purification. DNA fragments with a median size of about 200 bp were finally extracted and sequenced.

tag: strain

tag: tissue

value: spleen

tag: cell type

value: polyclonal rabbit IgG

tag: antibody vendor

value: Sigma


title: 25 ug/ml DRB-treated Jurkat cells, ChIP-Seq

city: Toronto

first: Qun

last: Pan

address: 160 College St.

country: Canada

institute: University of Toronto

description: ChIP-Seq

data_processing: The sequence reads were aligned to human genomic sequence (NCBI Genome Assembly Build 36) using Bowtie (Langmead et al., Genome Biol. 2009).

source_name: Jurkat T lymphoma cells with 25 ug/ml DRB treatment, pol II ChIP

growth_protocol: Jurkat cells were cultured in RPMI-1640 supplemented with 10% heat-inactivated fetal bovine serum (FBS).

extract_protocol: Genomic DNA was isolated by phenol/chloroform extraction and ethanol precipitation. ChIP-Seq was performed using an anti-pol II antibody (4H8) from Jurkat cells.The libary construction protocol is described in Schmidt D et al. Methods. (2009) Jul;48(3):240-8 (PMID 19275939).

treatment_protocol: Jurkat cells were treated with or without pol II elongation inhibitor 5,6-dichloro-1-β-D-ribofuranosyl-benzimidazole (DRB) at 10 and 25 ug/ml for 24 hours, followed by treatment with 20 ng/ml PMA for 6 hours.

tag: cell line

value: Jurkat

value: T lymphoma cells

tag: treatment

value: anti-pol II

tag: antibody catalog #

tag: antibody lot #


title: Th2_H3KAc

description: Chromatin IP against H3KAc.

source_name: Th2 cells, H3KAc

tag: antibody

value: Upstate Biotechnology


title: R13-E1-6h-Set2_GFP_ChIPSeq_1 and 2

tag: developmental stage

tag: fly

tag: antibody name

tag: manufacturer


title: 10 ug/ml DRB-treated Jurkat cells, ChIP-Seq

description: ChIP-Seq

source_name: Jurkat T lymphoma cells with 10 ug/ml DRB treatment, pol II ChIP


title: DMSO mock-treated Jurkat cells, ChIP-Seq

description: ChIP-Seq

source_name: Jurkat T lymphoma cells with DMSO mock treatment, pol II ChIP

value: DMSO


title: [E-MTAB-407] polyA+_spleen_6090097

description: Performer: LGTF

tag: material type

tag: organismpart


title: tribolium_embryos

status: Public on Dec 29 2010

city: Colchester

first: Antonio

last: Marco

address: Wivenhoe Park

department: School of Life Sciences

institute: University of Essex

description: Total endogenous small RNAs from Tribolium castaneum embryos.

data_processing: Reads were sequentially trimmed and mapped to the Tribolium reference genome (version 3.0) to a minimum size of 19 nucleotides using Bowtie, allowing one mismatch between the read and the genome. Potential rRNA and tRNA contaminants were filtered out. Unique sequences were merged, keeping track of the read count. For further information, refer to Marco et al. (2010) Genome Biol. Evol. 2:686-96 (PMID 20817720).

organism: Tribolium castaneum

source_name: Tribolium castaneum early embryos (0-5 days)

growth_protocol: Wild type beetles were cultured at 28ºC. RNA was extracted separately from 0-5 day embryos and adults.

extract_protocol: RNA was extracted with the miRVana miRNA isolation kit (Ambion). Molecules shorter than 40 nt were selected with the flashPAGE fractionator (Ambion) and purified with the flashPAGE reaction cleanup kit (Ambion). Libraries were constructed with the SOLiD small RNA Expression kit, as indicated by the manufacturer.

tag: genotype

value: wild type

value: embryo

tag: age

value: 0-5 days

tag: gender

value: male and female


title: [E-MTAB-407] polyA+_lung_1100350

description: Performer: LGTF

value: lung

tag: Sex

tag: biosourcetype

tag: biosourceprovider


title: tribolium_adults

description: Total endogenous small RNAs from Tribolium castaneum adults.

source_name: Tribolium castaneum adult individuals

value: adult


title: [E-MTAB-407] polyA+_testis_7060321

description: Performer: LGTF

value: testis

value: male


title: [E-MTAB-407] polyA+_liver_6100188

description: Performer: LGTF

value: liver

value: Clontech


title: [E-MTAB-407] polyA+_muscle_7040106

description: Performer: LGTF

value: muscle


title: [E-MTAB-407] polyA+_heart_7082801A

description: Performer: LGTF

value: heart


title: [E-MTAB-407] polyA+_brain_6120217

description: Performer: LGTF

value: brain


title: [E-MTAB-223] ZR751_input_lane2

description: Performer: CRUK-CRI

data_processing: scanning | The Eland software (Illumina) was used to align reads against the hg18 reference genome. The first 32 bases were aligned allowing a maximum of 2 mismatches. Reads aligning to multiple locations were discarded. The remaining un-aligned reads were shortened by 3 bases at the 3' end and the procedure repeated until the remaining aligned sequences were 23 bases long.

growth_protocol: grow | RPMI 1640 medium por DMEM supplemented with 10% inactivated FBS, l-glutamine and PEST at 37C with 5% CO2.

extract_protocol:
immunoprecipitate | Antibodies were from Santa Cruz Biotechnology (sc-543), abcam (ab5089 and ab23738) and Millipore (07-729) and ChIP was performed using 10e7-10e8 cells per reaction. Briefly, crosslinking of protein and DNA was done in room temperature for 10 minutes with serum free media containing 0,37 % formaldehyde. Cells were scraped and washed in PBS before treatment of cell lysis buffer with protease inhibitors on ice. Pelleted nuclei were resuspeded in RIPA buffer and a BioRuptor (Diagenode) was used to sonicate DNA. ChIP was performed with 10 micrograms of antibody. ChIP DNA was gel fractionated and amplified to create two size libraries corresponding to insert sizes of 200-300 bp. For DNase I hypersensitivity was performed as previously described (Eeckhoute et al., 2006). Digested DNA was run on a gel and the digested material (between 200bp and 500bp in size). For all ChIPs washing was done six times with RIPA buffer and once with TE-buffer and DNA-protein complexes were eluted from beads twice with 200 microlitres of 0.1 M NaHCO3 and 1 % SDS and treated with RNAseA at 65C for 6 hours and Proteinase K at 45C over night. Chromatin was amplified with Solexa protocol chromatin amplification.
sequencing | Sequencing was carried out by the genomic service of Cambridge Research Institute-CRUK using the Illumina genome analyzer II

tag: cellline

value: female

tag: diseasestate

value: breast cancer

tag: chip antibody


title: [E-MTAB-223] MCF7_input_lane2

description: Performer: CRUK-CRI


title: [E-MTAB-223] full_ER_ChIP_T47D_exp1_lane2

description: Performer: CRUK-CRI

value: ER


title: [E-MTAB-223] ZR751_input_lane1

description: Performer: CRUK-CRI


title: [E-MTAB-223] FAIRE_siFoxA1_E2_exp1_lane1

description: Performer: CRUK-CRI

value: not applicable


title: [E-MTAB-223] FAIRE_siFoxA1_veh_exp1_lane1

description: Performer: CRUK-CRI


title: [E-MTAB-223] FAIRE_siNT_E2_exp1_lane1

description: Performer: CRUK-CRI


title: [E-MTAB-223] full_ER_ChIP_T47D_exp2_lane1

description: Performer: CRUK-CRI


title: [E-MTAB-223] MCF7_input_lane1

description: Performer: CRUK-CRI


title: [E-MTAB-223] TAMr_input

description: Performer: CRUK-CRI


title: [E-MTAB-223] FAIRE_siNT_veh_exp1_lane1

description: Performer: CRUK-CRI


title: [E-MTAB-223] full_ER_ChIP_T47D_exp2_lane2

description: Performer: CRUK-CRI


title: [E-MTAB-223] full_ER_ChIP_T47D_exp1_lane3

description: Performer: CRUK-CRI


title: [E-MTAB-223] full_ER_ChIP_T47D_exp1_lane1

description: Performer: CRUK-CRI


title: [E-MTAB-223] full_ER_ChIP_ZR751_exp1_lane2

description: Performer: CRUK-CRI


title: [E-MTAB-223] full_ER_ChIP_ZR751_exp2_lane2

description: Performer: CRUK-CRI


title: Lung_96h_pooled 3 infected pigs (H96)

status: Public on Dec 05 2010

city: Guangzhou

first: Yaosheng

last: Chen

state: Guangdong

address: Beisanlu,Higher education megacenter

institute: Sun Yat-sen University

description: no additional information

data_processing: Image recognition, base calling and tag counting were performed using the Illumina Pipeline.

source_name: pooled lung of three pigs (96h pi infected with H-PRRSV)

growth_protocol: All animal procedures were performed according to guidelines developed by the China Council on Animal Care and protocol approved by Animal Care and Use Committee of Guangdong Province, P.R. China.

extract_protocol: Total RNA was extracted from frozen lungs using standard protocols (Trizol) and then treated with DNase to remove potential genomic DNA contamination according to the manufactures’s protocols. RNA integrity and concentration were evaluated by Agolent 2100 Bioanalyzer. For RNA library construction and deep sequencing, RNA samples were prepared as follows: for each time point of H-PRRSV-inoculated group and UNC group equal quantities of RNA isolated from three individual lungs were pooled. Approximately 6 μg of RNA representing each group were submitted to Solexa (now Illumina Inc.) for sequencing.

treatment_protocol: Pigs were inoculated with 6 ml viral suspension (4 ml intranasally and 2 ml intramuscularly) of H-PRRSV at a dose of 106.0 TCID50 ml-1 on day 0. Three infected pigs randomly chosen within each group were necropsied at each time point of 96 h pi and 168 h pi.

tag: tissue

tag: treatment group

value: 96h pi infected with H-PRRSV


title: [E-MTAB-223] full_ER_ChIP_ZR751_exp1_lane1

description: Performer: CRUK-CRI


title: Lung 168h pooled 3 infected pigs (N168)

data_processing: Image recognition,base calling and tag counting were performed using the Illumina Pipeline.

source_name: pooled lung of three pigs (168h pi infected with N-PRRSV)

extract_protocol: Total RNA was extracted from frozen lungs using standard protocols (Trizol) and then treated with DNase to remove potential genomic DNA contamination according to the manufactures’s protocols. RNA integrity and concentration were evaluated by Agolent 2100 Bioanalyzer.For RNA library construction and deep sequencing, RNA samples were prepared as follows: for each time point of H-PRRSV-inoculated group and UNC group equal quantities of RNA isolated from three individual lungs were pooled. Approximately 6 μg of RNA representing each group were submitted to Solexa (now Illumina Inc.) for sequencing.

treatment_protocol: Pigs were inoculated with 6 ml viral suspension (4 ml intranasally and 2 ml intramuscularly) of N-PRRSV at a dose of 106.0 TCID50 ml-1 on day 0.Three infected pigs randomly chosen within each group were necropsied at each time point of 96 h pi and 168 h pi.

value: 168h pi infected with N-PRRSV


title: [E-MTAB-223] full_ER_ChIP_MCF7_exp2_lane2

description: Performer: CRUK-CRI


title: Lung 96h pooled 3 infected pigs (N96)

source_name: pooled lung of three pigs (96h pi infected with N-PRRSV)

value: 96h pi infected with N-PRRSV


title: [E-MTAB-223] full_ER_ChIP_MCF7_exp2_lane1

description: Performer: CRUK-CRI


title: [E-MTAB-223] full_ER_ChIP_MCF7_exp1_lane3

description: Performer: CRUK-CRI


title: [E-MTAB-223] full_ER_ChIP_ZR751_exp2_lane1

description: Performer: CRUK-CRI


title: [E-MTAB-223] full_ER_ChIP__MCF7_exp1_lane2

description: Performer: CRUK-CRI


title: [E-MTAB-223] full_ER_ChIP_MCF7_exp1_lane1

description: Performer: CRUK-CRI


title: [E-MTAB-223] FoxA1_ChIP_TAMR_full_media

description: Performer: CRUK-CRI


title: [E-MTAB-223] Veh_CTCF_ChIP_lane1

description: Performer: CRUK-CRI

value: CTCF


title: [E-MTAB-223] E2_CTCF_ChIP_lane1

description: Performer: CRUK-CRI


title: [E-MTAB-223] E2_CTCF_ChIP_lane2

description: Performer: CRUK-CRI


title: Lung_pooled 3 control pigs (C)

description: no additional information

source_name: pooled lung of three pigs (uninfected negative control)

value: lung

value: control


title: [E-MTAB-223] FoxA1_ChIP_MCF7_exp2

description: Performer: CRUK-CRI


title: [E-MTAB-223] E2_ER_ChIP_siFoxA1_exp1_lane2

description: Performer: CRUK-CRI


title: [E-MTAB-223] FoxA1_ChIP_MCF7_exp1

description: Performer: CRUK-CRI


title: [E-MTAB-223] FoxA1_ChIP_MCF7_full_media

description: Performer: CRUK-CRI


title: [E-MTAB-223] Veh_CTCF_ChIP_lane2

description: Performer: CRUK-CRI


title: [E-MTAB-223] E2_ER_ChIP_siFoxA1_exp1_lane1

description: Performer: CRUK-CRI


title: [E-MTAB-223] TAMr_ER_ChIP_veh

description: Performer: CRUK-CRI


title: [E-MTAB-223] E2_ER_ChIP_siNT_exp1_lane1

description: Performer: CRUK-CRI


title: [E-MTAB-223] E2_ER_ChIP_siNT_exp1_lane2

description: Performer: CRUK-CRI


title: [E-MTAB-223] TAM_ER_ChIP_exp3

description: Performer: CRUK-CRI


title: HandDI_TagSeq

status: Public on Dec 06 2010

city: Shanghai

first: Zhe

last: Wang

address: 3663 Zhongshan North Road

institute: East China Normal University

description: NlaIII tag sequencing

data_processing: Image analysis, base calling, extraction of 21bp tags and tag counting were performed using the Illumina pipeline.

organism: Myotis pilosus

source_name: Embryonic forelimb digit I

extract_protocol: Total RNA were extracted from forelimb digit I, forelimb digits II-V, and all five hindlimb digits using TRIzol® Reagent (Invitrogen, Cat. No. 15596026, US), treated with DNase I (Roche, Cat. No. 04716728001, Germany) and converted to double stranded cDNA. Subsequently, the cDNA samples were digested using the restriction enzyme NlaIII, which recognizes and cuts the most 3’ “CATG”. Three digital expression libraries (Libraries Hand DI, Hand DII-V and Foot) from the three samples were constructed with each molecule in the library was a 21 bp tag derived from a single transcript with defined adapters attached to both ends (Illumina, Cat. No. FC-102-1005, US). Next, the molecules were amplified to create clusters on a flow cell using a Solexa Cluster Station (Illumina, Cat. No. FC-103-1004, US). Each library was amplified within one lane of the flow cell. Finally, massively parallel sequencing-by-synthesis was performed on the Illumina Genome Analyzer II.

value: embryo


title: Foot_TagSeq

description: NlaIII tag sequencing

source_name: Embryonic hindlimb digits I-V


title: [E-MTAB-223] TAM_ER_ChIP_exp2_lane2

description: Performer: CRUK-CRI


title: [E-MTAB-223] Veh_ER_ChIP

description: Performer: CRUK-CRI


title: HandDII-V_TagSeq

description: NlaIII tag sequencing

source_name: Embryonic forelimb digits II-V


title: [E-MTAB-223] TAM_ER_ChIP_exp2_lane1

description: Performer: CRUK-CRI


title: [E-MTAB-223] E2_ER_ChIP_exp3

description: Performer: CRUK-CRI


title: [E-MTAB-223] TAM_ER_ChIP_exp1_lane2

description: Performer: CRUK-CRI


title: [E-MTAB-223] TAM_ER_ChIP_exp1_lane1

description: Performer: CRUK-CRI


title: [E-MTAB-223] E2_ER_ChIP_exp2_lane1

description: Performer: CRUK-CRI


title: [E-MTAB-223] E2_ER_ChIP_exp1_lane2

description: Performer: CRUK-CRI


title: [E-MTAB-223] E2_ER_ChIP_exp2_lane2

description: Performer: CRUK-CRI


title: [E-MTAB-197] NA19238_5

data_processing: Used for pilot and lane correlation analysis

extract_protocol:
nucleic_acid_extraction | This method involves several steps that are designed to convert total RNA into a library of template molecules suitable for high throughput DNA sequencing.  The first step involves purifying the poly-A containing mRNA molecules using poly-T oligo-attached magnetic beads.  Following purification, the mRNA is fragmented into small pieces using divalent cations under elevated temperature. Then, the cleaved RNA fragments are copied into first strand cDNA using reverse transcriptase and a high concentration of random hexamer primers.  This is followed by second strand cDNA synthesis using DNA Polymerase I and RNaseH.  Finally, the short cDNA fragments are prepared for sequencing on the Illumina Genome Analyzer.
sequencing | 37 bp paired end RNA sequencing

value: cell

tag: population

value: HapMap YRI

tag: celltype

value: lymphoblastoid

tag: observation

value: yoruba

tag: individual


title: [E-MTAB-197] NA19238_7

tag: material type


title: [E-MTAB-197] NA19238_6


title: [E-MTAB-341] hNRNPC iCLIP

description: Center name: CRUK-CRI

library_source: synthetic

source_name: hNRNPC iCLIP

growth_protocol:
grow | HeLa cells grown in a 10 cm plate were covered with ice-cold PBS buffer and subjected to UV-C irradiation (100 mJ/cm2, Stratalinker 2400).
specified_biomaterial_action | Upon removal of PBS buffer, cells were scraped off and transferred into microtubes (2 ml each). Cells were precipitated by centrifugation for 1 min at 14000 rpm and shock-frozen on dry ice. For magnetic bead preparation, 50 ul of protein A-coated Dynabeads (Invitrogen) were washed 2x with 900 ul lysis buffer (50 mM Tris-HCl pH 7.4, 100 mM NaCl, 1 mM MgCl2, 0.1 mM CaCl2, 1 % NP-40, 0.1 % SDS, 0.5 % Na-Deoxycholate). Dynabeads were resuspended in 200 ul lysis buffer, and 10 ug of hnRNP C antibody (Santa Cruz H-105) were added. After rotation at room temperature for 30-60 min, Dynabeads were washed 2x with lysis buffer and kept in last wash until addition of crosslinked lysate. Pellets were resuspended in 1 ml lysis buffer and sonicated. For partial RNase digestion, RNase I (Ambion) was diluted 1:50 and 1:100 in lysis buffer for high and low RNase treatment, respectively. 10 ul RNase I dilution and 5 ul Turbo DNase (Ambion) were added to the crosslinked lysate and incubated for 3 min at 37C and 800 rpm. Cells were precipitated by two rounds of centrifugation at 4C and 14000 rpm for 10 min followed by careful collection of the supernatant. The supernatant was added to Dynabeads and incubated for 1 h or overnight at 4C and 800 rpm. Dynabeads were washed 2x with high-salt wash buffer (50 mM Tris-HCl pH 7.4, 1 M NaCl, 1 mM EDTA, 0,1 % SDS, 0.5 % Na-Deoxycholate, 1 % NP-40) and 1x with PNK wash buffer (20 mM Tris-HCl pH 7.4, 10 mM MgCl2, 0,2 % Tween-20). For dephosphorylation of 3 prime ends, Dynabeads were resuspended in 2 ul 10x Shrimp alkaline phosphatase buffer (Promega), 17.5 ul H2O and 0.1 ul Shrimp alkaline phosphatase (Promega) and incubated at 37C for 10 min with intermittent shaking (10 sec at 700 rpm followed by 20 sec pause). Samples were washed 2x with high-salt wash buffer, 1x with 900 ul PNK wash buffer and 1 x with 50 ul 1x RNA ligase buffer (NEB, freshly prepared from frozen stock). For RNA linker ligation, Dynabeads were resuspended in 15 ul L3 ligation mix (5 ul L3 RNA linker [5  prime- phosphate-UGAGAUCGGAAGAGCGGTTCAG-3 prime-Puromycin, 20 pM], 1.5 ul 10x RNA ligase buffer, 7.75 ul H2O, 0.5 ul RNasin [Promega], 0.25 ul RNA ligase [NEB]) and incubated overnight at 16C. Samples were mixed with 5 ul NuPAGE loading buffer (Invitrogen), incubated for 5 min at 70C and placed on a magnetic stand to collect the eluate. Samples were run on 9-well or 10-well Novex NuPAGE 4-12% Bis-Tris gels (Invitrogen) with 1x MOPS running buffer (Invitrogen). After gel electrophoresis, protein and covalently bound RNAs were transferred to a nitrocellulose membrane (Whatman) using a Novex wet transfer apparatus (Invitrogen). The nitrocellulose membrane was rinsed with 1x PBS, wrapped into cling film and exposed to a BioMax XAR Film (Kodak) at -80C.

extract_protocol:
nucleic_acid_extraction | For isolation of crosslinked RNAs, 2 mg/ml proteinase K (Roche) was pre-incubated in PK buffer (100 mM Tris-HCl pH 7.5, 50 mM NaCl, 10 mM EDTA) for 5 min at 37C. In order to recover different size fractions of RNAs, two fragments were cut out of the nitrocellulose membrane at different heights above the molecular weight of the protein (40 kDa). 200 ul proteinase K solution was added to each fragment and incubated for 30 min at 55C. Incubation was repeated after addition of 130 ul PK/7 M urea buffer (100 mM Tris-HCl pH 7,5, 50 mM NaCl, 10 mM EDTA, 7 M urea). Samples were cooled to 37C, mixed with 170 ul H2O and 600 ul RNA phenol/CHCl3 (Ambion) and incubated for 5 min at 37C and 1100 rpm. After centrifugation for 10 min at 13000 rpm and room temperature, 450ul of the aqueous phase were transferred into a new microtube and again subjected to centrifugation. 400 ul of supernatant were mixed with 0.5 ul Glycoblue (Ambion), 40 ul 3 M sodium acetate pH 5.5 and 1 ml 100 % EtOH and incubated overnight at -20C. RNAs were precipitated by centrifugation for 30 min at 15000 rpm and 4C, washed with 500 ul 80 % EtOH and resuspended in 12 ul H2O.
reverse_transcription | For reverse transcription, 1 ul RT primer (2 pmol/ul; the following three primers were used for replicates 1 to 3: 5 prime-phosphate- NNNCAAGATCGGAAGAGCGTCGTGGATCCTGAACCGCTC-3 prime; 5 prime-phosphate- NNNGAAGATCGGAAGAGCGTCGTGGATCCTGAACCGC-3prime; 5 prime-phosphate- NNNTGAGATCGGAAGAGCGTCGTGGATCCTGAACCGCTC-3 prime; NNN represents 3-nt random barcode and bold nucleotides mark 2-nt barcode used as an experiment identifier) and 1 ml 10 mM dNTP mix were added to the RNA, preheated for 5 min to 70C and then held at 42C. Once 6 ul RT mix (5 ul 5x RT buffer [Invitrogen], 1 ul 0.1 M DTT, 0.5 ul Superscript III reverse transcriptase [Invitrogen], 0.5 ul RNasin) were added and mixed by pipetting, reverse transcription was performed with the following program: 10 min at 42C, 40 min at 50C, 20 min at 55C, and hold at 4C. To remove RNA, samples were heated for 2 min to 95C, mixed with 1 ul RNase A (Ambion) and incubated for 20 min at 37C. cDNAs were precipitated by addition of 80 ul TE buffer, 0.5 ul Glycoblue, 10 ul 3 M sodium acetate pH 5.3 and 250 ul 100 % EtOH, incubation for 1 h on dry ice or overnight at -20C, and centrifugation for 30 min at 4C and 15000 rpm. Pellets were washed with 500 ul 80 % EtOH, dried for 3 min at room temperature and resuspended in 6 ul H2O. For size separation, cDNAs were mixed with 2 ul 2x TBE-urea loading buffer (Invitrogen) and incubated for 3 min at 70C. Samples were run on a 6 % TBE urea gel (Invitrogen) in 1x TBE buffer for 40 min at 180 V. In order to recover different size fractions, two bands were cut from the gel corresponding to a cDNA size of 100 - 175 nt and 175 - 350 nt. Gel fragments were mixed with 400 ml TE buffer, crushed with a 1 ml syringe plunger and incubated for 2 h at 37C and 1100 rpm. A Costar SpinX column (Corning Incorporated) was prepared by addition of two 1 cm glass wool pre-filters (Whatman 1823-101) and centrifugation for 1 min at 13000 rpm. After transfer of the supernatant to the column, 40ul 3 M sodium acetate pH 5.5 and 0.5ul glycogen were added. Columns were vortexed before adding 1 ml 100 % EtOH and incubating overnight at -20C. Columns were washed by addition of 500ul 80 % EtOH and centrifugation for 10 min at 15000 rpm and 4C. Pellets were dried for 3 min at room temperature and resuspended in 12ul H2O. In order to circularize the cDNAs, samples were mixed with 1.5ul 10x CircLigase buffer II (Epicentre), 0.75ul 50 mM MnCl2 and 0.75ul CircLigase II (Epicentre) and incubated for 1h at 60oC. For subsequent linearization, a primer (5 prime- GTTCAGGATCCACGACGCTCTTCAAAA-3 prime) complementary to the BamHI restriction site in the RT primer was annealed by adding 26ul H2O, 5ul FastDigest buffer (Fermentas) and 1ul 10uM primer and incubation with the following program: 2 min at 95C, 70 cycles starting for 1 min at 95C and reducing the temperature with every cycle by 1C. BamHI cleavage was performed by adding 3ul Fastdigest BamHI (Fermentas) and incubating for 30 min at 37C. Samples were mixed with 50ul TE buffer, 0.5ul Glycoblue, 10ul 3 M sodium acetate pH 5.5 and 250ul 100 % EtOH and incubated for 1 h on dry ice or overnight at -20C. cDNAs were precipitated by centrifugation for 30 min at 15000 rpm and 4C, washed with 500ul 80 % EtOH, dried for 3 min at room temperature and resuspended in 9ul H2O.
sequencing | For high-throughput sequencing, cDNAs were PCR-amplified by adding 0.3ul Illumina paired-end primer mix (10uM each; 5 '- CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCT-3 '; 5 '- AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3 ', oligonucleotide sequences  2006 and 2008 Illumina, Inc. All rights reserved) and 10ul 2x Immomix (Bioline) and incubation with the following program: 10 min at 95oC, 35 cycles of [10 sec at 95oC, 10 sec at 65oC, 20 sec at 72oC], 3 min at 72oC. In order to desalt the PCR products, a Microspin G-25 column (GE Healthcare) was resupended by vortexing and spinned for 1 min at 735 x g. Upon sample application, PCR products were re-eluted by centrifugation for 2 min at 735 x g, and sequenced on an Illumina GA2 flow cell.

value: HeLa

tag: antibody

value: hnRNP C antibody


title: [E-MTAB-197] NA19238_3


title: [E-MTAB-197] NA19238_4


title: r2d2[1] ovary total RNA

city: New York

first: Nicolas

last: Robine

state: NY

address: 1275 York Avenue, Box 252

department: Developmental Biology

institute: Memorial Sloan Kettering Cancer Center

data_processing: Raw sequences were clipped by 3' linker sequences recognition, and clipped sequences longer than 18 nt were selected for analysis.

source_name: r2d2[1] ovary total RNA

growth_protocol: Young adult flies were fed with fly food supplemented with yeast for 3-4 days. Aged flies were anesthetized with CO2 and dissected in PBS, and ovaries were collected in an eppendorf tube.The flies were frozen on dry ice and stored at -80C until use.

extract_protocol: total RNA extracted with Trizol (Invitrogen) from dissected ovaris, ~18-30nt RNAs isolated using PAGE, ligation to adapters requires 5' monophosphate and 3' OH

value: Ovary

tag: strain


title: [E-MTAB-223] FoxA1_ChIP_ZR751_full_media

description: Performer: CRUK-CRI

value: female

value: breast cancer


title: [E-MTAB-223] E2_ER_ChIP_exp1_lane1

description: Performer: CRUK-CRI

value: ER


title: [E-MTAB-197] NA19238


title: [E-MTAB-197] NA19238_2


title: Lung_168h_pooled 3 infected pigs (168)

description: no additional information

source_name: pooled lung of three pigs (168h pi infected with H-PRRSV)

value: 168h pi infected with H-PRRSV


title: [E-MTAB-197] NA12892_7

value: HapMap CEU

value: caucasian


title: [E-MTAB-197] NA12892_6


title: [E-MTAB-197] NA12892_5


title: [E-MTAB-197] NA12892_3


title: [E-MTAB-197] NA12892_4


title: [E-MTAB-197] NA12892

data_processing: Used in EQTL Analysis


title: [E-MTAB-197] NA12873


title: [E-MTAB-197] NA12874


title: [E-MTAB-197] NA12892_2


title: [E-MTAB-197] NA12813


title: [E-MTAB-197] NA12763


title: [E-MTAB-197] NA12812


title: [E-MTAB-197] NA12776


title: [E-MTAB-197] NA12891


title: [E-MTAB-197] NA12828


title: [E-MTAB-197] NA12872


title: [E-MTAB-197] NA12762


title: [E-MTAB-197] NA12760


title: [E-MTAB-197] NA12751


title: [E-MTAB-197] NA12761


title: [E-MTAB-223] TAMr_ER_ChIP_Tam

description: Performer: CRUK-CRI


title: [E-MTAB-197] NA12750


title: [E-MTAB-197] NA12717


title: [E-MTAB-197] NA12814


title: [E-MTAB-197] NA12815


title: [E-MTAB-197] NA12716


title: [E-MTAB-197] NA12489


title: [E-MTAB-197] NA12414


title: ago2[414] ovary total RNA

source_name: ago2[414] ovary total RNA


title: [E-MTAB-197] NA12234


title: [E-MTAB-197] NA12156


title: [E-MTAB-197] NA12154


title: [E-MTAB-197] NA12045


title: [E-MTAB-197] NA12155


title: [E-MTAB-197] NA12144


title: [E-MTAB-197] NA12249


title: [E-MTAB-197] NA12044


title: [E-MTAB-197] NA12003


title: [E-MTAB-197] NA12005


title: [E-MTAB-197] NA11993


title: [E-MTAB-197] NA11994


title: [E-MTAB-197] NA11995


title: [E-MTAB-197] NA12006


title: [E-MTAB-197] NA12004


title: [E-MTAB-197] NA11931


title: [E-MTAB-197] NA11920


title: [E-MTAB-197] NA11918


title: [E-MTAB-197] NA11840


title: [E-MTAB-197] NA11894


title: [E-MTAB-197] NA12287


title: [E-MTAB-197] NA11881


title: [E-MTAB-197] NA11829


title: [E-MTAB-197] NA11830


title: [E-MTAB-197] NA11831


title: [E-MTAB-197] NA12043


title: [E-MTAB-197] NA10851


title: [E-MTAB-214] Input DNA

description: Performer: EMBL_Heidelberg

source_name: Input DNA

growth_protocol:
grow | Chromatin was obtained from male 3rd instar larvae salivary glands as previously described (Legube et al, 2006). In brief, collected salivary glands (10 per ChIP) were fixed in 1 mL of fixing solution (50 mM HEPES at pH 7.6, 100 mM NaCl, 0.1 mM EDTA at pH 8, 0.5 mM EGTA at pH 8, 2% formaldehyde) for 15 min at room temperature on a rotating wheel. The glands were centrifuged at 2000 rpm for 1 min, washed once in PBS with 0.01% Triton X-100 and 0.125 M glycine; then washed for 10 mins each in 1 ml of buffer A (0.25% Triton X-100, 10 mM EDTA (pH 8), 0.5 mM EGTA (pH 8), 10 mM Tris (pH 8)) and buffer B (200 mM NaCl, 10 mM Tris (pH 8), 10 mM EDTA (pH 8), and 0.5 mM EGTA (pH 8)). The glands where then resuspended in 500 _L of sonication buffer (10 mM Tris at pH 8, 1 mM EDTA at pH 8, 0.5 mM EGTA at pH 8.0) and sonicated 8 min (pulsed eight times for 30 sec, paused for 30 sec, high) in a Bioruptor (Cosmo Bio).
specified_biomaterial_action | Immunoprecipitation was then performed on 500 _L of chromatin from salivary glands, according to Orlando et al. (1997), using 3 _L of a polyclonal antibody against NSL-1, MCRS2.

extract_protocol:
nucleic_acid_extraction | Chromatin was obtained from male 3rd instar larvae salivary glands as previously described (Legube et al, 2006). In brief, collected salivary glands (10 per ChIP) were fixed in 1 mL of fixing solution (50 mM HEPES at pH 7.6, 100 mM NaCl, 0.1 mM EDTA at pH 8, 0.5 mM EGTA at pH 8, 2% formaldehyde) for 15 min at room temperature on a rotating wheel. The glands were centrifuged at 2000 rpm for 1 min, washed once in PBS with 0.01% Triton X-100 and 0.125 M glycine; then washed for 10 mins each in 1 ml of buffer A (0.25% Triton X-100, 10 mM EDTA (pH 8), 0.5 mM EGTA (pH 8), 10 mM Tris (pH 8)) and buffer B (200 mM NaCl, 10 mM Tris (pH 8), 10 mM EDTA (pH 8), and 0.5 mM EGTA (pH 8)). The glands where then resuspended in 500 _L of sonication buffer (10 mM Tris at pH 8, 1 mM EDTA at pH 8, 0.5 mM EGTA at pH 8.0) and sonicated 8 min (pulsed eight times for 30 sec, paused for 30 sec, high) in a Bioruptor (Cosmo Bio). The fragments generated were 300 bp on an average. Immunoprecipitation was then performed on 500 _L of chromatin from salivary glands, according to Orlando et al. (1997), using 3 _L of a polyclonal antibody against NSL-1, MBD-R2, MCRS2 and MOF or a correspondent preimmune anti-serum (mock ChIP). The immunoprecipitated DNA was resuspended in 100 ml of nuclease free water. Since the ChIP derived DNA was already sufficiently fragmented, we did not perform a DNA fragmentation step.
sequencing  | Sequencing was carried out using the 1G Illumina Genome Analyser (Solexa) according to the manufacturer's instructions.

tag: developmentalstage

value: 3rd instar larvae

tag: organismpart

value: Input


title: [E-MTAB-197] NA07347


title: [E-MTAB-197] NA07051


title: [E-MTAB-197] NA06994


title: [E-MTAB-214] MCRS2

description: Performer: EMBL_Heidelberg


title: [E-MTAB-197] NA07346


title: [E-MTAB-223] T47D_input_lane2

description: Performer: CRUK-CRI

data_processing:
Sequences generated by the Illumina genome analyzer were aligned against NCBI Build 36.3 of the human genome using MAQ with default parameters
Uniquely aligning reads were converted to BED format using a custom script.
Peaks were called using MACS (version 1.4.0alpha2; Zhang et al, 2008) using only uniqely aligned reads
scanning | The Eland software (Illumina) was used to align reads against the hg18 reference genome. The first 32 bases were aligned allowing a maximum of 2 mismatches. Reads aligning to multiple locations were discarded. The remaining un-aligned reads were shortened by 3 bases at the 3' end and the procedure repeated until the remaining aligned sequences were 23 bases long.

treatment_protocol: Drug treatments were performed with Dox (Clontech) at 0.1 ug/ml, R5020 (Du Pont) at 10 nM or 17beta-estradiol (Sigma) at 10 mM. Dox-containing medium was changed every 24 hours to ensure optimum Dox activity. Adherent cells on tissue culture plates were incubated in fresh, warm, serum-free medium supplemented with 1% formaldehyde, at 37 C for 10 minutes. Cells were washed 2 times with cold PBS and scraped into 600 ul PBS with protease inhibitors (P8340, Sigma). Fixed cells were spun for 2 minutes at 6,000xg, washed as before and snap frozen in liquid nitrogen.

tag: cell line

tag: treatment

value: doxycycline

tag: timepoint

tag: Sex

tag: disease state


title: [E-MTAB-214] NSL1

description: Performer: EMBL_Heidelberg

tag: chip antibody


title: [E-MTAB-197] NA07357


title: [E-MTAB-197] NA06986


title: [E-MTAB-197] NA07037


title: [E-MTAB-197] NA06985


title: [E-MTAB-197] NA11992


title: [E-MTAB-305] testes RNA

description: Provider: TRON

source_name: testes RNA

extract_protocol:
nucleic_acid_extraction | We purchased total RNA from Ambion (Austin, USA). Each tissue samples was pooled from multiple donors. Librarys were prepared as in Armour CD, Castle JC, Chen R, Babak T, Loerch P, et al. (2009) Digital transcriptome profiling using selective hexamer priming for cDNA synthesis. Nat Methods 6: 647-649.
sequencing | We generated an average of 50 million sequence reads per tissue using an Illumina GA-II sequencer, with sequence lengths of 36 nt (adipose, hypothalamus, liver) and 50 nt (colon, heart, kidney, lung, ovary, skeletal muscle, spleen, testes). We trimmed reads to a common length of 28 nt to avoid aligning sequenced amplification primers.

value: testes


title: [E-MTAB-305] spleen RNA

description: Provider: TRON

source_name: spleen RNA

value: spleen


title: [E-MTAB-305] skeletal muscle RNA

description: Provider: TRON

source_name: skeletal muscle RNA

value: skeletal muscle


title: [E-MTAB-305] ovary RNA

description: Provider: TRON

source_name: ovary RNA

value: ovary


title: [E-MTAB-197] NA11832

value: cell

value: lymphoblastoid


title: [E-MTAB-305] lung RNA

description: Provider: TRON

source_name: lung RNA

value: lung


title: [E-MTAB-308] Zebrafish embryo 2 dpf mRNA 3' end

description: Provider: SANGER

organism: Danio rerio

source_name: Zebrafish embryo 2 dpf mRNA 3' end

growth_protocol: grow | Zebrafish embyos or tissues were collected from a Tuebingen strain incross and grown at 28 C.   Collected samples were snap frozen on dry ice and stored at -70 C.

extract_protocol:
nucleic_acid_extraction | Total RNA was extracted using Trizol Reagent (Invitrogen) following the manufacturer's instructions. Pellets were resuspended RNase-free 10 mM Tris pH 7.5 and the RNA was quantified using a NanoDrop ND-1000 Spectrophotometer (Axon Instruments).
specified_biomaterial_action | 20 µg of total RNA was fragmented using RNA Fragmentation Reagent (Ambion) for 5 minutes at 70 C and ethanol precipitated with glycogen and LiCl.   RNA was annealed to the oligo stBPM1polyT22 (biotin-GGCCAGTCCTGGAGTTTTTTTTTTTTTTTTTTTTTTVN) and bound to streptavidin  magnetic beads.   After washing by pull down on a magnet, the bound RNA was reverse transcribed with SuperScript II (Invitrogen) and a second strand synthesised with DNA polymerase I (Promega) and RNase H (NEB).   After further washing, the double strand cDNA was released from the beads with BpmI (NEB).   The cDNA was recovered with the QIAgen PCR Purification Kit and made into a standard Illumina library following the manufacturerÕs protocol with a fragment size of 250 to 300 bp.
scanning | Base calling was performed using Illumina pipeline 1.3 (Bustard 1.3.4) with phred quality scores at 64 levels using Illumina pipeline 1.3 (Gerald).    There were 152 cycles of sequencing with 2 reads per cluster, a forward read starting at base 1 and a reverse read starting at base 77.
sequencing | Sequencing was performed using the Illumina Genome Analyzer II

tag: strainorline

value: Tuebingen

tag: age

value: 2 days

tag: initialtimepoint

value: fertilization

value: embryo


title: [E-MTAB-308] Zebrafish embryo 3 dpf mRNA 3' end

description: Provider: SANGER

source_name: Zebrafish embryo 3 dpf mRNA 3' end

value: 3 days


title: [E-MTAB-197] NA10847

tag: population

value: HapMap CEU

tag: celltype


title: [E-MTAB-305] liver RNA

description: Provider: TRON

source_name: liver RNA

value: liver


title: [E-MTAB-305] kidney RNA

description: Provider: TRON

source_name: kidney RNA

value: kidney


title: [E-MTAB-308] Zebrafish embryo 1 dpf mRNA 3' end

description: Provider: SANGER

source_name: Zebrafish embryo 1 dpf mRNA 3' end

value: 1 day


title: [E-MTAB-197] NA07000

tag: observation

value: caucasian

tag: individual


title: [E-MTAB-305] hypothalamus RNA

description: Provider: TRON

source_name: hypothalamus RNA

tag: material type

value: hypothalamus


title: [E-MTAB-308] Zebrafish adult male head mRNA 3' end

description: Provider: SANGER

source_name: Zebrafish adult male head mRNA 3' end

value: adult

value: head

value: male


title: [E-MTAB-223] T47D_input_lane1

description: Performer: CRUK-CRI

value: doxycycline

value: female

value: breast cancer


title: [E-MTAB-308] Zebrafish embryo 5 dpf mRNA 3' end

description: Provider: SANGER

source_name: Zebrafish embryo 5 dpf mRNA 3' end

value: Tuebingen

value: 5 days


title: [E-MTAB-305] colon RNA

description: Provider: TRON

source_name: colon RNA

value: colon


title: [E-MTAB-305] adipose RNA

description: Provider: TRON

source_name: adipose RNA

value: adipose