title: FLAG-HA ZFP36

city: Aurora

first: Neelanjan

last: Mukherjee

state: CO

department: BMG/RBI

institute: University of Colorado School of Medicine

description: ZFP36 crosslinked ribonucleoprotein complexes

data_processing:
Library strategy: PAR-CLIP
Fastq reads were trimmed using cutadapt with the following parameters: -a TCGTATGCCGTCTTCTGCTTG -g AATGATACGGCGACCACCGACAGGTTCAGAGTTCTACAGTCCGACGATC -b CGTACGCGGGTTTAAACGA -b CGTACGCGGAATAGTTTAAACTGT -n 1 -m 20
Trimmed fastq was converted to fasta and redundant reads were collapsed with readID indicating copy number using perl scripts
Reads were mapped to hg19 using bowtie version 0.12.7 and the following parameters:  -v 1 -m 10 --all --best --strata
Sites were determined using PARalyzer v1.5 with the specified parameters: BANDWIDTH=3  CONVERSION=T>C  MINIMUM_READ_COUNT_PER_GROUP=2  MINIMUM_READ_COUNT_PER_CLUSTER=2  MINIMUM_READ_COUNT_FOR_KDE=3  MINIMUM_CLUSTER_SIZE=11  MINIMUM_CONVERSION_LOCATIONS_FOR_CLUSTER=2  MINIMUM_CONVERSION_COUNT_FOR_CLUSTER=2  MINIMUM_READ_COUNT_FOR_CLUSTER_INCLUSION=1  MINIMUM_READ_LENGTH=20  MAXIMUM_NUMBER_OF_NON_CONVERSION_MISMATCHES=0
Genome_build: hg19
Supplementary_files_format_and_content: bed

source_name: FlpIn T-Rex Human embryonic kidney 293 stable cell line

growth_protocol: FlpIn T-Rex HEK293 stable cell lines were cultured in DMEM (11995, Invitrogen) supplemented with 10% Fetal bovine serum, 2 mM L-Glutamine, 50 units of Penicillin, 50 µg/ml Streptomycin, 100 µg/ml Hygromycin, and 15 µg/ml Blasticidin

extract_protocol: Cell pellets were lysed in NP40 lysis buffer, centrifuged, and the resulting supernatants used in subsequent steps. The cleared supernatants were incubated with Rnase T1 (Fermentas) at  22ºC, for 15 min, then cooled on ice. FLAG-antibody conjugate paramagnetic beads (Dynabeads, Invitrogen) were added to each supernatant to begin immunoprecipitation. Immunoprecipitates were subjected to dephosphorylation using calf-intestinal alkaline phosphatase (NEB), followed by three buffer washes, then treatment with T4 polynucleotide kinase (NEB) using gamma radiolabelled - 32P ATP. Radiolaballed immunoprecipitates were then fractionated by SDS-PAGE. The immunoprecipitated bands of interest were excised, electroeluted, and treated with Proteinase K to isolate crosslinked RNAs. Crosslinked RNAs were taken through a standard adapter-mediated small RNA cloning protocol and sequenced using Illumina GAII platform.

treatment_protocol: Expression of stable transgenes were induced by incubation with 1 µg/ml doxycyline concurrent with 100 µM 4-Thiouridine (4SU, Sigma) for 16 h. For harvesting, cells were rinsed with ice-cold PBS, irradiated with 365 nM UV light (0.15 J/sq cm), collected and pelleted, and finally snap-frozen (-80 deg C)

tag: photoactivatable ribonucleoside and crosslinking wavelength

value: Sigma-Aldrich anti-FLAG M2

tag: immunoprecipitated protein

value: FLAG-HA ZFP36


title: leaf

status: Public on Dec 23 2013

type: SRA

city: New York

first: Huan

last: Wang

state: New York

address: 1230 York Avenue

country: USA

institute: The Rockefeller University

library_source: transcriptomic

data_processing:
Sequenced reads were mapped to Arabidopsis whole genome (TAIR10) using Tophat.
Genome_build: Arabidopsis Thaliana TAIR10

molecule: total RNA

organism: Arabidopsis thaliana

source_name: WT leaf

growth_protocol: Col-0 seedlings were grown on MS plates for two weeks and transferred to soil under long day conditions (16 hours light). 3 week old seedlings were bombarded with uncoated 0.6 um gold particles using a Biolistic PDS-1000/He Particle Delivery System (Bio-Rad), to simulate plasmid transformation, although no plasmid was used for these samples. Plants were grown for 18 days after bombarding. Leaves, flowers (inflorescences) and siliques were collected from these 5.5 week old plants and immediately frozen in liquid nitrogen.

extract_protocol: RNA was extracted using the Qiagen RNeasy Plant Mini kit. Extracted RNA was treated with Turbo DNase (Life Technologies AM2238) according to product specification. DNase treated RNA was then applied again to a RNeasy spin column with 0.5 volumes of ethanol, washed and eluted according to kit instructions.

tag: ecotype

tag: tissue

value: leaf


title: DNase-Seq (DNCHD4) 2

status: Public on Dec 24 2013

city: Bethesda

first: Songjoon

last: Baek

state: MD

address: 41 Library Drive

department: CCR

institute: NCI / NIH

description: DNase-Seq (-Tet, dominant-negative CHD4 )

library_source: genomic

data_processing:
Illumina Casava 1.6 software was used for basecalling.
Areas of enrichment were identified using DNase2Hotspots software(https://sourceforge.net/projects/dnase2hotspots) software with FDR 0%.
Raw signal density graphs were generated by counting tags at each genomic coordinate after extending the length of sequences to 150bp by strands.
Genome_build: mm9
Supplementary_files_format_and_content: comma-delimited (CSV)  text files include genomic locations (Chromosome, start,  end ), z-score, p-value and the maximum density of hotspots.

molecule: genomic DNA

organism: Mus musculus

source_name: 3134-tet (DNCHD4)

growth_protocol: The 3134 murine mammary epithelial cell line, originally derived from a subclone of 904.13, was maintained in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS, Atlanta Biologicals, Lawrenceville, GA), 1 mM sodium pyruvate, 0.1 mM non-essential amino acids, 2 mM L-glutamine, and 5 mg/ml penicillin-streptomycin in a 37°C incubator with 5% C02.

extract_protocol: Expression of dominant-negative variant proteins was induced by removal of tetracycline for 48 hrs prior to harvest by trypsinization. Nuclei from harvested cells were isolated and digested with 60-80 U/ml DNase I (Roche, Indianapolis, IN) for 3 min at 37°C. Digested DNA were incubated at 55°C with 10 μg/ml RNase A (Roche) for a few hours to overnight followed by addition of 25 μg/ml Proteinase K (Ambion, Austin, TX) and incubation at 55°C for at least 4 hrs. DNA fragments were purified by phenol/chloroform extraction and ultracentrifugation through a sucrose gradient. After purification, fragments (between 100 and 500 bp in size) were then pooled, precipitated and assembled into libraries for sequencing.

treatment_protocol: To generate dominant-negative variant cell lines, the coding sequence of human SNF2H (hSNF2H) fused to the FLAG tag sequence at the N-terminus in the pCI-neo vector was received as a gift from David Picketts (U. of Ottawa). Using BamHI and SalI restriction enzyme sites, FLAG-hSNF2H was cloned into a tetracycline-inducible retroviral vector (pRevTRELink,  followed by site-directed mutagenesis of lysine 211 to arginine using the QuikChange XL Site Directed Mutagenesis Kit according to the manufacturer’s instructions (Stratagene, La Jolla, CA). This dominant-negative variant of FLAG-hSNF2H (dnSnf2h) was then stably integrated into a cell line containing the tetracycline transactivator regulatory system (3134Tet, 7110).  Similarly, the coding sequence of mouse Chd4 (a gift from John Svaren (U. of Wisconsin)) was cloned into pRevTRELink containing a triple FLAG tag sequence using NotI/SalI restriction enzyme sites followed by site-directed mutagenesis of lysine 250 to cysteine and integration into the 3134Tet cell line. . Constructs were fully sequenced to confirm accuracy prior to cell line integrations. All tetracycline-regulated cell lines were maintained in DMEM and 5 μg/ml tetracycline to repress expression of the dominant-negative proteins. For experiments, cells were plated in DMEM supplemented with 10% charcoal-dextran treated FBS with or without tetracycline for 48 hrs.

tag: cell line

tag: treatment


title: 0320 CSR-1 IP gfp::cdk-1

city: Worcester

first: Craig

last: Mello

state: MA

address: 368 Plantatoin Street, Suite AS5-2047

department: Program in Molecular Medicine

institute: University of Massachusetts Medical School

description: 0320 CSR-1 IP gfp::cdk-1

data_processing:
Remove 5' barcode and 3' barcode (or called linker)
Map the reads to WS215 genome and annotation
A custom PERL pipeline is used to summarize reads and generate histogram at single nt level.
Generic genome browse is used to visualize the data
Genome_build: WormBase WS215
Supplementary_files_format_and_content: Tab delimited txt format file containing sequence and read number used in the analysis with the first line as header

organism: Caenorhabditis elegans

source_name: neSi11 gfp::cdk-1; neSi23 flag::csr-1(IP)

growth_protocol: C. elegans worm grown at 20 degree

extract_protocol:
small RNA cloning, 5' and 3' ligation dependent
Total small RNA cloning/Small RNA cloning by CSR-1 IP

treatment_protocol: Small RNAs of 10-40nt from total RNAs were resolved on a 15% polyacrylamide 7M Urea Gel, along with 2 picomoles of RNA standard (18- and 40-nt) in separate lanes. SYBR gold staining was used to visualize the RNA standards. A gel fragment was excised from the sample lanes in the migration range between the two standards. RNA was eluted from the gel fragment in (0.3M NaCl-TE pH7.5) solution overnight and isopropanol-precipitated with 20mg of glycogen as the carrier. The gel purified RNA was incubated with 0.25 U/µl TAP (Epicentre) and  1U/µl SuperRNaseIN (Ambion) in 10µl reaction containing 1X buffer at 37 oC for 1 hr, followed by phenol-chloroform extraction and isopropanol precipitation. The TAP-treated samples were then incubated with 5µM of 3’-end linker (1, 2, 3 from IDT DNA Technologies), 1 Unit/µl of SuperRNaseIN, 10% DMSO and 1 U/µl T4 RNA ligase (Takara) in 10µl ligation buffer (50mM Tris-Cl pH7.5, 10mM MgCl2,10mg/mL BSA, 10mM DTT).  The 3’ ligated products were gel purified, eluted, precipitated and then incubated with 5 µM of 5’ adapter oligonucleotide with 4nt barcode at the 5' end, 1 Unit/µl SuperRNaseIN (Ambion) and 2 U/µl of T4 RNA ligase in ligation buffer (50mM Tris-HCI pH7.5), 10mM MgCI2, 10mM DTT, 1mM ATP) and 10% DMSO. The ligated products were gel purified and reverse transcribed in a standard 20µl reaction (SuperScript III, Invitrogen). The cDNA was amplified by PCR and purified in a 8% acrylamide gel. PCR products generated for all the samples were sequenced on a Solexa sequencing HiSeq platform (Illumina, Inc.) and single-end 50 nt reads were obtained.

tag: 5' barcode

value: 5' barcode (D) ATCA

tag: 3' barcode

value: 3' barcode (2) CACTCGGGCACCAAGGA

tag: rna subset

value: small RNA


title: RNAPol2_Jurkat_THZ-2-149_6hr_ChipSeq

city: Cambridge

first: Richard

middle: A

last: Young

address: 9 Cambridge Center

institute: Whitehead Institute for Biomedical Research

description: Chromatin IP against Total RNAPol2 in Jurkat_THZ-2-149_6hr

data_processing:
Images analysis and base calling was done using the solexa pipeline.
For all samples reads were aligned to their indicated build using bowtie with parameters -e 70 -k 2 -m 2 -n 2 --best --concise. Seed length (-l) was set to read length for each dataset.
Genome_build: hg18
Supplementary_files_format_and_content: WIG files: For all samples, reads were aligned with bowtie 0.12.9 to the mm9 genome.  Aligned sequences were processed with MACS 1.4.2 with parameters -w -S --space=50 --keep-dup=auto -p 1e-9. Counts were normalized to reads per million.

organism: Homo sapiens

source_name: T-ALL cell line

growth_protocol: Cells grown in RPMI1640+10%FBS

extract_protocol: Whole cell extracts were sonicated to solubilize the chromatin. The chromatin extracts containing DNA fragments with an average size of 500 bp were immunoprecipitated using different antibodies. Purified immunoprecipitated DNA were prepared for sequencing according to a modified version of the Solexa Genomic DNA protocol. Fragmented DNA was end repaired and subjected to 18 cycles of LM-PCR using oligos provided by Illumina. Amplified fragments between 150 and 300bp (representing shear fragments between 50 and 200nt in length and ~100bp of primer sequence) were isolated by agarose gel electrophoresis and purified.

treatment_protocol: Treated with 250nM of THZ-2-149 for 6 hours (competitive Cdk7 inhibitor - non-covalent analog of THZ-2-102).

tag: chip antibody

value: RNA Pol II (Rpb1 N-terminus)

tag: antibody catalog number

value: Santa Cruz SC-899

value: Jurkat

tag: concentration

tag: drug

tag: duration of treatment

value: 6 hr


title: RNAPol2_Jurkat_THZ-2-102_r1_6hr_ChipSeq

description: Chromatin IP against Total RNAPol2 in Jurkat_THZ-2-102 6hr rep1

treatment_protocol: Treated with 250nM THZ-2-102 for 6 hours (Cdk7 covalent inhibitor - N. Gray lab).  Replicate 1.

tag: replicate


title: 0320 total oma-1::gfp csr-1

description: 0320 total oma-1::gfp csr-1

source_name: neSi22 oma-1::gfp; csr-1(tm892)

value: 3' barcode (1) CTGTAGGCACCATCAAT


title: 0320 CSR-1 IP oma-1::gfp

description: 0320 CSR-1 IP oma-1::gfp

source_name: neSi22 oma-1::gfp; neSi23 flag::csr-1(IP)

value: 5' barcode (B) CAGT


title: 0320 input for CSR-1 IP gfp::cdk-1

description: 0320 input for CSR-1 IP gfp::cdk-1

source_name: neSi11 gfp::cdk-1; neSi23 flag::csr-1(input)

value: 3' barcode (3) TTTAACCGCGAATTCCAG


title: 0320 input for CSR-1 IP oma-1::gfp

description: 0320 input for CSR-1 IP oma-1::gfp

source_name: neSi22 oma-1::gfp; neSi23 flag::csr-1(Input)


title: 0320 total oma-1::gfp

description: 0320 total oma-1::gfp

source_name: neSi22 oma-1::gfp (RNAa)


title: 0320 total oma-1::gfp rde-3

description: 0320 total oma-1::gfp rde-3

source_name: neSi22 oma-1::gfp; rde-3(ne3370)

value: 5' barcode (C) GCTG


title: RNAPol2_Jurkat_DMSO_6hr_ChipSeq

description: Chromatin IP against Total RNAPol2 in Jurkat DMSO 6hr

treatment_protocol: Cells treated with DMSO/vehicle for 6 hours.  Equivalent DMSO concentration to the matched set of drug treatments (flavopiridol, THZ-2-102, THZ-2-149)

value: DMSO


title: CDD410

city: Sariyer

first: Cory

middle: David

last: Dunn

state: Istanbul

address: Rumelifeneri Yolu

department: Molecular Biology and Genetics

country: Turkey

institute: Koç University

description:
Ot5374
Transcriptome of whole cells.
Paired-end prep sample, but only one end (R2) analyzed bioinformatically.

data_processing:
Uploaded to Galaxy server (https://usegalaxy.org/) for analysis.
Ran FASTQ Groomer to prepare sequence reads for mapping.
Mapped against reference genome sacCer3 using Tophat 2.0.6.
Expression level and statistical analysis determined for all samples by cuffdiff 2.1.1 with geometric library normalization, a blind dispersal estimation method, and a 0.05 false discovery rate with a minimum alignment count of 10 for FDR assignment.  In addition, we used the 'multi-read correct' setting and bias correction based on the sacCer3 genome.
Transcript assignments using cuffdiff 2.1.1 were made using the Ensembl EF4 GTF file for Saccharomyces cerevisiae.
Genome_build: sacCer3
Supplementary_files_format_and_content: Tab-delimited text files: Transcript_FPKM.txt contains FPKM read counts, and Transcript_Diff_Exp.txt contains statistical analysis (FDR) of differential expression.

organism: Saccharomyces cerevisiae

source_name: whole cells, mrpl16∆ rho-minus

growth_protocol: YEPD medium, logarithmic phase.

extract_protocol:
Nucleospin RNA II kit.
PolyA cDNA preparation performed by Otogenetics Corporation.

treatment_protocol: For those strains lacking mtDNA, mtDNA was removed by deletion of mitochondrial ribosomal subunit Mrpl16p. Blocking translation in mitochondria was previously shown to cause mtDNA loss. Rho-minus status of the cells lacking MRPL16 was confirmed by mating assay.

tag: strain/background

tag: genotype/variation

value: mrpl16∆ rho-minus

tag: cell type

value: whole cells


title: CDD452

description:
Ot5377
Transcriptome of whole cells.
Paired-end prep sample, but only one end (R2) analyzed bioinformatically.

source_name: whole cells, sap155∆ sap190∆ mrpl16∆ rho-minus

value: sap155∆ sap190∆ mrpl16∆ rho-minus


title: CDD463

description:
Ot5666
Transcriptome of whole cells.
Paired-end prep sample, but only one end (R2) analyzed bioinformatically.

source_name: whole cells, rho-plus

value: rho-plus


title: CDD450

description:
Ot5376
Transcriptome of whole cells.
Paired-end prep sample, but only one end (R2) analyzed bioinformatically.

source_name: whole cells, sap4∆ sap190∆ mrpl16∆ rho-minus

value: sap4∆ sap190∆ mrpl16∆ rho-minus


title: CDD415

description:
Ot5375
Transcriptome of whole cells.
Paired-end prep sample, but only one end (R2) analyzed bioinformatically.

source_name: whole cells, sit4∆ mrpl16∆ rho-minus

value: sit4∆ mrpl16∆ rho-minus


title: smRNA sequencing of of H1 derived Mesendoderm Cultured cells; smRNA-seq_h1-mesendoderm_r2a

city: La Jolla

first: UCSD AND

last: SALK

state: CA

address: Health Sciences Drive

institute: University of California, San Diego

description:
sample_term_id: NTR_0000856
assay_term_id: OBI_0001271
nucleic_acid_term_id: SO_0000871
Design description: smRNA sequencing of of H1 derived Mesendoderm Cultured cells
Library name: smRNA-seq_h1-mesendoderm_r2a
EDACC Genboree Experiment Page:
http://genboree.org/java-bin/project.jsp?projectName=XML%20Submissions%2FUCSD%2FEXPERIMENT%2FEDACC.18998
EDACC Genboree Sample Page:
http://genboree.org/java-bin/project.jsp?projectName=XML%20Submissions%2FUCSD%2FSAMPLE%2FEDACC.10253
****************
For data usage terms and conditions, please refer to:
http://www.drugabuse.gov/funding/funding-opportunities/nih-common-fund/epigenomics-data-access-policies
****************

data_processing: Various levels of processed data files will be made available as this project proceeds.

source_name: H1 cell line treated with BMP4 and Activin A biological replicate 2 (r2); smRNA-seq_h1-mesendoderm_r2a

extract_protocol: Library construction protocol: Small RNAs were isolated using the miRNeasy Mini Kit (QIAGEN) as per manufacturer's instructions. Approximately one microgram of total RNA was used as input. Each sequencing library was constructed using the TruSeq Small RNA Sample Prep Kit (Illumina, San Diego, CA) according to the manufacturer's instructions. The completed library was then gel size selected to approximately 145-160 bp using the QIAquick Gel Extraction Kit (QIAGEN). Libraries were pooled, and then sequenced using the Illumina HiSeq 2000 (Illumina) instrument as per manufacturer's instructions.

tag: sra sample accession

tag: disease

value: Thomson Laboratory

value: Cell Line

tag: line

tag: lineage

value: embryonic stem cell differentiated into mesendoderm cells

value: H1 cells differentiated in +5ng/mL BMP4 +25ng/mL Activin A medium

tag: passage

tag: medium

value: TeSR

tag: Sex

value: Male

tag: batch

value: Replicate 2

value: mRNA-Seq

value: Qiagen RNeasy mini kit, performed as per manufacturer's instructions

value: 1 µg

value: Dynabeads oligo(dt)25 (Invitrogen)

value: PolyA RNA resuspended in 2x Superscript III buffer supplemented with 10 mM DTT was incubated at 94°C for 4 min

value: SuperScript III (Invitrogen)

value: Purification with RNAClean XP beads (Agencourt)

value: DNA Polymerase I (E. coli) (New England Biolabs)

value: dNTP mix made from 10 mM dATP, dCTP, dGTP, dUTP

value: Purification with AMPure XP beads (Agencourt)

value: TruSeq Multiplexed Adapters (Illumina)

value: 16°C for 16 hours with T4 DNA ligase (New England Biolabs)

value: Two rounds of purification with AMPure XP beads (Agencourt)

value: One half of adapter-ligated cDNA digested with uracil DNA glycosylase (Enzymatics) and used for subsequent steps.

value: TruSeq PCR Master Mix (Illumina)

value: 98°C 30 sec; 10 cycles of 98°C 10 sec, 60°C 30 sec, 72°C 30 sec; 72°C 10 min

value: TruSeq PCR Primer Cocktail (Illumina)

value: Dynabeads oligo(dt)25 protocol

value: Random hexamers

value: First-strand Superscript III (Invitrogen) RT protocol. RNAClean XP purification of first strand product. Second-strand synthesis using dUTP-containing dNTPS and DNA Polymerase 1 (E. coli) (New England Biolabs)

value: total RNA

value: 5 ng/µL

value: 5' CAAGCAGAAGACGGCATACGAGAT 3'


title: smRNA sequencing of of H1 BMP4 derived Trophoblast Cultured cells; smRNA-seq_h1-bmp4_r1a

description:
sample_term_id: UBERON_0000088
assay_term_id: OBI_0001271
nucleic_acid_term_id: SO_0000871
Design description: smRNA sequencing of of H1 BMP4 derived Trophoblast Cultured cells
Library name: smRNA-seq_h1-bmp4_r1a
EDACC Genboree Experiment Page:
http://genboree.org/java-bin/project.jsp?projectName=XML%20Submissions%2FUCSD%2FEXPERIMENT%2FEDACC.18995
EDACC Genboree Sample Page:
http://genboree.org/java-bin/project.jsp?projectName=XML%20Submissions%2FUCSD%2FSAMPLE%2FEDACC.12102
****************
For data usage terms and conditions, please refer to:
http://www.drugabuse.gov/funding/funding-opportunities/nih-common-fund/epigenomics-data-access-policies
****************

source_name: H1 BMP4 Derived Trophoblast Cultured Cell Line, biological replicate 1 (r1); smRNA-seq_h1-bmp4_r1a

value: Thomson Laboratory

value: Cell Line

value: embryonic stem cell differentiated by treatment with fibronectin and human collagen

value: H1 cells were cultured on plates coated with fibronectin and human collagen

value: 50% StemLine II serum-free HSC expansion medium (HSFEM; Sigma), 50% ESFM, GlutaMAX (1/100 dilution), Ex-Cyte supplement (1/2000 dilution), 100 mM MTG, and 10 ng/ml FGF2.

value: Male

value: Replicate 1

value: mRNA-Seq

value: Qiagen RNeasy mini kit, performed as per manufacturer's instructions

value: 1 µg

value: Dynabeads oligo(dt)25 (Invitrogen)

value: PolyA RNA resuspended in 2x Superscript III buffer supplemented with 10 mM DTT was incubated at 94°C for 4 min

value: SuperScript III (Invitrogen)

value: Purification with RNAClean XP beads (Agencourt)


title: smRNA sequencing of of H1 derived Neuronal Progenitor Cultured cells; smRNA-seq_h1-npc_r1a

description:
sample_term_id: CL_0000047
assay_term_id: OBI_0001271
nucleic_acid_term_id: SO_0000871
Design description: smRNA sequencing of of H1 derived Neuronal Progenitor Cultured cells
Library name: smRNA-seq_h1-npc_r1a
EDACC Genboree Experiment Page:
http://genboree.org/java-bin/project.jsp?projectName=XML%20Submissions%2FUCSD%2FEXPERIMENT%2FEDACC.18993
EDACC Genboree Sample Page:
http://genboree.org/java-bin/project.jsp?projectName=XML%20Submissions%2FUCSD%2FSAMPLE%2FEDACC.6199
****************
For data usage terms and conditions, please refer to:
http://www.drugabuse.gov/funding/funding-opportunities/nih-common-fund/epigenomics-data-access-policies
****************

source_name: H1 cell line differentiated into neural progenitor cells, biological replicate 1 (r1); smRNA-seq_h1-npc_r1a

value: embryonic stem cell differentiated into neural progenitor cells

value: H1 cells were differentiated by culturing in insulin, SB, Noggin

value: TeSR

value: DNA Polymerase I (E. coli) (New England Biolabs)

value: dNTP mix made from 10 mM dATP, dCTP, dGTP, dUTP

value: Purification with AMPure XP beads (Agencourt)

value: TruSeq Multiplexed Adapters (Illumina)

value: 16°C for 16 hours with T4 DNA ligase (New England Biolabs)

value: Two rounds of purification with AMPure XP beads (Agencourt)

value: One half of adapter-ligated cDNA digested with uracil DNA glycosylase (Enzymatics) and used for subsequent steps.

value: TruSeq PCR Master Mix (Illumina)

value: 98°C 30 sec; 10 cycles of 98°C 10 sec, 60°C 30 sec, 72°C 30 sec; 72°C 10 min

value: TruSeq PCR Primer Cocktail (Illumina)

value: Dynabeads oligo(dt)25 protocol

value: Random hexamers

value: First-strand Superscript III (Invitrogen) RT protocol. RNAClean XP purification of first strand product. Second-strand synthesis using dUTP-containing dNTPS and DNA Polymerase 1 (E. coli) (New England Biolabs)

value: total RNA


title: smRNA sequencing of of H1 derived Mesendoderm Cultured cells; smRNA-seq_h1-mesendoderm_r1a

description:
sample_term_id: NTR_0000856
assay_term_id: OBI_0001271
nucleic_acid_term_id: SO_0000871
Design description: smRNA sequencing of of H1 derived Mesendoderm Cultured cells
Library name: smRNA-seq_h1-mesendoderm_r1a
EDACC Genboree Experiment Page:
http://genboree.org/java-bin/project.jsp?projectName=XML%20Submissions%2FUCSD%2FEXPERIMENT%2FEDACC.18997
EDACC Genboree Sample Page:
http://genboree.org/java-bin/project.jsp?projectName=XML%20Submissions%2FUCSD%2FSAMPLE%2FEDACC.10252
****************
For data usage terms and conditions, please refer to:
http://www.drugabuse.gov/funding/funding-opportunities/nih-common-fund/epigenomics-data-access-policies
****************

source_name: H1 cell line treated with BMP4 and Activin A biological replicate 1 (r1); smRNA-seq_h1-mesendoderm_r1a

value: Thomson Laboratory

value: Cell Line

value: embryonic stem cell differentiated into mesendoderm cells

value: H1 cells differentiated in +5ng/mL BMP4 +25ng/mL Activin A medium

value: Male

value: Replicate 1

value: mRNA-Seq

value: Qiagen RNeasy mini kit, performed as per manufacturer's instructions

value: 1 µg

value: Dynabeads oligo(dt)25 (Invitrogen)

value: PolyA RNA resuspended in 2x Superscript III buffer supplemented with 10 mM DTT was incubated at 94°C for 4 min

value: SuperScript III (Invitrogen)

value: Purification with RNAClean XP beads (Agencourt)

value: 5 ng/µL

value: 5' CAAGCAGAAGACGGCATACGAGAT 3'


title: smRNA sequencing of of H1 cells; smRNA-seq_h1_r2a

description:
sample_term_id: EFO_0003042
assay_term_id: OBI_0001271
nucleic_acid_term_id: SO_0000871
Design description: smRNA sequencing of of H1 cells
Library name: smRNA-seq_h1_r2a
EDACC Genboree Experiment Page:
http://genboree.org/java-bin/project.jsp?projectName=XML%20Submissions%2FUCSD%2FEXPERIMENT%2FEDACC.18992
EDACC Genboree Sample Page:
http://genboree.org/java-bin/project.jsp?projectName=XML%20Submissions%2FUCSD%2FSAMPLE%2FEDACC.1106
****************
For data usage terms and conditions, please refer to:
http://www.drugabuse.gov/funding/funding-opportunities/nih-common-fund/epigenomics-data-access-policies
****************

source_name: H1 Cell Line, biological replicate 2 (r2); smRNA-seq_h1_r2a

value: embryonic stem cell

value: TeSR

value: DNA Polymerase I (E. coli) (New England Biolabs)

value: dNTP mix made from 10 mM dATP, dCTP, dGTP, dUTP

value: Purification with AMPure XP beads (Agencourt)

value: TruSeq Multiplexed Adapters (Illumina)

value: 16°C for 16 hours with T4 DNA ligase (New England Biolabs)

value: Two rounds of purification with AMPure XP beads (Agencourt)

value: One half of adapter-ligated cDNA digested with uracil DNA glycosylase (Enzymatics) and used for subsequent steps.

value: TruSeq PCR Master Mix (Illumina)

value: 98°C 30 sec; 10 cycles of 98°C 10 sec, 60°C 30 sec, 72°C 30 sec; 72°C 10 min

value: TruSeq PCR Primer Cocktail (Illumina)

value: Dynabeads oligo(dt)25 protocol

value: Random hexamers

value: First-strand Superscript III (Invitrogen) RT protocol. RNAClean XP purification of first strand product. Second-strand synthesis using dUTP-containing dNTPS and DNA Polymerase 1 (E. coli) (New England Biolabs)


title: WCE_Input_ChipSeq

description:
Chromatin IP against WCE_Input
barcode (already removed): TGACCA

data_processing:
Images analysis and base calling was done using the solexa pipeline.
For all samples reads were aligned to their indicated build using bowtie with parameters -e 70 -k 1 -m 1 -n 2 --best --concise. Seed length (-l) was set to read length for each dataset.
Genome_build: hg18
Supplementary_files_format_and_content: WIG files: For all samples aligned sequences were extended 150bp upstream and 0bp downstream (with respect to read strand) and allocated into 25bp bins. Counts were normalized to reads per million, and bins with at least 1 read per million are shown.

source_name: T-ALL cell line

growth_protocol: Human Jurkat cells were purchased from the American Type Culture Collection (ATCC), Manassas, VA. Cells were maintained under typical conditions in RPMI media with 10% Bovine Calf Serum. For location analysis, cells were grown to a density of 1 million per ml and 99% viability prior to cross-linked with formaldehyde for 20 min.

tag: chip antibody

value: None WCE

tag: cell line

value: Jurkat


title: CDD454

description:
Ot5378
Transcriptome of whole cells.
Paired-end prep sample, but only one end (R2) analyzed bioinformatically.

source_name: whole cells, pph21∆ pph22∆ mrpl16∆ rho-minus

value: pph21∆ pph22∆ mrpl16∆ rho-minus

value: whole cells


title: smRNA sequencing of of H1 derived mesenchymal cells; smRNA-seq_h1-msc_r1a

description:
sample_term_id: EFO_0000586
assay_term_id: OBI_0001271
nucleic_acid_term_id: SO_0000871
Design description: smRNA sequencing of of H1 derived mesenchymal cells
Library name: smRNA-seq_h1-msc_r1a
EDACC Genboree Experiment Page:
http://genboree.org/java-bin/project.jsp?projectName=XML%20Submissions%2FUCSD%2FEXPERIMENT%2FEDACC.18989
EDACC Genboree Sample Page:
http://genboree.org/java-bin/project.jsp?projectName=XML%20Submissions%2FUCSD%2FSAMPLE%2FEDACC.11127
****************
For data usage terms and conditions, please refer to:
http://www.drugabuse.gov/funding/funding-opportunities/nih-common-fund/epigenomics-data-access-policies
****************

source_name: H1-Mesenchymal Stem Cell Line, biological replicate 1 (r1); smRNA-seq_h1-msc_r1a

value: Thomson Laboratory

value: Cell Line

value: embryonic stem cell differentiated by treatment with fibronectin and human collagen

value: H1 cells were cultured on plates coated with fibronectin and human collagen

value: 50% StemLine II serum-free HSC expansion medium (HSFEM; Sigma), 50% ESFM, GlutaMAX (1/100 dilution), Ex-Cyte supplement (1/2000 dilution), 100 mM MTG, and 10 ng/ml FGF2.

value: Male

value: Replicate 1

value: mRNA-Seq

value: Qiagen RNeasy mini kit, performed as per manufacturer's instructions

value: 1 µg

value: Dynabeads oligo(dt)25 (Invitrogen)

value: PolyA RNA resuspended in 2x Superscript III buffer supplemented with 10 mM DTT was incubated at 94°C for 4 min

value: SuperScript III (Invitrogen)

value: Purification with RNAClean XP beads (Agencourt)

value: DNA Polymerase I (E. coli) (New England Biolabs)

value: total RNA

value: 5 ng/µL

value: 5' CAAGCAGAAGACGGCATACGAGAT 3'


title: smRNA sequencing of of H1 derived Neuronal Progenitor Cultured cells; smRNA-seq_h1-npc_r2a

description:
sample_term_id: CL_0000047
assay_term_id: OBI_0001271
nucleic_acid_term_id: SO_0000871
Design description: smRNA sequencing of of H1 derived Neuronal Progenitor Cultured cells
Library name: smRNA-seq_h1-npc_r2a
EDACC Genboree Experiment Page:
http://genboree.org/java-bin/project.jsp?projectName=XML%20Submissions%2FUCSD%2FEXPERIMENT%2FEDACC.18994
EDACC Genboree Sample Page:
http://genboree.org/java-bin/project.jsp?projectName=XML%20Submissions%2FUCSD%2FSAMPLE%2FEDACC.6200
****************
For data usage terms and conditions, please refer to:
http://www.drugabuse.gov/funding/funding-opportunities/nih-common-fund/epigenomics-data-access-policies
****************

source_name: H1 cell line differentiated into neural progenitor cells, biological replicate 2 (r2); smRNA-seq_h1-npc_r2a

value: Thomson Laboratory

value: Cell Line

value: embryonic stem cell differentiated into neural progenitor cells

value: H1 cells were differentiated by culturing in insulin, SB, Noggin

value: TeSR

value: Replicate 2

value: dNTP mix made from 10 mM dATP, dCTP, dGTP, dUTP

value: Purification with AMPure XP beads (Agencourt)

value: TruSeq Multiplexed Adapters (Illumina)

value: 16°C for 16 hours with T4 DNA ligase (New England Biolabs)

value: Two rounds of purification with AMPure XP beads (Agencourt)

value: One half of adapter-ligated cDNA digested with uracil DNA glycosylase (Enzymatics) and used for subsequent steps.

value: TruSeq PCR Master Mix (Illumina)

value: 98°C 30 sec; 10 cycles of 98°C 10 sec, 60°C 30 sec, 72°C 30 sec; 72°C 10 min

value: TruSeq PCR Primer Cocktail (Illumina)

value: Dynabeads oligo(dt)25 protocol

value: Random hexamers

value: First-strand Superscript III (Invitrogen) RT protocol. RNAClean XP purification of first strand product. Second-strand synthesis using dUTP-containing dNTPS and DNA Polymerase 1 (E. coli) (New England Biolabs)


title: smRNA sequencing of of H1 cells; smRNA-seq_h1_r1a

description:
sample_term_id: EFO_0003042
assay_term_id: OBI_0001271
nucleic_acid_term_id: SO_0000871
Design description: smRNA sequencing of of H1 cells
Library name: smRNA-seq_h1_r1a
EDACC Genboree Experiment Page:
http://genboree.org/java-bin/project.jsp?projectName=XML%20Submissions%2FUCSD%2FEXPERIMENT%2FEDACC.18991
EDACC Genboree Sample Page:
http://genboree.org/java-bin/project.jsp?projectName=XML%20Submissions%2FUCSD%2FSAMPLE%2FEDACC.1105
****************
For data usage terms and conditions, please refer to:
http://www.drugabuse.gov/funding/funding-opportunities/nih-common-fund/epigenomics-data-access-policies
****************

source_name: H1 Cell Line, biological replicate 1 (r1); smRNA-seq_h1_r1a

value: Thomson Laboratory

value: Cell Line

value: embryonic stem cell

value: Male

value: mRNA-Seq

value: Qiagen RNeasy mini kit, performed as per manufacturer's instructions

value: 1 µg

value: Dynabeads oligo(dt)25 (Invitrogen)

value: PolyA RNA resuspended in 2x Superscript III buffer supplemented with 10 mM DTT was incubated at 94°C for 4 min

value: SuperScript III (Invitrogen)

value: Purification with RNAClean XP beads (Agencourt)

value: DNA Polymerase I (E. coli) (New England Biolabs)

value: total RNA

value: 5 ng/µL

value: 5' CAAGCAGAAGACGGCATACGAGAT 3'


title: WT

description: wide type

data_processing:
base-call using Illumina's RTA package
adapter sequence was trimmed by Perl script
smRNA reads were aligned to Arabidopsis TAIR9 genome using C program
read counts are normalized to per million mapped reads
Genome_build: TAIR9
Supplementary_files_format_and_content: tab-delimited text files include smRNAs derived from TMM promoter region with their clone numbers

source_name: seedlings

growth_protocol: Plants were grown in a growth chamber under long-day (16 hour of light/8 hour of dark) condition illuminated by cool-white fluorescent light (100 μmol/m2/s) until flowering. Standard floral dip transformation was performed.

extract_protocol: Total RNA was extracted from 2-week-old seedlings by Trizol Reagent (Invitrogen) following the manufacturer’s instructions. Small RNA libraies were constructed by TruSeq Small RNA Sample Prep Kit (Illumina) following the manufacturer’s instructions. Briefly, 1 µg total RNA or purified small RNA was ligated with 3’ and 5’ adaptors and used as a template for RT-PCR. After PCR amplification, 6 µL of each sample were pooled and separated on a 6% polyacrylamide Gel. Gel slices corresponding to ~20-30nt small RNAs were recovered.

tag: tissue

value: seedlings

tag: silencer


title: AS10NI

description: antisense silencer 10 segregated by genetic crosses


title: IR8NI

description: inverted repeat silencer 8 segregated by genetic crosses


title: S26

description: sense silencer 26

value: sense silencer 26


title: AS10

description: antisense silencer 10

value: antisense silencer 10


title: AS4

description: antisense silencer 4

value: antisense silencer 4


title: S21

description: sense silencer 20

value: sense silencer 20


title: H3K27ac_ChipSeq

description:
Chromatin IP against H3K27ac
barcode (already removed): CGATGT

tag: antibody catalog number

value: Abcam AB4729

value: Jurkat


title: Pol2_Jurkat_DMSO_12hr_ChipSeq

description: Chromatin IP against Pol2_Jurkat_DMSO_12hr

treatment_protocol: Cells treated with DMSO/vehicle for 12 hours. Equivalent DMSO concentration to the drug treatment with THZ-2-102.

value: RNA Pol II (Rpb1 N-terminus)

value: Santa Cruz SC-899

tag: treatment

value: DMSO


title: Jurkat_DMSO_WCE_ChipSeq

description: Chromatin IP against Jurkat_DMSO_WCE

treatment_protocol: Treated with DMSO control.

value: None WCE


title: EBNA3C_rep2

status: Public on Dec 22 2013

city: Boston

first: Hufeng

last: Zhou

address: 181 Longwood Ave,

department: Department of Microbiology and Molecular Genetics

institute: Harvard Medical School

description:
Sample 2
ChIP-seq

data_processing:
Basecalls performed by the Harvard Medical School Biopolymer facility as per Illumina recommendations
ChIP-seq reads were aligned to the hg18 genome assembly using bowtie 0.12.7 with the following parameters: -k 1 -m 1
Transcription factor peaks were called using spp 1.10.1 and an IDR cutoff of 0.01
Genome_build: hg18
Supplementary_files_format_and_content: Peaks files were generated using spp

source_name: Lymphoblast Cell Lines

growth_protocol: Cells were grown in RPMI media supplemented witth 10% FetalPlex, 2 mM glutamine, penicillin and streptomycin.

extract_protocol:
Cells were fixed with 1% formaldehyde. After wash with PBS, the cells were lyzed in ChIP lysis buffer. Lysates were sonicated and cleared by centrifugation. After pre-clear with protein A beads, HA antibody (ab9100, Abcam) was incubated with the lysate overnight at 40C. Protein A was used to capture the protein DNA complexed. The eluted protein0DNA complexes were reverse crosslinked and DNA was purified by a Qiagen PCR purification column.
Libraries were prepared using PrepX BeadX kit from IntegenX. Briefly, DNA was end-repaired. 3' A base was added and ligated to adaptors. After PCR amplification, DNA was purified and analyzed with Illumina HiSeq.

value: EBV-infected B Lymphocytes

value: Anti-HA (ab9100, Abcam)


title: EBNA3C_rep1

description:
Sample 1
ChIP-seq


title: IR8

description: inverted repeat silencer 8

value: inverted repeat silencer 8


title: 430ppm - II

city: Esch-Belval

first: Patrick

last: May

state: Luxembourg

address: 7, avenue des Hauts-Fourneaux

department: Luxembourg Centre for Systems Biomedicine

country: Luxembourg

institute: University of Luxembourg

description: small RNA

data_processing:
Bustard 1.96 software (GAIIx)
3'adapter trimming using Novolalign V2.05.04
polyA/N/G/T sequences were discarded using custom Perl scripts
Reads smaller than 15nt were discarded
Supplementary_files_format_and_content: unnormalized read counts after filtering steps

source_name: Arabidopsis thaliana wild-type 21 days rossette leaves

growth_protocol: Seeds of Arabidopsis thaliana (ecotype Col-0) were imbibed at 4°C with distilled water for four days to facilitate uniform germination.  The seeds were re-suspended in 0.05% agarose after the cold treatment and planted in 1m3 mesocosms, where low (430 ± 50µmol mol-1 or ppm) and high (810 ± 50µmol mol-1 or ppm) carbon dioxide concentrations were maintained and monitored daily.  The temperature was set to 24˚C/18˚C (day/night), natural lighting was augmented on overcast days with sodium halide lamps, and humidity was maintained at natural conditions. All environmental parameters were monitored every hour daily.  After five self-seeding generations, leaf samples of the sixth generation were taken during vegetative growth phase.  A sample consists of a pool of 100 leaves that were chosen at random from the mesocosm population using a point-grid system.  Tissue was sampled onto liquid nitrogen and stored at -80°C until further analysis.

extract_protocol: Total RNAs were extracted from frozen leaves using TRIzol Reagent (Life Technologies Cat# 15596-026).  The small RNA libraries were prepared using a Digital Gene Expression for Small RNA Sample Prep Kit purchased from Illumina, Inc. (P/N: 1002398) following strictly the Manufacturer’s Instructions.

treatment_protocol: Arabidopsis thaliana wildtype ecotype Columbia (Col-0) seeds were soaked in water before being planted into soils that were previously wet in KORD 3.0 Sq Grn 504 growing pots (Cat#: 50-1210; Griffin Greenhouse & Nursery Supplies, Inc.).  The growing pots were then moved into a 4°C cold room for three days before being transferred into GCW30 Arabidopsis chambers (Environmental Growth Chambers, Inc) set with 22 ± 0.5°C, and 28 ± 0.5°C, 65% humidity, and long-day lightening (16 hours light/8 hours dark).  Rosette leaves at the bolting stage were collected and frozen immediately in liquid nitrogen for storage.

tag: ecotype

value: Columbia (Col-0)

tag: growth condition

value: 430 ± 50ppm CO2 concentration


title: 810ppm - II

description: small RNA

value: 810 ± 50ppm CO2 concentration


title: 22oC - I

description: small RNA

source_name: Arabidopsis thaliana wild-type rossette leaves at bolting stage

value: 22 ± 0.5oC temperature


title: 28oC - I

description: small RNA

value: 28 ± 0.5oC temperature


title: 810ppm - I

description: small RNA


title: smRNA sequencing of of H1 derived mesenchymal cells; smRNA-seq_h1-msc_r2a

description:
sample_term_id: EFO_0000586
assay_term_id: OBI_0001271
nucleic_acid_term_id: SO_0000871
Design description: smRNA sequencing of of H1 derived mesenchymal cells
Library name: smRNA-seq_h1-msc_r2a
EDACC Genboree Experiment Page:
http://genboree.org/java-bin/project.jsp?projectName=XML%20Submissions%2FUCSD%2FEXPERIMENT%2FEDACC.18990
EDACC Genboree Sample Page:
http://genboree.org/java-bin/project.jsp?projectName=XML%20Submissions%2FUCSD%2FSAMPLE%2FEDACC.11128
****************
For data usage terms and conditions, please refer to:
http://www.drugabuse.gov/funding/funding-opportunities/nih-common-fund/epigenomics-data-access-policies
****************

source_name: H1-Mesenchymal Stem Cell Line, biological replicate 2 (r2); smRNA-seq_h1-msc_r2a

value: Thomson Laboratory

value: Cell Line

value: embryonic stem cell differentiated by treatment with fibronectin and human collagen

value: H1 cells were cultured on plates coated with fibronectin and human collagen

value: 50% StemLine II serum-free HSC expansion medium (HSFEM; Sigma), 50% ESFM, GlutaMAX (1/100 dilution), Ex-Cyte supplement (1/2000 dilution), 100 mM MTG, and 10 ng/ml FGF2.

value: Male

value: Replicate 2

value: mRNA-Seq

value: Qiagen RNeasy mini kit, performed as per manufacturer's instructions

value: 1 µg

value: Dynabeads oligo(dt)25 (Invitrogen)

value: PolyA RNA resuspended in 2x Superscript III buffer supplemented with 10 mM DTT was incubated at 94°C for 4 min

value: SuperScript III (Invitrogen)

value: Purification with RNAClean XP beads (Agencourt)

value: DNA Polymerase I (E. coli) (New England Biolabs)

value: dNTP mix made from 10 mM dATP, dCTP, dGTP, dUTP

value: Purification with AMPure XP beads (Agencourt)

value: TruSeq Multiplexed Adapters (Illumina)

value: 16°C for 16 hours with T4 DNA ligase (New England Biolabs)

value: Two rounds of purification with AMPure XP beads (Agencourt)

value: One half of adapter-ligated cDNA digested with uracil DNA glycosylase (Enzymatics) and used for subsequent steps.

value: TruSeq PCR Master Mix (Illumina)

value: 98°C 30 sec; 10 cycles of 98°C 10 sec, 60°C 30 sec, 72°C 30 sec; 72°C 10 min

value: TruSeq PCR Primer Cocktail (Illumina)

value: Dynabeads oligo(dt)25 protocol

value: Random hexamers

value: First-strand Superscript III (Invitrogen) RT protocol. RNAClean XP purification of first strand product. Second-strand synthesis using dUTP-containing dNTPS and DNA Polymerase 1 (E. coli) (New England Biolabs)

value: total RNA

value: 5 ng/µL

value: 5' CAAGCAGAAGACGGCATACGAGAT 3'


title: CDK7_ChipSeq

description:
Chromatin IP against CDK7
barcode (already removed): ATCACG

value: Abcam AB9516

value: Jurkat


title: BMDC 6hr LPS JunB

city: South San Francisco

first: Jason

last: Hackney

address: 1 DNA Way

department: Department of Bioinformatics and Computational Biology

institute: Genentech, Inc.

description: Anti-BATF antibody was generated in-house by immunizing rabbits with a mixture of two peptides (1-22 aa and 108-125 aa), followed by affinity purification.

data_processing:
Illumina Casava1.7 software was used for basecalling and generating eland extended files mapped onto mm9.
Sequenced reads for the BMDCs were mapped to the mm9 whole genome using gsnap with parameter s-d mm9 -Q -n 2 -A sam.
QuEST was used for peak calling on the aligned eland extended or SAM files with the recommended 30-fold enrichment used as threshold.
Genome_build: MGSCv37
Supplementary_files_format_and_content: wig files indicating the reads aligned to the mm9 genome.

source_name: BMDCs, LPS, 6 hrs, JunB ChIP

extract_protocol: 20000000 cells were harvested, crosslinked with formaldehyde, sonicated, IPed with appropriate antibodies and reverse-crosslinked to obtain 300-500 bp DNA fragments. These DNA fragments were ligated to NuGen Ovation multiplexing adapters for sequencing on the Illumina HiSeq 2000.

treatment_protocol: Bone marrow-derived dendritic cells were stimulated with LPS for 6hrs.

tag: strain/background

tag: cell type

value: bone marrow-derived dendritic cells

value: LPS

tag: timepoint

value: 6 hrs

value: anti-JunB (Santa Cruz Biotechnology, sc-73x (210x), Lot# D1210)


title: 430ppm - I

description: small RNA

value: Columbia (Col-0)

value: 430 ± 50ppm CO2 concentration


title: BMDC 6hr LPS BATF

source_name: BMDCs, LPS, 6 hrs, BATF ChIP

value: anti-BATF (in-house)


title: RNA-seq_WT-2

first: Junjie

last: Guo

institute: Whitehead Institute

data_processing:
Pair-end reads (97 bp) of cDNA sequences were aligned to mouse reference genome mm9 by TopHat.
The relative abundances of each transcript were estimated by Cufflinks using Ensembl gene annotation (Build NCBIM37).
Transcript fpkm values were averaged across three replicates.
Genome_build: mm9
Supplementary_files_format_and_content: tab-delimited file containing transcript annotation and averaged fpkm values

molecule: polyA RNA

source_name: Dentate gyrus

growth_protocol: Adult mice (8- to 10-weeks-old, male, C57BL/6 background) were used for analysis in accordance with protocols approved by the Institutional Animal Care and Use Committee.

extract_protocol:
Dentate gyri were rapidly micro-dissected bilaterally from adult mice. Total RNA was extracted using the RNeasy kit (Qiagen) with on-column DNaseI-treatment.
Poly(A)-containing mRNA was purified from 1 µg DNase-treated total RNA. Following the purification, the mRNA was fragmented into small pieces using divalent cations under elevated temperature. Reverse transcriptase and random primers were used to generate the first-strand cDNA. The second-strand cDNA was synthesized using DNA polymerase I and RNaseH. These cDNA fragments went through an end-repair process using T4 DNA polymerase, T4 PNK and Klenow DNA polymerase, and the addition of a single ‘A’ base using Klenow Fragment (3' to 5' exo minus), then the ligation of the Illumina PE adapters using T4 DNA ligase. An index (up to 12) was inserted into Illumina adapters so that multiple samples can be sequenced in one lane of 8-lane flow cell if necessary. These products were then purified and enriched with PCR to create the final cDNA library for Illumina HiSeq2000 sequencing.

tag: strain

value: Male

tag: age

value: 8-10 weeks


title: RNA-seq_WT-3


title: Ter119_2_Capture-C

status: Public on Dec 21 2013

city: oxford

first: Jim

last: Hughes

address: Weatherall Institute Of Molecular Me

department: MHU

country: United Kingdom

institute: University of Oxford

data_processing:
Paired-end sequences, were mapped as single-end reads to overcome the built in assumptions of the relative positioning of paired-end sequences in the sequence alignment programs.  The sequences were mapped to an in silico Dpn II digested version of MM9 (UCSC Genome browser NCBI build 37).  Reads which did not align in the first pass were tested to see if they contained a Dpn II restriction site and if so were assumed to be a potential non-alignable junction fragment.  These fragments where split at the Dpn II site and if the smallest resulting fragment was >= 20 bp the individual fragments were remapped.  The paired-end relationship between the reads were reestablished and quantified using the custom script Frag2Arc.pl, excluding read pairs which map to the same Dpn II fragment and stored the resultant ligation events in a GFF3 file.
Genome_build: mm9
Supplementary_files_format_and_content: gff

source_name: Ter119+ Cells

extract_protocol: The protocol used for generation of the 3C libraries was similar to previous methods with minor modifications. Briefly, 107 cells were crosslinked with 2% formaldehyde (10 minutes, room temperature); quenched with cold glycine; washed in phosphate buffered saline; resuspended in cold lysis buffer (tris 10mM, NaCl 10mM, NP40 0.2%, complete proteinase inhibitor (Roche)) and snap frozen to -80.  Cells were thawed on ice, washed in milliq dH2O and dounce homogenised on ice (x 40 strokes). Cells were then resuspended with 0.25% SDS and restriction enzyme buffer and incubated at 37oC for 1h at 1400rpm on a Comfort Thermomixer (Eppendorf) followed by a further incubation of 1h following the addition of triton X100 (final concentration 1.67%). An overnight digestion was performed using Dpn II (500U /ml (NEB) at 37 degrees / 1400 rpm). The digested chromatin was ligated overnight (Fermentas HC Ligase final concentration 10U/ml) at 16 degrees at 1400 rpm on the Thermomixer. The samples were then decrosslinked overnight at 65 oC with Proteinase K (Roche) followed by a 30 min incubation at 37 oC with RNAse (Roche). Phenol/Chloroform extraction was then performed followed by an Ethanol precipitation and a wash with 70% Ethanol. Digestion efficiencys were assessed by gel electrophoresis (1% agarose) and RT-PCR (Taqman), which showed digestion efficiencies in excess of 80%.  DNA content of the Dpn II 3C libraries were quantitated using a Qubit fluorometer (Life technologies) and 5ug of each library was sheared using a Covaris S2 in milliq dH2O. Covaris settings used were: duty cycle 10%, Intensity 5, Cycles/burst 200, Time 6 cycles of 60seconds, Set Mode Frequency sweeping Temperature 4 to 7 degrees. Following shearing DNA was purified using AMPureXP beads (Agencourt) and DNA quality assessed on a Bioanalyser 2100 using a DNA High Sensitivity Chip (Agilent). DNA end repair and adapter ligation was performed using the NEB Next DNA sample preparation reagent kit using the standard protocol. Excess adapters were removed using AMpureXP beads. 500ng of each adapter ligated library were used as input for the SureSelect target enrichment kit using the recommended protocol and the custom designed Capture-C library. The quality of the resultant captured library was assessed by Bioanalyser using the high sensitivity Chip. The generated libraries fell within the expected guidelines for output of the SureSelect target enrichment kit and were submitted for Hi-Seq paired sequencing (50 bp read length).

treatment_protocol: Mouse primary erythroid cells were sorted from the spleens of acetylphenylhydrazine-treated mice based on the expression of Ter119 (Spivak et al., 1973; Vernimmen et al., 2009).

value: Ter119+ Cells


title: Normal human serum

city: Nanjing

first: Zhen

state: Jiangsu

address: 22th,Hankou Road

department: School of Lifesciences

country: China

institute: Nanjing University

description: Individuals were recruited from a large pool of individuals seeking a routine health checkup at the Healthy Physical Examination Centre of Jinling Hospital

data_processing: Sequence reads were obtained by Illumina pipeline. Reads of low quality were removed and adaptor sequence were trimed by our Perl scripts.

source_name: 43 healthy Chinese serum

extract_protocol: Briefly, after PAGE purification of small RNA molecules (<30 nucleotides) and ligation of a pair of Solexa adaptors to the RNA 5′ and 3′ ends, RNA molecules were amplified using primers to the adaptor regions for 17 cycles. Fragments that were approximately 90 bp (small RNA+adaptors) were isolated from an agarose gel. Purified DNA was used for cluster generation and sequencing analysis using an Illumina Solexa Sequencer according to the manufacturer’s instructions. Image files were generated by the sequencer and processed to produce digital-quality data. After masking the adaptor sequences and  removing contaminated reads, clean reads were processed for in silico analysis.

treatment_protocol: For Solexa sequencing, equal volumes of sera from 43 healthy human were pooled to  form sample pools. Total RNA was extracted from pooled samples using Trizol  Reagent (Invitrogen).

value: serum


title: Bisulfite-seq_WT-1

data_processing:
Reads mapped to in silico bisulfite-converted genome by Bismark (version 0.7.5) using default parameters, with no mismatch or multiple alignment allowed
Methylation read-out values extracted from SAM files using Bismark methylation extractor (version 0.7.5) in single-end mode
Coverage and methylation values for each genomic location were calculated using customized Perl scripts
Low coverage cytosines were filtered out.
Genome_build: mm9
Supplementary_files_format_and_content: Tab-delimited files containing C/T read counts and methylation values of high-coverage genomic locations. Each sample has one file for each of three contexts of cytosines.

extract_protocol:
Dentate gyri were rapidly micro-dissected bilaterally from adult mice.  Genomic DNA was extracted using the DNeasy Kit (Qiagen).
Three µg genomic DNA spiked-in with 0.5% unmethylated lambda DNA (Promega) from each sample was fragmented to ~ 300 bp DNA fragments using Covaris Acoustic System. The ends of fragmented DNA were repaired with T4 DNA polymerase, Klenow DNA polymerase, and T4 PNK were used to convert the overhangs into phosphorylated blunt ends. Klenow Fragment (3’ to 5’ exo minus) was used to add an ‘A’ base to the 3’ end of the blunt phosphorylated DNA fragments. Following ligation of adapters (TruSeq index, 12 indexes), bisulfite conversion was performed using an EZ DNA Methylation-Direct kit (Zymo). PCR was then used to enrich bisulfite-converted DNA fragments to obtain the DNA library suitable for HiSeq2000 sequencing.


title: RNA-seq_WT-1


title: mES_Capture-C

source_name: E14-TG2a.IV ES cells

value: E14-TG2a.IV ES cells


title: Su(H) Kc167 late embryonic D.melanogaster ChIP Rep3

city: Toronto

first: DCC

last: modENCODE

state: Ontario

address: MaRS Centre, South Tower, 101 College Street, Suite 800

country: Canada

institute: Ontario Institute for Cancer Research

description: ChIP DNA; Antibody information listed below: official name: Su(H);target name: fly genes:Su(H);host: Rabbit;antigen: Epitope corresponding to amino acids 259-594;clonal: Polyclonal;purified: Protein A/G;company: Santa Cruz Biotechnology Inc.;catalog: sc-25761;reference: http://www.scbt.com/datasheet-25761-suppressor-of-hairless-d-300-antibody.html;short description: Santa Cruz antibody agains Suppressor of Hairless;input DNA

data_processing: Solexa_Alignment protocol. Raw image files were processed following the following Illumina-compatible pipeline:1.  The Raw images were first processed with the Casava 1.7.0.2.  Base-called sequences with confidence metrics were obtained using OLB-1.9.0.  All multiplexed samples were demultiplexed using Casava 1.7.0.3. Finally the BWA program was used to align the sequence reads to the Drosophila melanogaster (v5.32) genome, allowing up to 2 edit distance in the seed region and 3 in the full read length. Tags that aligned to more than one  location were excluded from our analysis for both ChIP sample and the corresponding control sample (input chromatin). Only properly aligned reads with mapping quality more than 30 were kept. MACS_Peak-calling protocol. Peak calls were determined by using Model-based Analysis of ChIP-Seq (MACS) software, version 2 (MACS2). MACS2 was supplied with the following parameters:tagSize = 50mfold = 2genomeSize = 120000000bandwidth = 100pvalue = 1e-5  Processed data are obtained using following parameters: genome version is flybase BDGPv5

organism: Drosophila melanogaster

source_name: Su(H) Kc late embryo ChIP Rep.3

extract_protocol:
Cell lines (S2, Kc167...) are grown as described by the DGRC and the Cherbas Lab. After expanding the cell line, cells are transfered in a plate. Once confluent, 1.8% formaldehyde is added to the cell culture. Cells are harvested in presence of the formaldehyde with the help of a cell scrapper. After 15 minutes of incubation at room temperature, the cross-linking reaction is quenched with glycine for 5 minutes. Cells pellet are then washed 3 times with lysis buffer. Regular chromatin extraction before sonication is then performed.
Formaldehyde Cross-linking of Chromatin from any Drosophila tissue (Embryos, Larvae, Pupae, Adult flies).1. Homogenize the material (about 150-200 mg of embryos, pupae, adµlts, larvae, sufficient for four-five independent immuno-precipitations) in 5 ml of buffer A1 + 1.8% formaldehyde (290 µl of 37% solution) at room temperature using first a Potter homogenizer and then a Douncer with type A pestle (3 strokes). Wait 15? (total time starting from beginning of homogenization). Add glycin solution to 225 mM (540 µl of 2.5 M solution for 6 ml of cross-linked mixture), mix and incubate 5?. Put on ice. Keep on ice unless specified.2. Transfer the homogenate to a 15 ml tube. Centrifuge 5 min, at 4000 g at 4°C. Discard supernatant. Add 3 ml of buffer A1, resuspend pellet and spin down the same way. Repeat washing step 3 times.3. Wash once in 3 ml of lysis buffer without SDS. Spin down, 5 min at 4000g.4. Resuspend cross-linked material in 0.5 ml of lysis buffer and add SDS to 0.1% (2.5 µl) and N-lauroylsarcosine to 0.5% (12.5 µl). Incubate 10 min at 4°C in a rotating wheel.5. Sonicate in Polypropylene 15ml Falcon tubes according to .Diagenode instructions. The settings are: High power, 15 minutes, 30 seconds off, 30 seconds on. The water bath shoµld be kept cold either in a cold room or by a cold water pump.6. Rotate 10'. Transfer to eppendorf tubes and centrifuge 5', room temperature at maximum speed. Transfer supernatant to the new tube. Add another 0.5 ml of lysis buffer to the pellet, rotate for 10 min. Repeat centrifugation and combine supernatants. Centrifuge the combined supernatant 2x10 min at max speed. The supernatant from this stage will be referred as chromatin extract. Chromatin can be stored at ?80°C for several months at this stage. Add sodium azide to 0.02% for storage.7. [Optional : Purification of chromatin. Put chromatin extract in Centricon YM-100 column (4212), centrifuge 3x40' (or more) at 1000 g while adding lysis buffer. At least 3 vol. of lysis buffer should pass through column. Bring the final volume of chromatin extract to 1 ml by lysis buffer + protease inhibitors.][edit]
Solutions and MaterialsLysis buffer: (see above)5mg/ml RNaseA (DNase free)PAS suspension: 100 mg of CL-4B (Amersham, 17-0780-01) PAS shoµld be resuspended in 1 ml of lysis buffer + protease inhibitors + 0.1 mg/ml BSA for 50 %v suspension. Wash in lysis buffer 2-3 times, overall equilibration time 1h. Store up to one week at 4 C.TE: 10 mM Tris-HCl pH 8.0, 1 mM EDTAElution buffer1: 10 mM EDTA, 1% SDS, 50 mM Tris-Cl pH 8.Elution buffer2: TE+0.67% SDS.LiCL 4M? Protocol 1. To an amount of chromatin corresponding to 150 mg of biological material, suspended in a final volume of chromatin extract of 1 ml (in lysis buffer + protease inhibitors), add 100 µl of PAS suspension for preincubation. Incubate several hours or overnight at 4°C, then remove PAS. Crosslinked chromatin at this stage can be stored several days at 4°C or frozen at ?70°C.2. [Optional : Check for amount of DNA. From the 1 ml solution above, take a 100 µl aliquot, Add proteinase K up to 100 ug/ml and SDS to 1%, incubate 6h at 60°C, then 20 min at 70°C, add RNAse to 50 ug/ml and incubate additional 2h at 37°C. Phenol-chlorophorm extract, ethanol precipitate. Run on an agarose gel to check amount and size of DNA.]3. Separate chromatin sample into 4x250 µl aliquots (one aliquot is enough for one IP but the amount may be decreased further). Immunoprecipitate chromatin by adding the antibody (Ab) of interest (amount of Ab shoµld be determined empirically, usually the same concentration than for a successfµl IF experiement). Keep a control sample without Ab IP, named "Mock".4. Incubate 4 h at 4°C on a head to head rotating wheel, then add 50 µl of PAS suspension and incubate 4 h or overnight. Spin down PAS and proceed to washes.5. Wash PAS 4x with lysis buffer, followed by 2x with TE (without protease inhibitors). Each wash is for 5 min at 4°C, using 1 ml of solution.6. Elution of precipitated material. Spin down PAS. Add 100 µl of elution buffer, mix and incubate 10 min at 65°C. Spin down PAS and transfer supernatant to new tube. Add 150 µl of elution buffer 2 to PAS, mix, centrifuge at fµll speed and transfer eluate to a tube together with the eluate from the first centrifugation. The combined material is the "chromatin precipitate" (approx. 250 µl).7. Incubate precipitate 6 h (or overnight) at 65 C to reverse cross-links. Add 250 µl of Proteinase K solution, incubate at 50°C 2-3 h.8. Add 55 µl of 4M LiCl and 500 µl of phenol-chlorophorm. Mix and microfuge at fµll speed at RT. Transfer aqueous phase to a new tube and precipitate with 1 ml of 100% ethanol. Wash with 750 µl of 70% ethanol. Spin down and dry precipitate.9. Dissolve in 25 µl of water. This is the chromatin immunoprecipitate or ?Native ChIP? sample.
DNA fragments recovered following chromatin IP prepared for Illumina sequencing using the Epicentre Nextera DNA Sample Prep Kit (Cat. # GA0911).  After a final gel purification step the DNA is loaded into a flow cell for multiplexed sequencing.

value: embryo-derived cell-line

tag: developmental stage

value: late embryonic stage

tag: genotype

value: se/e

value: Female


title: NFE2_Ter119_Positive

description: Chip-seq

data_processing:
Paired-end reads from ChIP-Seq and input samples were aligned to the mouse genome build using Bowtie (version 0.12.3) using standard parameters
Paired-end reads from ChIP-Seq  were aligned to the mouse genome build using Bowtie (version 0.12.3) using standard parameters  Wig files of genomic read distribution were produced using a sliding 300bp window moving at 30 bp increments
Genome_build: MM9
Supplementary_files_format_and_content: Wig files of genomic distribution of aligned reads

extract_protocol:
ChIPs were performed according to the Millipore ChIP protocol, as described previously (De Gobbi et al., 2007). For ChIP-Seq experiments, Ter119+ cells were fixed with 1% formaldehyde for 10 minutes at RT and chromatin was sonicated to a size <500 bp. Immunoprecipitations were performed, after an overnight incubation with the appropriate antibody, with protein A agarose (Millipore). A sample containing no antibody was used as a negative control. Input and immunoprecipitated DNA were purified by phenol and chloroform extraction followed by ethanol precipitation. Subsequently the material was analysed by real time PCR (ABI Prism 7000 Sequence Detection System, Applied Biosystems) using a series of PCR amplicons and 5'FAM-3'TAMRA probes across the alpha-globin locus.
Libraries were constructed using the NEBNext DNA Sample Prep Kit (NEB) with the following modifications: End Repair was performed with 1µl of Enzyme mix and adapters were ligated using 4µl of Ligase. We used 10µl of a 1 in 100 dilution of Illumina Adapters (Multiplexing Sample Preparation Oliogonucleotide Kit) for 10ng of DNA and amplified with 25 µM each of the following custom primers: PCR primer 1.0 5'-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCT TCCGATCT-3' Index primer 5'-CAAGCAGAAGACGGCATACGAGAT[INDEX]CAGTGACTGGAGTTCA GACGTGTGCTCTTCCGATCT-3' *Indexes were according to the six bases tags developed by Illumina. The amplified libraries were purified using Ampure beads and size selected using 2% E-Gel® EX (Invitrogen), the distribution of fragments in the purified fraction was determined using a High Sensitivity Bioanalyzer kit and a 2100 Bioanalyzer Instrument system (Agilent). For equimolar pooling of the libraries, the relative concentration of the libraries was determined by realtime using Agilent QPCR Library Quantification Kit and a MX3005P instrument (Agilent). Finally, in order to determine the volume to use for sequencing, a second realtime was performed to measure the relative concentration of the pool compared to a previously sequenced ChIP library. Sequencing was performed as 50bp paired end on a GAIIx according to Illumina specifications using Cluster Generation Kit v4 and Sequencing Kit v4 in combination with illumina SCS 2.6/ RTA 1.6 software.

tag: antibody vendor/catalog

value: SC-477 from Santa Crus

tag: antibody


title: CTCF_Ter119_Positive

description: Chip-seq

value: 07-729 from Millipore

value: CTCF


title: Bisulfite-seq_WT-2

tag: Sex


title: Su(H) Kc167 late embryonic D.melanogaster Input

description: input DNA

source_name: Su(H) Kc late embryo Input

tag: cell line


title: Ter119_1_Capture-C


title: RNA-seq of E14-TG2a.IV ES cells

data_processing:
Bcl conversion to Qseq files using OLB v1.8.0 Gerald- using Eland alignment to produce sequence and export files (carried out by CASAVA 1.7.0) Reads were mapped to the mm9 mouse genome build using TopHat 1.1.1b
Genome_build: mm9
Supplementary_files_format_and_content: bedGraph file of read densities from tophat coverage output.

growth_protocol: E14-TG2a.IV ES cells were grown as previously described (Nichols et al., 1990).

extract_protocol:
RNA was isolated with Tri-Reagent (Sigma). The RNA quality was assessed using Agilent Bioanalyser. Only RNA with overall RIN score >9 was used for further procedures. Subsequently, RNA was treated with DNAseI (DNA-free, Ambion) according to the manufacturer's protocol kit according to the protocol. Good quality and DNA-free RNA was subjected to polyA fractionation using PolyA selection kit (Promega) using the standard kit protocol.  The RNA fraction was precipitated overnight and resuspended with water. The quality of obtained RNA samples was assessed using PicoChip (Agilent). A single end sequencing library was generated using the standard Illumina prtocol.
Samples were quantified using Nanodrop (Invitrogen) and quality controlled on a 6000 Nano Bioanalyzer chip (Agilent). mRNA enrichment was achieved by processing 6µg of total RNA using the Magnetic mRNA Isolation Kit from NEB (S1550S) with minor modifications.  Generation of double stranded cDNA and library construction were performed using NEBNext® mRNA Sample Prep Reagent Set 1 (E6100L) according to manufacturer specifications.  Upon ligation of Illumina Adapters (Multiplexing Sample Preparation Oliogonucleotide Kit) each library was size selected using a 2% E-Gel® EX (Invitrogen).  The following custom primers (25 µM each) were used for the PCR enrichment  step:  Multiplex PCR primer 1.0  5'-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTC CGATCT-3'  Index primer  5'-CAAGCAGAAGACGGCATACGAGAT[INDEX]CAGTGACTGGAGTTCAGAC GTGTGCTCTTCCGATCT-3'  Indices were according to the eight bases tags developed by WTCHG (manuscript in preparation).  The amplified library was purified using Ampure beads (Beckman/Agencourt) and size distribution determined using Agilent DNA 1000 Kit and a 2100 Bioanalyzer Instrument system (Agilent). Libraries were quantified by realtime using Agilent QPCR Library Quantification Kit and a MX3005P instrument (Agilent) and pooled accordingly. Finally, a second realtime was performed to measure the relative concentration of the pool compared to a previously sequenced mRNA library in order to determine the volume to use for sequencing. . Sequencing was performed as 50bp single read on a GAIIx according to Illumina specifications using Cluster Generation Kit v4 and Sequencing Kit v4.


title: Su(H) Kc167 late embryonic D.melanogaster ChIP Rep2

source_name: Su(H) Kc late embryo ChIP Rep.2

tag: tissue


title: Su(H) Kc167 late embryonic D.melanogaster ChIP Rep1

source_name: Su(H) Kc late embryo ChIP Rep.1


title: dMyc S2-DRSC late embryonic D.melanogaster ChIP Rep2

description: ChIP DNA; Antibody information listed below: official name: MycN;target name: fly genes:dm;host: Rabbit;antigen: raised against amino acids 46-507 of Drosophila Myc;clonal: Polyclonal;purified: Protein A/G;company: Santa Cruz Biotechnology Inc.;catalog: sc-28208;reference: http://www.scbt.com/datasheet-28208-mycn-d46-507-antibody.html;input DNA

source_name: dMyc S2 late embryo ChIP Rep.2


title: dMyc S2-DRSC late embryonic D.melanogaster Input

description: input DNA

source_name: dMyc S2 late embryo Input


title: BMDC 6hr LPS PU.1

source_name: BMDCs, LPS, 6 hrs, PU.1 ChIP

value: bone marrow-derived dendritic cells

value: LPS

value: 6 hrs

tag: chip antibody

value: anti-PU.1 (Santa Cruz Biotechnology, sc-352x (T-21x), Lot# J2411)


title: dMyc S2-DRSC late embryonic D.melanogaster ChIP Rep1

source_name: dMyc S2 late embryo ChIP Rep.1


title: Hr78-GFP L3 D.melanogaster ChIP Rep2

description: ChIP DNA; Antibody information listed below: official name: GFP ab290;target name: Green Fluorescent Protein;host: Rabbit;antigen: entire GFP sequence;clonal: Polyclonal;purified: Crude;company: Abcam;catalog: ab290;reference: abcam website Jungreis et al. Genome Res. 2011 Dec;21(12):2096-113.;short description: This antibody is reactive against all variants of Aequorea victoria GFP such as S65T-GFP RS-GFP YFP and EGFP.;input DNA

source_name: Hr78-GFP L3 ChIP Rep.2

extract_protocol:
1. y; bw cn sp flies are cultivated in standard fly food covered with yeast powder.2. Flies are removed from the bottles after 24 hours and eggs are allowed to grow at 25 C for several days until they reach the desired growth stage.3. Wandering third instal larvae are collected from the side of the bottles using a brush. They are rinsed with EWB and cross-linked in 1.8% formaldehyde at room temperature for 15 min.4. Chromatin collection is performed as described in The ChIP-chip protocol from the White lab.
Solutions and MaterialsLysis buffer: (see above)5mg/ml RNaseA (DNase free)PAS suspension: 100 mg of CL-4B (Amersham, 17-0780-01) PAS shoµld be resuspended in 1 ml of lysis buffer + protease inhibitors + 0.1 mg/ml BSA for 50 %v suspension. Wash in lysis buffer 2-3 times, overall equilibration time 1h. Store up to one week at 4 C.TE: 10 mM Tris-HCl pH 8.0, 1 mM EDTAElution buffer1: 10 mM EDTA, 1% SDS, 50 mM Tris-Cl pH 8.Elution buffer2: TE+0.67% SDS.LiCL 4M? Protocol 1. To an amount of chromatin corresponding to 150 mg of biological material, suspended in a final volume of chromatin extract of 1 ml (in lysis buffer + protease inhibitors), add 100 µl of PAS suspension for preincubation. Incubate several hours or overnight at 4°C, then remove PAS. Crosslinked chromatin at this stage can be stored several days at 4°C or frozen at ?70°C.2. [Optional : Check for amount of DNA. From the 1 ml solution above, take a 100 µl aliquot, Add proteinase K up to 100 ug/ml and SDS to 1%, incubate 6h at 60°C, then 20 min at 70°C, add RNAse to 50 ug/ml and incubate additional 2h at 37°C. Phenol-chlorophorm extract, ethanol precipitate. Run on an agarose gel to check amount and size of DNA.]3. Separate chromatin sample into 4x250 µl aliquots (one aliquot is enough for one IP but the amount may be decreased further). Immunoprecipitate chromatin by adding the antibody (Ab) of interest (amount of Ab shoµld be determined empirically, usually the same concentration than for a successfµl IF experiement). Keep a control sample without Ab IP, named "Mock".4. Incubate 4 h at 4°C on a head to head rotating wheel, then add 50 µl of PAS suspension and incubate 4 h or overnight. Spin down PAS and proceed to washes.5. Wash PAS 4x with lysis buffer, followed by 2x with TE (without protease inhibitors). Each wash is for 5 min at 4°C, using 1 ml of solution.6. Elution of precipitated material. Spin down PAS. Add 100 µl of elution buffer, mix and incubate 10 min at 65°C. Spin down PAS and transfer supernatant to new tube. Add 150 µl of elution buffer 2 to PAS, mix, centrifuge at fµll speed and transfer eluate to a tube together with the eluate from the first centrifugation. The combined material is the "chromatin precipitate" (approx. 250 µl).7. Incubate precipitate 6 h (or overnight) at 65 C to reverse cross-links. Add 250 µl of Proteinase K solution, incubate at 50°C 2-3 h.8. Add 55 µl of 4M LiCl and 500 µl of phenol-chlorophorm. Mix and microfuge at fµll speed at RT. Transfer aqueous phase to a new tube and precipitate with 1 ml of 100% ethanol. Wash with 750 µl of 70% ethanol. Spin down and dry precipitate.9. Dissolve in 25 µl of water. This is the chromatin immunoprecipitate or ?Native ChIP? sample.
DNA fragments recovered following chromatin IP prepared for Illumina sequencing using the Epicentre Nextera DNA Sample Prep Kit (Cat. # GA0911).  After a final gel purification step the DNA is loaded into a flow cell for multiplexed sequencing.

value: Unknown

tag: transgene

value: Hr78 genomic coding region


title: Hr78-GFP L3 D.melanogaster ChIP Rep1

source_name: Hr78-GFP L3 ChIP Rep.1


title: dMyc Kc167 late embryonic D.melanogaster Input

description: input DNA

source_name: dMyc Kc167 late embryo Input


title: Hr78-GFP L3 D.melanogaster Input

description: input DNA

source_name: Hr78-GFP L3 Input


title: H Kc167 late embryonic D.melanogaster ChIP Rep3

description: ChIP DNA; Antibody information listed below: official name: KW3-h-D1;target name: Hairy;host: Rabbit;antigen: PMLVSMPQRTASTGSASSHSSAGYESAPGSSSSCSYAPPSPANSSYEPMDIKPSVIQRVPMEQQPLSLVIKKQIKEEEQPWRP;clonal: Polyclonal;purified: Protein A/G;company: Other;catalog: Aviva-KW3-h-D1;short description: Antisera raised against a peptide from the Hairy protein. Produced commercially for the modENCODE Project by K. White's group.;input DNA

source_name: H Kc late embryo ChIP Rep.3


title: H Kc167 late embryonic D.melanogaster ChIP Rep2

source_name: H Kc late embryo ChIP Rep.2


title: H Kc167 late embryonic D.melanogaster ChIP Rep1

source_name: H Kc late embryo ChIP Rep.1


title: HP1 8-16h embryonic D.melanogaster Input

description: input DNA

source_name: HP1 8-16h embryo Input

extract_protocol:
1. y; bw cn sp flies are cultivated in cages with apple juice agar plates covered with yeast powder.   2. For a 8-16 hours egg laying, new plates are added after a 2 hours pre-egglaying at 12 pm and flies are allowed to lay eggs for 8 hours.   3. Embryos are collected at 7.45 am using a filter mesh and a brush. They are rinsed with EWB and cross-linked at 8am in 1.8% formaldehyde at room temperature for 15 min.
Solutions and MaterialsLysis buffer: (see above)5mg/ml RNaseA (DNase free)PAS suspension: 100 mg of CL-4B (Amersham, 17-0780-01) PAS shoµld be resuspended in 1 ml of lysis buffer + protease inhibitors + 0.1 mg/ml BSA for 50 %v suspension. Wash in lysis buffer 2-3 times, overall equilibration time 1h. Store up to one week at 4 C.TE: 10 mM Tris-HCl pH 8.0, 1 mM EDTAElution buffer1: 10 mM EDTA, 1% SDS, 50 mM Tris-Cl pH 8.Elution buffer2: TE+0.67% SDS.LiCL 4M? Protocol 1. To an amount of chromatin corresponding to 150 mg of biological material, suspended in a final volume of chromatin extract of 1 ml (in lysis buffer + protease inhibitors), add 100 µl of PAS suspension for preincubation. Incubate several hours or overnight at 4°C, then remove PAS. Crosslinked chromatin at this stage can be stored several days at 4°C or frozen at ?70°C.2. [Optional : Check for amount of DNA. From the 1 ml solution above, take a 100 µl aliquot, Add proteinase K up to 100 ug/ml and SDS to 1%, incubate 6h at 60°C, then 20 min at 70°C, add RNAse to 50 ug/ml and incubate additional 2h at 37°C. Phenol-chlorophorm extract, ethanol precipitate. Run on an agarose gel to check amount and size of DNA.]3. Separate chromatin sample into 4x250 µl aliquots (one aliquot is enough for one IP but the amount may be decreased further). Immunoprecipitate chromatin by adding the antibody (Ab) of interest (amount of Ab shoµld be determined empirically, usually the same concentration than for a successfµl IF experiement). Keep a control sample without Ab IP, named "Mock".4. Incubate 4 h at 4°C on a head to head rotating wheel, then add 50 µl of PAS suspension and incubate 4 h or overnight. Spin down PAS and proceed to washes.5. Wash PAS 4x with lysis buffer, followed by 2x with TE (without protease inhibitors). Each wash is for 5 min at 4°C, using 1 ml of solution.6. Elution of precipitated material. Spin down PAS. Add 100 µl of elution buffer, mix and incubate 10 min at 65°C. Spin down PAS and transfer supernatant to new tube. Add 150 µl of elution buffer 2 to PAS, mix, centrifuge at fµll speed and transfer eluate to a tube together with the eluate from the first centrifugation. The combined material is the "chromatin precipitate" (approx. 250 µl).7. Incubate precipitate 6 h (or overnight) at 65 C to reverse cross-links. Add 250 µl of Proteinase K solution, incubate at 50°C 2-3 h.8. Add 55 µl of 4M LiCl and 500 µl of phenol-chlorophorm. Mix and microfuge at fµll speed at RT. Transfer aqueous phase to a new tube and precipitate with 1 ml of 100% ethanol. Wash with 750 µl of 70% ethanol. Spin down and dry precipitate.9. Dissolve in 25 µl of water. This is the chromatin immunoprecipitate or ?Native ChIP? sample.
DNA fragments recovered following chromatin IP prepared for Illumina sequencing using the Epicentre Nextera DNA Sample Prep Kit (Cat. # GA0911).  After a final gel purification step the DNA is loaded into a flow cell for multiplexed sequencing.

value: Y cn bw sp

value: Embryo 8-16h


title: H Kc167 late embryonic D.melanogaster Input

description: input DNA

source_name: H Kc late embryo Input


title: NPCRingIB_ChIPSeq

city: Barcelona

first: Luciano

last: Di Croce

address: Dr. Aiguader, 88

country: Spain

institute: Centre for Genomic Regulation

description: Chromatin IP against Ring1B

data_processing:
Basecalls performed using Illumina Pipeline
ChIP-seq reads were aligned to the mm9 genome assembly using bowtie version 0.12.7 with the --best option and allowing 2 mismatches in the seed
peaks were called by using MACS version 1.4.2
Genome_build: mm9
Supplementary_files_format_and_content: text files containing peak coordinates

source_name: Neural Progenitor cell (NPC)

growth_protocol: NPCs were derived by ESC (E14TG2a strain) according to this protocols (Colombo et al, Stem Cells, 2006). ESC-derived NPC were grown in N2 medium supplemented with EGF and FGF2

extract_protocol:
NPC were crosslinked in 1% formaldehyde for 10 min at room temperature. Crosslinking was quenched with 0.125 M glycine for 5 min. Pelleted cells were lysed in 1 ml ChIP buffer and sonicated for 12 min in a Bioruptor. ChIP-Seq experiments were performed as previously described (Morey et al, Cell Stem Cell. 2012, 10: 47-62)
It was performed using the NEBNext DNA Sample Prep Reagents Set 1

value: Ring1B Ab generated in Di Croce´s lab

value: Neural Progenitor cell (NPC)


title: HP1 8-16h embryonic D.melanogaster ChIP Rep1

description: ChIP DNA; Antibody information listed below: official name: HP1 antibody (ab24726);target name: HP1;host: Rabbit;antigen: Recombinant full length protein (Fruit fly (Drosophila melanogaster)).;clonal: Polyclonal;purified: Affinity;company: Abcam;catalog: ab24726;short description: Rabbit polyclonal to HP1;input DNA

source_name: HP1 8-16h embryo ChIP Rep.1


title: GATA1_Ter119_Positive

description: Chip-seq

source_name: Ter119+ Cells

value: ab11852 from Abcam


title: NPCZRF1_ChipSeq

description: Chromatin IP against ZRF1

value: Zrf1 Ab generated in Di Croce´s lab


title: Syn02182011 R1.1

status: Public on Dec 20 2013

city: Richland

first: Lye Meng

last: Markillie

state: WA

address: 902 Battelle Blvd

department: Biosystems Dynamics & Simulation

institute: Environmental Molecular Sciences Laboratory

data_processing:
The 50-base short read sequences produced by the 5500XL SOLiD  sequencer were mapped in color space using SOLiD LifeScope software version 2.5 using the default parameters
Genome_build: Shewanella sp. W3-18-1: ASM1518v1; NC_008750
Genome_build: Synechococcus 7002: ASM1948v1;  NC_010474, NC_010475, NC_010476, NC_010477, NC_010478, NC_010479 and NC_010480
Supplementary_files_format_and_content: tab-delimited text files include RPKM values for each Sample

organism: Picosynechococcus sp. PCC 7002

growth_protocol: Shewanella W3-18-1 cultures were maintained at 30oC in tryptic soy broth or M1 defined medium amended with 20 mM D,L-Na lactate and 10 ml each of 10X Wolfe's vitamin and 10X mineral solutions (Kieft et al 1999). In chemostat cultures, Shewanella W3-18 was grown using 5 mM D,L-Na lactate as the sole source of C . Synechococcus 7002 was routinely grown in modified A+ media (Stevens and Porter 1980) at room temperature in 1-L Roux bottles sparged with 2% CO2-enriched air under continuous 50 µmolom-2os-1 illumination. Chemostat cultivation of Synechococcus 7002 was carried out using 8 mM NaHCO3 as the sole source of C in a white-light photobioreactor

extract_protocol:
RNA was extracted using Trizol following by DNAseI treatment using Qiagen RNAseay mini column
Template cDNA was prepared using the Applied Biosystems SOLiD® Total RNA-Seq Kit (Life Technologies, Carlsbad, CA) according to manufacturer's protocol. Sequencing was carried using the SOLiDTM 5500XL protocol (Life Technologies).

treatment_protocol: The co-cultivation experiments were carried out in a modified A+ medium which was contained 17 mM NH4Cl and 10 µm FeCl3·6H2O. D,L-Na lactate or NaHCO3 were used as the sole source of carbon at 5 mM and 8 mM concentrations

tag: lactate and nahco3 as carbon source

value: Syn 7002 OG1+LK1

tag: condition

value: Low light / low O2


title: Syn02182011 R1.3 [AB3_4]

value: High light / low O2


title: dMyc Kc167 late embryonic D.melanogaster ChIP Rep1

source_name: dMyc Kc167 late embryo ChIP Rep.1

value: embryo-derived cell-line

value: late embryonic stage

value: se/e

value: Female


title: RBM10 CLIP-Seq Replicate 2

city: Budapest

first: Endre

last: Sebestyén

address: Üllői út 26.

department: 1st Department of Pathology and Experimental Cancer Research

country: Hungary

institute: Semmelweis University

data_processing:
Basecalling was performed using Illumina pipeline versions 1.6.0 within within SCS 2.6 for the RBM6 and RBM10 data, Illumina pipeline versions 1.7.0 within within SCS 2.8 for the RBM5 data.
Reads were mapped to the human genome using GEM (Marco-Sola et al. 2013), allowing for up to 2 mismatches and retaining only sequences that mapped uniquely to the genome.
All the unmapped reads were then trimmed 1 base from the 3’ end and remapped. This sequential trimming and mapping was continued up to 21nt length reads (15 bases of trimming).
Additional filtered datasets and analysis description can be found here: http://regulatorygenomics.upf.edu/Data/RBMs/
Genome_build: hg18
Supplementary_files_format_and_content: BED files containing all mapped reads.

source_name: HeLa cells

growth_protocol: HeLa cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and penicillin/streptomycin.

extract_protocol:
HeLa cells were grown in 150mm plates, rinsed once with ice-cold PBS and placed in a Stratalinker without the cover plate and irradiated once with an energy of 400mJ/cm. Cells were then scraped off the palte and centrifuged at 2500rpm for 5 min at 4°C and the pellet re-suspended in 3x the cells volume of PBS and distributed in two separate eppendorf tubes. Each tube of crosslinked lysate was resuspended in 700 μl of 1X PXL (PBS (tissue culture grade without Mg++ or Ca++) 0.1% SDS 0.5% deoxycholate 0.5% NP-40, supplemented with protease inhibitors and with 15 μl RNAsin (Promega)) and kept on ice for 10 min. 30 μl of RQ1 DNAse (Promega, M6101) were added to each tube and extracts were incubated at 37° for 5 min, centrifuged at 1000 rpm, followed by an incubation at 37° for 5 minutes with RNAse I. Extracts were the centrifuged at 12000 rpm for 10’ and then clarified on protein A Dynabeads  for 2 hours at 4°C,  before immunoprecipitation using protein A pre-adsorbed with either rabbit IgGs or EWS antibody (5μg) at 4 °C under constant rotation. After incubation for 3 hours, stringent washes were performed with ice-cold buffers as follows: 2 washes with 1X PXL, 2 washes with 5X PXL (High-salt Wash Buffer) and 2 washes with PNK Buffer (50 mM Tris-Cl pH 7.4 10 mM MgCl2 0.5% NP-40). Alkaline phosphatase (Roche) treatment (3μl) was performed at 37°C for 20’ in Thermomixer, followed by 4 washes in PNK buffer (one wash including 20mM EGTA). 3’ RNA linker ligation was carried out overnight at 16°C on beads using T4 RNA ligase (Fermentas) and radioactively-labeled linkers. Beads were extensively washed (1x PXL buffer, 1x 5XPXL buffer, 3x PNK buffer), before and after PNK treatment (T4, NEB, [40 units]) for 20’ at 37°C.  Finally, beads were resuspended in 30ml of PNK buffer, supplemeted with Novex loading buffer and loaded on a Novex NuPAGE 10% Bis-Tris gel. The gel was run at 175V in the cold room and subsequently transfered to nitrocellulose using the Novex wet transfer apparatus, for 1 h at 30V in NuPAGE Transfer Buffer with 10% methanol. After transfer, the nitrocellulose filter was rinsed in 1X PBS, and exposed to autoradiogram. The radioactively labeled RNA-protein complexes were excised and proteinase K treatment was performed (20’ at 37°C) before RNA extraction with Phenol-Chloroform and preciiptation overnight with 3M NaOAc pH 5.2, glycogen (Ambion, 9510) and 1:1 EtOH:isopropanol.
The RNA was resuspended in RNase free water and 5’ end linker ligation (containing also barcode sequences) was carried out with T4 RNA ligase at 16°C for 5 hours. RNAs were reverse-transcribed, PCR amplified with barcoded primers and sequenced using Solexa (Illumina) technology.

value: HeLa

value: Sigma Aldricht RBM10 (HPA034972) antibody


title: SYN03282011R1.0

value: Synechococcus 7002 OG1+LK1

value: low light / high O2


title: dMyc Kc167 late embryonic D.melanogaster ChIP Rep2

source_name: dMyc Kc167 late embryo ChIP Rep.2


title: NPCIgG_ChIPSeq

description: Chromatin IP against IgG

value: Rabbit IgG Abcam, ab37415


description: These are co-culture experiments, the raw data are shared with SS020812 R1.0 co-culture [Synechococcus] and SS020812 R1.0 co-culture [Synechococcus]

source_name: SS020812 R1.0 co-culture

value: Synechococcus 7002 (OG1+LK1)

value: Shew W3-18 co-culture 5mM lactate


title: RBM10 CLIP-Seq Replicate 1


title: RBM10 Control IgG CLIP-Seq

value: Non-specific rabbit IgG antibody


title: RBM6 CLIP-Seq Replicate 1

value: Sigma Aldricht RBM6 (HPA026272) antibody


title: RBM5 CLIP-Seq Replicate 1

value: In-house generateted rabbit polyclonal RBM5 antibody


title: RBM5 CLIP-Seq Replicate 2


title: ChIP-seq_P13-WTMECs-H3K4me3

city: Cheonan

first: Keunsoo

last: Kang

state: Chungnam

address: Dandae-ro 119

department: Microbiology

country: South Korea

institute: Dankook University

data_processing:
Basecalls performed using the CASAVA software.
Sequenced reads were aligned to the mm9 genome assembly using the Bowtie program (Langmead et al., Genome Biol., 2009). Total mapped reads per sample were normalized to 10 million and bedGraph files were generated by using the HOMER program (Heinz et al., Mol Cell, 2010).
Genome_build: MGSCv37
Supplementary_files_format_and_content: BedGraph files.

source_name: MECs (mammary epithelial cells), p13, H3K4me3 ChIP

growth_protocol: Animals were handled and housed in accordance with the guidelines of NIH and all experiments were approved by the Animal Care and Use Committee of NIDDK.

extract_protocol: The ChIP DNA fragments were blunt-ended and ligated to the Illumina Indexed DNA adaptors and sequenced using the Illumina HiSeq 2000.

treatment_protocol: Frozen-stored mammary tissues from p13 and L1 were broken into powder with mortar and pestle, and MECs were isolated from mammary tissues of the day 13 of pregnancy. Then, cells were cross-linked with 1% formaldehyde for 10 min at RT. Nuclei were extracted in Farnham lysis buffer (5mM PIPES PH8.0, 85mM KCl, 0.5% NP-40 and protease inhibitor). Chromatin was fragmented to 200 to 300 bp by sonication using MSONIX Sonicator 3000 (QSonica, Newtown, CT). Antibodies against STAT5 (Santa Cruz Biotechnology, 10ug) and H3K4me3 (Millipore, 5ug) were used for ChIP.

tag: genotype/variation

value: wild type

value: MECs (mammary epithelial cells)

value: day 13 of pregnancy


title: ChIP-seq_P13-WT-STAT5A

source_name: Mammary gland tissues, p13, STAT5A ChIP

tag: strain/background

value: mammary gland

value: STAT5A (Santa Cruz, sc-1081, lot# J3007)


title: ChIP-seq_P13-WT-Input

source_name: Mammary gland tissues, p13, input


description: These are co-culture experiments, the raw data are shared with SS020812 R1.2 co-culture [Shewanella] and SS020812 R1.2 co-culture [Synechococcus]

source_name: SS020812 R1.2 co-culture

value: Synechococcus 7002 (OG1+LK1G)

value: Shew W3-18 co-culture 8mM NaHCO3, C-limited


title: RBM6 CLIP-Seq Replicate 2

value: HeLa


title: RBM6 Control IgG CLIP-Seq


title: ChIP-seq_L1-WT-STAT5A-rep2

source_name: Mammary gland tissues, L1, STAT5A ChIP

value: at parturition


title: ChIP-seq_L1-WT-Input-rep2

source_name: Mammary gland tissues, L1, input


title: RNA-seq_WT_L1_oneLane

data_processing:
Basecalls performed using the CASAVA software.
Sequenced reads were aligned to the mm9 genome assembly using the TopHat program (Trapnell et al., Bioinformatics, 2009).
Genome_build: MGSCv37
Supplementary_files_format_and_content: Tab-delimited text file with FPKM values.

source_name: Mammary gland tissues, L1

extract_protocol:
Total RNA was extracted using TRIzol reagent, followed by a cleanup step using the RNeasy Plus Mini Kit (QIAGEN). Poly(A) RNA was purified twice from 1 ug total RNA and cDNA was synthesized using SuperScript II (Invitrogen).
Libraries were prepared using TruSeq RNA Sample Preparation Kit (Illumina Inc., San Diego, CA), and sequenced using the illlumina HiSeq 2000.

treatment_protocol: Mammary tissues without lymph nodes were collected, frozen in liquid nitrogen, and stored at -80 °C.


title: ChIP-seq_L1-WT-H3K4me3-rep2

source_name: Mammary gland tissues, L1, H3K4me3 ChIP


title: ESCell_94_TACAACG_amplified

city: Stockholm

first: Sten

last: Linnarsson

address: Scheeles väg 2

department: MBB

country: Sweden

institute: Karolinska Institutet

description: PolyA RNA

data_processing:
Read quality filtering and barcode extraction: Every read that was considered valid by the Illumina HiSeq control software was processed and filtered as follows: a) any 3' bases with a quality score of 'B' were removed; b) the well-identifying barcode was extracted from the 5' end of the read; c) if the read ended in a poly(A)-sequence leaving less than 25 transcript-derived bases, the read was discarded; d) the UMI was extracted and if any of the UMI bases had a Phred score less than 17 the read was discarded; e) a maximum of nine template-switching generated Gs were removed from the 5' end of the transcript-derived sequence; f) if the remaining sequence consisted of less than six non-A bases, or a dinucleotide repeat with less than six other bases at either end, the read was discarded.
Alignment: The reads were aligned to the UCSC  mm10 genome using the Bowtie aligner, allowing for up to three mismatches and up to 24 alternative mappings for each read. The genome included an artifical chromosome, containing a concatamer of the ERCC control sequences. Any reads with no alignments were re-aligned against another artifical chromosome, containing all possible splice junctions arising from the exons defined by the known transcript variants. Reads mapping within these splice junctions were translated back to the corresponding actual genomic positions. The UCSC transcript models22 were used for the expression level calculation. If a locus had several transcript variants, the exons of these were merged to a combined model that represented all expression from the locus. To account for incompete cap site knowledge, the 5' ends of all models were extended by 100 bases, but not beyond the 3' end of any upstream nearby exon of another gene of the same orientation.
Annotation and quantitation: The annotation step was performed barcode-by-barcode. For every unique mapping (genomic position and strand), the number of reads in each UMI was counted. Any multiread that had one or more repeat mappings that was outside exons, was assigned randomly as one of these repeats and contributed to the summarized read count of that repeat class. Else, if it had one or more mappings to exons, it was assigned at the exon where it was closest to the transcript model 5' end, even if the sequence was repeat-like. If it had no exon mapping, it was assigned randomly at one of the mappings. After assigning reads, the number of molecules at each mapping position was determined by the number of distinct UMIs observed. To account for UMIs that stem from PCR-induced mutations or sequencing errors, any UMI that had fewer reads than 1/100 of the average of the non-zero UMIs was excluded. The raw UMI count was corrected for the UMI collision probability (important only at high UMI counts) as described. The expression level of each transcript model was taken as the total number of molecules at all its possible mapping positions.
Genome_build: GRCm38
Supplementary_files_format_and_content: Tab-delimited text file containing counts of detected cDNA molecules for each transcript model in each of the 96 cells, using data from all three sequencing lanes.

source_name: R1 mouse embryonic stem cells, amplified

growth_protocol: R1 mouse embryonic stem cells were grown in ES1 medium on irradiated MEF feeder cells and harvested at confluency. After trypzinisation, the feeder cells were given time to settle in order to obtain a pure R1 ESC suspension. To reduce contamination with dead and dying cells, dead cells were subsequently stained with Red Fixable Dead Cell Stain (Life Technologies) and dead and early-apoptotic cells were depleted with AnnexinV-conjugated microbeads (Miltenyi Biotec). The cells were resuspended at a concentration of 400 cells/μl in ES1 medium with 10% DMSO, aliquoted and frozen at -80°C.

extract_protocol:
A 30 μL aliquot of ~12000 cells was thawed and 20 μL C1 Suspension Reagent was added (all "C1" reagents were from Fluidigm, Inc.). 5 μL of this mix was loaded according to the manufacturer's protocol on a C1 Single-Cell AutoPrep IFC microfludic chip designed for 10-17 μm cells, and the chip was then processed on a Fluidigm C1 instrument using the "mRNA Seq: Cell Load (1772x/1773x)" script. This captured one cell in each of up to 96 capture chambers, and took approximately 30 minutes. The plate was then transferred to an automated microscope (Nikon TE2000E) and an image was acquired from each site using μManager (http://micro-manager.org), which took less than 15 minutes.
20 μL lysis buffer (0.15% Triton X-100, 1 U/μL TaKaRa RNase inhibitor, 4 μM reverse transcription primer C1-P1-T31, 5% C1 Loading Reagent and 1:50,000 Life Technologies ERCC Spike-In Mix 1), reverse transcription mix (1x SuperScript II First-Strand Buffer supplemented with 3 mM MgCl2, 1.5 mM dNTP, 4 mM DTT, 3.3% C1 Loading Reagent, 1.8 μM template-switching oligo C1-P1-RNA-TSO, 1.5 U/μL TaKaRa RNase inhibitor and 18 U/μL Life Technologies Superscript II reverse transcriptase) and PCR mix (1.1x Clontech Advantage2 PCR buffer, 440 μM dNTP, 530 nM PCR primer C1-P1-PCR-2, 5% C1 Loading Reagent and 2x Advantage2 Polymerase Mix) were added to the designated wells according to the manufacturer's instructions, but using the indicated mixes in place of the corresponding commmercial reagents. The plate was then returned to the Fluidigm C1 and the "mRNA Seq: RT + Amp (1772x/1773x)" script was executed, which took about 8.5 hours and included lysis, reverse transcription and 21 cycles of PCR. When the run finished, the amplified cDNA was harvested in a total of 13 μL C1 Harvesting Reagent and quantified on an Agilent BioAnalyzer. The typical yield was 1 ng/μL. Amplified cDNA was simultaneously fragmented and barcoded by 'tagmentation', i.e. using Tn5 DNA transposase to transfer adapters to the target DNA. 96 different 10x transposome stocks (6.25 μM barcoded adapter C1-TN5-x, 40% glycerol, 6.25 μM Tn5 transposase, where x denotes a well-specific barcode) were prepared, each with a different barcode sequence. 6 μL harvested cDNA was mixed with 5 μL tagmentation buffer (50 mM TAPS-NaOH pH 8.5, 25 mM MgCl2 and 50% DMF), 11.5 μL nuclease-free water and 2.5 μL 10x transposome stock. The mix was incubated for 5 minutes at 55°C then cooled on ice. 100 μL Dynabeads MyOne Streptavidin C1 beads were washed in 2xBWT (10 mM Tris HCl pH 7.5, 1 mM EDTA, 2 M NaCl, 0.02% Tween-20) then resuspended in 2 mL 2xBWT. 20 μL beads was added to each well and incubated at room temperature for 5 minutes. All fractions were pooled, the beads were immobilized and the supernatant removed (thus removing all internal fragments, and retaining only the 5'- and 3'-most fragments). The beads were then resuspended in 100 μL TNT (20 mM Tris pH 7.5, 50 mM NaCl, 0.02% Tween), washed in 100 μL Qiagen Qiaquick PB, then twice in 100 μL TNT. The beads were then resuspended in 100 μL restriction mix (1x NEB NEBuffer 4, 0.4 U/μL PvuI-HF enzyme), designed to cleave 3'  fragments carrying the PvuI recognition site. The mix was incubated for one hour at 37°C, then washed three times in TNT. Finally, the single-stranded library was eluted by resuspending the beads in 100 μL 100 mM NaOH, incubating for 5 minutes, removing the beads, then adding 100 μL 100 mM HCl and 50 μL Neutralization buffer (200 mM Tris pH 7.5, 0.05% Tween-20). At this point, the typical yield was 1-5 nM single-stranded library. Aliquots from these were run on the HiSeq 2000. Another aliquot was further amplified by 9 cycles of PCR to produce a double-stranded library as described in Islam, S. et al. Nat. Protoc. 7, 813-828 (2012). These were also run on the HiSeq 2000.

tag: cell type

value: mouse embryonic stem cells

tag: barcode

value: TACAACG


title: ESCell_95_TTCTCTG_amplified

description: PolyA RNA

value: TTCTCTG


title: ESCell_93_CACCTTT_amplified

description: PolyA RNA

value: CACCTTT


title: ESCell_91_TTAGCGG_amplified

description: PolyA RNA

value: TTAGCGG


title: ESCell_92_GGACCTT_amplified

description: PolyA RNA

value: GGACCTT


title: ESCell_86_GTTGTCC_amplified

description: PolyA RNA

value: GTTGTCC


title: ESCell_90_GATAAGT_amplified

description: PolyA RNA

value: GATAAGT


title: ESCell_88_GCTTAAC_amplified

description: PolyA RNA

value: GCTTAAC


title: ESCell_87_GGGAATA_amplified

description: PolyA RNA

value: GGGAATA


title: ESCell_85_CAGCAAG_amplified

description: PolyA RNA

value: CAGCAAG


title: SYN03282011R1.3 [AB3_1]

value: Synechococcus 7002 OG1+LK1

value: high light / high O2

tag: strain


title: ESCell_84_CGACTAT_amplified

description: PolyA RNA

value: CGACTAT


title: ESCell_82_AGCAAGC_amplified

description: PolyA RNA

value: AGCAAGC


title: ESCell_81_GCGTCTA_amplified

description: PolyA RNA

value: GCGTCTA


title: ESCell_89_GAGGCGT_amplified

description: PolyA RNA

value: GAGGCGT


title: ESCell_96_CCAAATG_amplified

description: PolyA RNA

value: mouse embryonic stem cells

value: CCAAATG


title: ESCell_80_TATGCTC_amplified

description: PolyA RNA

value: TATGCTC


title: ESCell_78_ATGCCTG_amplified

description: PolyA RNA

value: ATGCCTG


title: ESCell_77_AGTTGTG_amplified

description: PolyA RNA

value: AGTTGTG


title: ESCell_79_GACACAA_amplified

description: PolyA RNA

value: GACACAA