title: MCF7 GROseq nutlin 1

city: Amsterdam

first: Koos

last: Rooijers

address: Plesmanlaan 121

department: Gene Regulation

country: Netherlands

institute: Netherlands Cancer Institute

data_processing:
Library strategy: GROseq
rRNA reads were removed from the data by alignment to a rRNA index compiled from Ensembl annotations (“rRNA”, “rRNA_pseudogene” and “Mt_rRNA”) using bowtie2 (v.2.0.6, parameters “--seed 42 --end-to-end -N1 -L20 -i C,1 -D5 -R5”) and keeping the unmapped reads.
Data was aligned to the human genome using bowtie2 (v. 2.0.6) using parameters “--seed 42 --sensitive”.
Reads that had SAM flag 0x256 (indicating non-primary alignment) or mapping quality < 10 were removed before generating WIG files / further processing
Genome_build: hg19
Supplementary_files_format_and_content: WIG files: genomic coverage based on the alignments, taking only primary alignments with mapQ >= 10. Count data: tables generated by htseq-count, mode 'intersection-strict', taking only primary alignments with mapQ >= 10.

source_name: mammary gland/breast

growth_protocol: MCF-7cells were cultured in DMEM containing 10% FBS, penicillin and streptomycin at 37ºC and 5% CO2.

extract_protocol:
GRO-Seq was performed as described before (Core, L.J., Waterfall, J.J., and Lis, J.T., Science 322, 1845-1848 (2008)) with minor modifications. Briefly, 5 million nuclei per condition were isolated and incubated 5 min at 30°C with equal volume of reaction buffer (10 mM Tris-Cl pH 8.0, 5 mM MgCl2, 1mM DTT, 300 mM KCL, 20 units of SUPERase In, 1% sarkosyl, 500 µM ATP, GTP, and Br-UTP, 0.2 µM CTP+32P CTP) for the nuclear-run on.  The reaction was stopped and RNA extracted with Trizol LS (Invitrogen) according to the manufacturer’s instructions. Base hydrolysis was performed using RNA fragmentation Reagents (Ambion) and the reaction was purified through p-30 RNAse-free spin column (BioRad). BrU labeled nascent transcripts were immunoprecipitated with anti-BrU agarose beads (Santa Cruz Biotech), washed once in binding buffer, once in low salt buffer (0.2X SSPE, 1 mM EDTA, 0.05% Tween-20), once in high salt buffer (0.25X SSPE, 1 mM EDTA, 0.05% Tween-20, 137.5 mM NaCl) and twice in TET buffer (TE with 0.05% Tween-20). Then, BrU-incorporated RNA was eluted with elution buffer (20 mM DTT, 300 mM NaCl, 5 mM Tris-Cl pH 7.5, 1 mM EDTA and 0.1% SDS) and RNA isolated with Trizol LS.  After the binding step, the BrU-RNAs were treated with tobacco acid pyrophosphatase (TAP; Epicenter) to remove 5’-methyl guanosine caps, then with T4 polynucleotide kinase (PNK; NEB) to remove 3’-phosphate group at low pH. The BrU-RNAs were treated with T4 PNK again at high pH in the presence of ATP to add 5’-phosphate group. The reaction was stopped and RNA was extracted with Trizol LS.
Sequencing library was prepared using TruSeq Small RNA kit (Illumina) following manufacturers instructions. Briefly, end-repaired RNA was ligated to RNA 3’ and 5’ adapters, followed by RT-PCR amplification. cDNA was purified using Agencourt AMPure XP (Beckman Coulter) and PCR amplified during 12 cycles. Final libraries were cleaned-up and size-selected by two times Agencourt AMPure XP and sequenced according to Illumina’s protocol in a HiSeq 2000 platform.

treatment_protocol: Nutlin samples were treated with 8μM Nutlin-3a for 16h


title: MCF7 GROseq nutlin 2

status: Public on Mar 24 2015

type: SRA

library_source: transcriptomic

molecule: total RNA

organism: Homo sapiens

tag: cell line


title: MCF7 GROseq control 1


title: IW_0.5-1.5_MPI_1

city: Tuebingen

first: Stefan

last: Czemmel

address: Auf der Morgenstelle 10

department: University of Tuebingen

country: Germany

institute: Quantitative Biology Center (QBiC)

data_processing:
Illumina Casava 1.9 software used for basecalling
Raw fastq files were generated for all 12 libraries for both lanes. Fastq files for each sample from both lanes (L001 and L002) were combined in one fastq file. This leads to a total of 12 fastq files for downstream analysis.
Quality control of fastq raw data was performed using Fastqc (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). As sequences showed overall high quality no filtering step was applied on the reads.
Sequences were aligned to the 12x grape genome (IGGP_12x, European Nucleotide Archive (ENA) accession number: GCA_000003745.2) using Tophat including Bowtie aligner with default parameters. Raw read counts for all annotated features were obtained with HTSeq (v0.5.4p5) and the script htseq-count using the default option 'union'. This mode calls reads as ambigous if they map to more than one gene and subsequently exclude them from counting for any feature/gene. If reads do not map to any feature/gene, the read was counted as no_feature.
Normalization of read counts was performed within the DESeq package (v1.10) by normalizing for effective library sizes using the function estimateSizeFactors and by estimating variance using the function estimateDispersions (with default option 'pooled'). This gives the normalized read count text file which was used as input to test for differential expression
For inference, a multifactoral design was chosen and two models were specified within DESeq: The full model regresses gene expression based on two factors, time (levels: 0 MPI and 0.5-1.5 MPI) and treatment (levels: Non-infected wounded (NIW) and infected wounded (IW). The reduced model regresses gene expression only on the factor time. For each gene, a generalized linear models (GLMs) was fitted according to the two models, and then compared in order to infer whether the additional specification of the factor treatment has significant effect on gene expression.
Genome_build: IGGP_12x
Supplementary_files_format_and_content: tab-delimited text files, which include raw and normalized read counts for each sample and gene as produced by HTSeq and DESeq, respectively.

organism: Vitis vinifera

growth_protocol: The experiment was conducted in the greenhouse on plants, which were propagated in 10 x 10 cm pots from green cuttings 12 months before inoculation. After inoculation, plants were arranged in a completely randomized design on the central bench of a greenhouse with lighting for 14 hours per day.

extract_protocol:
For each RNA extraction, frozen leaves from three plants were mixed and grinded to a fine powder using mortar and pistil under liquid nitrogen. Total RNA was isolated using a rapid cetyltrimethylammonium bromide (CTAB)-based extraction method with minor modifications (Gambino et al., 2008).  DNA was removed by RNase-Free DNase (Qiagen) digestion in solution and then purified using RNAeasy Mini Kit (Qiagen).  Primers for leucoanthocyanidin reductase 1 gene were used to check all RNA preparations for genomic DNA contamination (Bogs et al., 2005).  RNA quality was analyzed using Experion RNA StdSens Chips (BioRad) to verify intact ribosomal bands [rRNA ratio (25s/18s) = 1.8-2.0] and RNA Quality Indicator (RQI) values > 8. For 0 MPI samples, 2 ug of the extracted total RNA was used to make the library. For the mixed second time point (0.5-1.5 MPI) 0.66 ug of extracted RNA from 0.5 MPI, 1 MPI and 1.5 MPI were mixed to give a total of 2ug RNA which was used to make the library. RNA extraction and library preparation was performed on biological triplicates for each of the four treatment time combination (IW 0 MPI, IW 0.5-1.5 MPI, NIW 0 MPI, NIW 0.5-1.5 MPI) to give a total of 12 libraries for RNA-Seq.
RNA libraries for sequencing were prepared from 2ug of total RNA using a modified version of the Illumina TruSeq RNA Library Prep Protocol which is accessible at the website of the DNA Technologie Core, UC Davis (http://dnatech.genomecenter.ucdavis.edu/wp-content/uploads/2013/11/TruSeq_RNA_Sample_Prep_Sept2012.pdf). TruSeq Adapter used were AGTCAA (TruSeq Adapter, Index 13) for IW_0_MPI_1, AGTTCC (TruSeq Adapter, Index 14) for IW_0_MPI_2, ATGTCA (TruSeq Adapter, Index 15) for IW_0_MPI_3, CCGTCC  (TruSeq Adapter, Index 16) for IW_0.5-1.5_MPI_1, GTCCGC  (TruSeq Adapter, Index 18) for IW_0.5-1.5_MPI_2, GTGAAA  (TruSeq Adapter, Index 19) for IW_0.5-1.5_MPI_3, GTGGCC  (TruSeq Adapter, Index 20) for NIW_0_MPI_1, GTTTCG  (TruSeq Adapter, Index 21) for NIW_0_MPI_2, CGTACG  (TruSeq Adapter, Index 22) for NIW_0_MPI_3, GAGTGG  (TruSeq Adapter, Index 23) for NIW_0.5-1.5_MPI_1, ACTGAT  (TruSeq Adapter, Index 25) for NIW_0.5-1.5_MPI_2, ATTCCT (TruSeq Adapter, Index 27) for NIW_0.5-1.5_MPI_3. The 12 libraries were pooled, sequenced on two lanes of the same flow cell of a HiSeq2500 and de-multiplexed to produce 24 raw fastq files. TruSeq Adapter Index sequences are incorporated in the names of the raw fastq files.

treatment_protocol: For IW plants, 20 μl of a homogenized, 3-day liquid culture of N. parvum was pipetted into the wound in the woody part of the plant that was sealed with Vaseline and Parafilm (Travadon et al., 2013).  NIW plants were ‘mock-inoculated’ with 20 µl of sterile water. The third leaf of each plant, counted from the shoot tip, was collected with flame-sterilized forceps at 10 min. post inoculation, which served as the first sampling point (0 MPI) and also at subsequent time points (0.5 MPI, 1 MPI and 1.5 MPI). Leaf samples were immediately frozen in liquid Nitrogen, and then stored at -80°C for RNA extraction.

tag: tissue

value: asymptomatic young leaf

tag: pathogen

value: Neofusicoccum parvum

tag: time post inoculation

value: mix of late time points:0.5 - 1.5 months post inoculation

tag: genotype

value: Cabernet Sauvignan


title: MCF7 p21ekd siep21e 1

data_processing:
(for ChIRP data) Adapter (GATCGGAAGAGCACACGTCTGAACTCCAGTCAC) sequences were clipped from 3' ends of reads (using cutadapt v1.1, parameters -O 12 -e 0.2 -m 12)
(for ChIRP data) Data was aligned to the human genome using bowtie2 (v. 2.0.6) using parameters “--seed 42 -N 1”.
(for ChIRP data) Peak detection was run using macs2 (v. 2.0.10.20130501)using parameters “ -g hs -B -p 0.1”. Peaks with a -log10(q-value) >= 5 and an enrichment >= 4 with respect to the input were kept, and peaks found in the odd and even samples were intersected. Overlapping peaks in both samples that had a position-wise Pearson correlation of abundance >= 0.2 and at least 25 reads in both samples were merged.
(for GROseq data) The first 6 nucleotides (index sequences) were clipped from reads. rRNA reads were removed from the data by alignment to a rRNA index compiled from Ensembl annotations (“rRNA”, “rRNA_pseudogene” and “Mt_rRNA”) using bowtie2 (v.2.0.6, parameters “--seed 42 --end-to-end -N1 -L20 -i C,1 -D5 -R5”) and keeping the unmapped reads.
(for GROseq data) Data was aligned to the human genome using bowtie2 (v. 2.0.6) using parameters “--seed 42 --sensitive”.
A transcript annotation was assembled by merging annotations from Ensembl (v. 37.65), RefSeq (obtained from UCSC Tables 9AUG2012) and the Broad lincRNA catalog (doi:10.1101/gad.17446611), using gffread (supplied with cufflinks) parameters (-M -L -F -G -T).
(for samples “MCF7 control siScramble 1”, “MCF7 control siScramble 2”, “MCF7 nutlin siScramble 1”, “MCF7 nutlin siScramble 2”, “MCF7 control totalRNA”, “MCF7 nutlin totalRNA”) Data was aligned using TopHat (v.2.0.3, parameters “-m1 -F0.0 --segment-length 21 --segment-mismatches 1”) in conjunction with bowtie2 (v.2.0.0-beta6) and the aforementioned transcript annotations.
(for all other RNAseq samples) Data was aligned using TopHat (v.2.0.7, parameters “-m1 -F0.0 --segment-length 25 --segment-mismatches 1 --no-novel-juncs --no-novel-indels --no-coverage-search”) in conjunction with bowtie2 (v.2.0.6) and the aforementioned transcript annotations.
For all samples, reads that had SAM flag 0x256 (indicating non-primary alignment) or mapping quality < 10 were removed before generating WIG files / further processing
Genome_build: hg19
Supplementary_files_format_and_content: WIG files: genomic coverage based on the alignments, taking only primary alignments with mapQ >= 10. Count data: tables generated by htseq-count, mode 'intersection-strict', taking only primary alignments with mapQ >= 10.

molecule: polyA RNA

extract_protocol: RNA libraries were prepared for sequencing using standard Illumina protocols.

treatment_protocol: si-p21e, treated with 8μM Nutlin-3a for 16h


title: MCF7 p21ekd siep21e 2


title: MCF7 p21ekd siScramble 2

treatment_protocol: siScramble, treated with 8μM Nutlin-3a for 16h


title: MCF7 p21ekd siScramble 1


title: MCF7 LEDkd siLED 2

treatment_protocol: siLED, treated with 8μM Nutlin-3a for 16h


title: MCF7 LEDkd siScramble 2


title: MCF7 LEDkd siScramble 1


title: MCF7 LEDkd siLED 1


title: MCF7 control totalRNA


title: MCF7 ChIRP input

library_source: genomic

molecule: genomic DNA

extract_protocol: LED DNA probes were designed using an online designer at http://www.singlemoleculefish.com. All probes were biotinylated at the 3’ end (C3 biotin). MCF7 cells grown in DMEM were rinsed with room temperature PBS. For chemical crosslinking, cells were fixed on plate with appropriate amounts of 1% glutaraldehyde in PBS for 10 min at room temperature. Crosslinking was then quenched with 0.125 M glycine for 5 min. Cells were rinsed again with PBS, scraped into Falcon tubes, and pelleted 2500 g for glutaraldehyde crosslinking. To prepare chromatin, cell pellets were quickly thawed in 37°C and resuspended in Swelling Buffer. Nuclei was lysed in nuclear lysis buffer on ice for 10 min, and sonicated until DNA reaches size range of 100–500 bp. Chromatin is diluted in two times volume of hybridization buffer probes of 100 pmol were added to 3 ml of diluted chromatin, which was mixed by end-to-end rotation at 37°C for 4 hr. Streptavidin magnetic C1 beads were washed in nuclear lysis buffer, blocked with BSA at room temperature, and washed in nuclear lysis buffer. Blocked C1 beads were added per 100 pmol of probes, and incubated 30 min at 37°C. Beads:biotin-probes:RNA:chromatin were captured and washed with washing buffer. Beads were resuspended in DNA elution buffer and eluted with a cocktail of RNase A and RNase H. Elution was carried out at 37°C with rotation and eluent from both steps was combined. Eluted chromatin was pK treated at 65°C for 45 min. DNA was then extracted with phenol:chloroform:isoamyl and precipitated with ethanol overnight. Eluted DNA was subject to Illumina’s high- throughput sequencing protocol.


title: MCF7 ChIRP IP even probe pool

tag: chirp pool probe sequences

value: "CCCTCTTTGGCCAATATGAG", "GGGAACTCAGACCTTCCTTC", "GATCTTCCCTGTGACTTTCC", "GTTTTCAGTCCCAGGAACTG", "TGTCACTCTTGTCCCATAGA", "TGACACCAGGGTTTGAAAGG", "CGCAACTTTATCCCAGTAGG", "ACCGCCTAGAAAACAGACTG", "ACACAGTGAATCACACACAG", "ACAAGAAGACGTGACAACAC", "CTCTGAACAGGAAACACGGT", "CGCCAGACTCTAAAAAGGAA", "CTGTGTCGAGTCTGTTTCAG", "GATTCTGCAGTAACCTTGCT", "TCTGATGGGACAGAATTTGC", "CATTAGCACCAGTGCGTTTG", "GGGGATAAAGAGGACCATCT", "TTTTCCTCTGTAGCAGGGAT", "CGTATTCACTGCTTACAAGC", "CACTGGTAGCTCAGGAAAAG", "GGATTTCTTCTGGACCTCAC", "AAGACAGGTAGACCCATCTC", "ATGTATTTGCTCTAGTGCCC", "GCGGAATCAGATTTCAAAGT"


title: MCF7 nutlin totalRNA

treatment_protocol: Treated with 8μM Nutlin-3a for 16h


title: MCF7 ChIRP IP odd probe pool

value: "GTAGTTTCTGGAGCCCGATG", "CAAATGCCAATGCCTTTCTG", "CAAGGAAAGCGACATTCCTA", "AGCATTGTACACCAGTTAGC", "CAGCTTCATTCAGGACCTGA", "ATCTGTGCATCAGACACAAG", "CTCAGCAAAGAGCTTTCCTC", "GACTTCCGTGACCCTCTTAT", "GTATGTCATTCCGGTCAGTG", "AAACAAGAACAGCTCTCTGG", "GCCTCCAGTTGGGATTTTAG", "CAGGTCAAGTCGGTTCTCAG", "CCCTCAGGATTCTACTGACT", "GAGAGGAGTCTCAGTGCTAC", "GCCAGGTCCTTTATCTTCTC", "CTCCTTGTCAGGATGCTCTG", "CAAGGACTGGGGTCCATTTC", "ATCTTGCTTTGCCAAGTCAT", "TAAAAACTCCAGGTTGTGCA", "TTCTTCTTGGGGCTCTGTTT", "GGTCATCTTCCATCTTTGCT", "CATTTGGAGAAGGCTCTCAG", "GAAACTTCCCACTTAGCTGC", "GGTGCACACAAAGTGAAAAT"


title: MCF7 nutlin siScramble 2


title: MCF7 GROseq control 2


title: MCF7 control siScramble 2

treatment_protocol: siScramble


title: MCF7 control siScramble 1


title: MSC_rep2

status: Public on Mar 23 2015

city: Ranica

first: Paraskevas

last: Iatropoulos

state: BG

address: Via G.B. Camozzi, 3

country: Italy

institute: IRCCS Istituto di Ricerche Farmacologiche Mario Negri

data_processing:
Data extraction, base calling and trimming for adaptor sequences were performed using TorrentSuite™ Software 3.6
Alignment to whole genome (version hg19) was performed using TorrentSuite™ Software 3.6 with default parameters.
To count reads overlapping with known exon coordinates (RefSeq, UCSC) we used the HTSeq software through a modified version of the FeatureCounter plugin that allowed to discard reads with mapping quality score <5. Regions of two or more overlapping genes that lay on the same strand were analyzed as different transcipts. Genes/trascribed regions with mean read count <5 in the 2 replicates have been filtered out.
Genome_build: hg19
Supplementary_files_format_and_content: tab-delimited text files include RPKM values for each Sample.

source_name: mesenchymal stem cells

growth_protocol: HK2 cells and /or CL17 cells were cultured in Keratinocyte Serum Free medium (Clonetics, Life Technologies) supplemented with Bovine Pituitary Extract (BPE) and Epidermal Growth Factor (EGF). MSC were cultured in DMEM low glucose, supplemented with 5% human platelet lysate according to (Capelli et al; BM Transpl, 2007).

extract_protocol:
Total RNA was prepared from MSC, HK2 cells and five of the clones obtained by limiting dilution, using TRIzol reagent according to the manufacturer’s instructions.
PolyA+ RNA was obtained from 20µg of total RNA using Dynabeads® mRNA DIRECT™ Micro Kit after adding external RNA spike-in controls (ERCC). Libraries were prepared using the Ion Total RNA-Seq Kit v2. Template preparation and sequencing were performed using the Ion PGM™ Template OT2 200 Kit and the Ion PGM™ Sequencing 200 Kit v2. Each replicate was sequenced on the Ion PGM™ Sequencer using a Ion 318™ Chip.

tag: cell type

value: bone marrow mesenchymal stem cells (Capelli et al; BM Transpl, 2007)


title: MCF7 nutlin siScramble 1


title: CL17_rep2

source_name: reprogrammed MSC; Clone 17

value: reprogammed MSC-proximal tubular cells; clone 17


title: MSC_rep1


title: BT247(1:10)_12h_rep3

city: Kiel

first: Hinrich

last: Schulenburg

address: Am Botanischen Garten 1-9

department: Evolutionary Ecology and Genetics, Zoological Institute

description: Diluted by E. coli OP50 at 1:10

data_processing:
Illumina Casava1.8 software used for basecalling.
Sequenced reads were trimmed for adaptor sequence, and masked for low-quality sequence, then mapped to WBcel235 whole genome using tophat-2.0.8 with parameters --no-coverage-search  --b2-very-sensitive
Reads Per Kilobase per Megabase of library size (RPKM) were calculated using cufflinks-2.0.2
Genome_build: WBcel235
Supplementary_files_format_and_content: tab-delimited text files include RPKM values for each Sample ...

organism: Caenorhabditis elegans

source_name: whole body

extract_protocol:
RNA was harvested using Trizol reagent. Illumina TruSeq RNA Sample Prep Kit (Cat#FC-122-1001) was used with 1 ug of total RNA for the construction of sequencing libraries.
RNA libraries were prepared for sequencing using standard Illumina protocols

treatment_protocol: Worms were exposed to BT at the stage L4. The BT strains were diluted in either 1:2 or 1:10 (E.coli OP50). The experiments with BT were performed at 20°C.

tag: strain

value: N2/Wide type

tag: treatment

tag: timepoint

tag: medium

value: PFM

tag: temperature


title: BT247(1:10)_12h_rep2

description: Diluted by E. coli OP50 at 1:10


title: BT247(1:10)_6h_rep1

description: Diluted by E. coli OP50 at 1:10


title: BT247(1:10)_6h_rep2

description: Diluted by E. coli OP50 at 1:10


title: BT247(1:10)_6h_rep3

description: Diluted by E. coli OP50 at 1:10


title: BT247(1:10)_12h_rep1

description: Diluted by E. coli OP50 at 1:10


title: BT247(1:2)_12h_rep1

description: Diluted by E. coli OP50 at 1:2


title: BT247(1:2)_6h_rep3

description: Diluted by E. coli OP50 at 1:2


title: BT247(1:2)_12h_rep3

description: Diluted by E. coli OP50 at 1:2


title: BT247(1:2)_12h_rep2

description: Diluted by E. coli OP50 at 1:2


title: BT247(1:2)_6h_rep2

description: Diluted by E. coli OP50 at 1:2


title: BT247(1:2)_6h_rep1

description: Diluted by E. coli OP50 at 1:2


title: BT407_12h_rep3


title: BT407_12h_rep2


title: BT407_12h_rep1


title: OP50_12h_rep3


title: BT407_6h_rep2


title: CL17_rep1


title: HK2_rep2

source_name: proximal tubular cells

value: CRL-2190; ATCC


title: BT407_6h_rep3


title: OP50_12h_rep2


title: BT407_6h_rep1


title: HK2_rep1


title: OP50_6h_rep3


title: OP50_6h_rep1


title: OP50_6h_rep2


title: OP50_12h_rep1


title: 4C_EScells_WT_Hoxa9

city: Lausanne

first: Marion

last: LELEU

address: EPFL-SV-ISREC-UPDUB- Station 19

department: School of Life Sciences

country: Switzerland

institute: EPFL

data_processing:
De-multiplexing, mapping and 4C-seq were done through HTSstation (David et al, 2014 PLoS ONE and http://htsstation.epfl.ch/).
Reads were mapped to the mm9 (NCBIM37) Mouse genome assembly with bowtie version 0.12.7 (Langmead et al, 2009 Genome Biology) with parameters -n 2 --best -strata -m 5.
Mapped reads were translated to restriction fragments using the HTSstation (David et al, 2014 PLoS ONE and http://htsstation.epfl.ch/).
4C figures were made using a running mean algorithm with a window size of eleven fragments.
Genome_build: mm9
Supplementary_files_format_and_content: Signal/restriction fragment (bigwig) and smoothed (bedGraph), one per viewpoint, per cell type. Signal/restriction fragment (bigwig) with inversed genomic fragment, one per viewpoint, for Inv(Itga6_HoxDVRVIII) cells

organism: Mus musculus

source_name: ES cells

extract_protocol:
Multiplexed samples were sequenced on the Illumina HiSeq system using 100 bp single-end reads according to the manufacturer’s specifications. 4C-seq reads were sorted, aligned, and translated to restriction fragments using the 4C-seq pipeline of the BBCF HTSstation (David et al. 2014, Plos ONE and available at http://htsstation.epfl.ch). Samples were mapped to the ENSEMBL Mouse assembly NCBIM37 (mm9).
Illumina adapters (single end reads) were included in the inverse 4C PCR primers used for 4C-seq library preparation
Cells were isolated in PBS supplemented with 10% Fetal Calf Serum. Tissue sample were further made single cell by collagenase treatment and applying a cell-strainer cap (BD Biosciences). Chromatin was crosslinked in the presence of 2% formaldehyde.
PCR amplification using primers with included adapter sequences (for Illumina HiSeq2000)

treatment_protocol:
4C–seq libraries were constructed as described before (Noordermeer et al. 2011, Science). ES cell 4C-seq libraries were prepared from samples consisting of 20 million cells. The primary restriction enzyme used was NlaIII (New England Biolabs, R0125L) and the secondary restriction enzyme was DpnII (New England Biolabs, R0543M). For each viewpoint approximately 1 μg of the 4C-seq library was amplified using 16 individual PCR reactions with inverse primers containing Illumina Solexa adapter sequences.
Circular Chromosome Conformation Capture (4C) technique was performed as described (Noordermeer et al, 2011 Science). Cross-linked chromatin was digested with NlaIII, ligated under diluted conditions, cross links were reversed and DNA was further digested by DpnII and again ligated under diluted conditions.

value: Embryonic stem cells

tag: age

value: WT


title: 4C_EScells_WT_Hoxa4


title: 4C_EScells_WT_Hoxc9


title: 4C_EScells_WT_Hoxb4


title: 4C_EScells_WT_Pax6


title: 4C_E10.5_Mx_WT_Hoxd13

source_name: Maxillary arch E10.5

extract_protocol:
Multiplexed samples were sequenced on the Illumina Genome HiSeq 2000 system using 100 bp single-end reads according to the manufacturer’s specifications. 4C-seq reads were sorted, aligned, and translated to restriction fragments using the 4C-seq pipeline of the BBCF HTSstation (David et al. 2014, Plos ONE and available at http://htsstation.epfl.ch). Samples were mapped to the ENSEMBL Mouse assembly NCBIM37 (mm9).
Illumina adapters (single end reads) were included in the inverse 4C PCR primers used for 4C-seq library preparation
Cells were isolated in PBS supplemented with 10% Fetal Calf Serum. Tissue sample were further made single cell by collagenase treatment and applying a cell-strainer cap (BD Biosciences). Chromatin was crosslinked in the presence of 2% formaldehyde.
PCR amplification using primers with included adapter sequences (for Illumina HiSeq2000)

treatment_protocol:
4C–seq libraries were constructed as described before (Noordermeer et al. 2011, Science). 4C-seq libraries were prepared from 76 E10.5 maxillary arches. The primary restriction enzyme used was NlaIII (New England Biolabs, R0125L) and the secondary restriction enzyme was DpnII (New England Biolabs, R0543M). For each viewpoint approximately 1 μg of the 4C-seq library was amplified using 16 individual PCR reactions with inverse primers containing Illumina Solexa adapter sequences.
Circular Chromosome Conformation Capture (4C) technique was performed as described (Noordermeer et al, 2011 Science). Cross-linked chromatin was digested with NlaIII, ligated under diluted conditions, cross links were reversed and DNA was further digested by DpnII and again ligated under diluted conditions.

value: Maxillary arch cells


title: 4C_E10.5_Jct_WT_Hoxd13

source_name: Junction E10.5

treatment_protocol:
4C–seq libraries were constructed as described before (Noordermeer et al. 2011, Science). 4C-seq libraries were prepared from 76 E10.5 junctions. The primary restriction enzyme used was NlaIII (New England Biolabs, R0125L) and the secondary restriction enzyme was DpnII (New England Biolabs, R0543M). For each viewpoint approximately 1 μg of the 4C-seq library was amplified using 16 individual PCR reactions with inverse primers containing Illumina Solexa adapter sequences.
Circular Chromosome Conformation Capture (4C) technique was performed as described (Noordermeer et al, 2011 Science). Cross-linked chromatin was digested with NlaIII, ligated under diluted conditions, cross links were reversed and DNA was further digested by DpnII and again ligated under diluted conditions.

value: Junction cells


title: 4C_E10.5_FB_InvItga6_HoxDVRVIII_Pdk1

source_name: Forebrain E10.5

treatment_protocol:
4C–seq libraries were constructed as described before (Noordermeer et al. 2011, Science). 4C-seq libraries were prepared from 10 E10.5 forebrains. The primary restriction enzyme used was NlaIII (New England Biolabs, R0125L) and the secondary restriction enzyme was DpnII (New England Biolabs, R0543M). For each viewpoint approximately 1 μg of the 4C-seq library was amplified using 16 individual PCR reactions with inverse primers containing Illumina Solexa adapter sequences.
Circular Chromosome Conformation Capture (4C) technique was performed as described (Noordermeer et al, 2011 Science). Cross-linked chromatin was digested with NlaIII, ligated under diluted conditions, cross links were reversed and DNA was further digested by DpnII and again ligated under diluted conditions.

value: Forebrain cells


title: 4C_E10.5_AT_WT_Dlx1

source_name: Anterior trunk E10.5

treatment_protocol:
4C–seq libraries were constructed as described before (Noordermeer et al. 2011, Science). 4C-seq libraries were prepared from 10 E10.5 anterior trunks. The primary restriction enzyme used was NlaIII (New England Biolabs, R0125L) and the secondary restriction enzyme was DpnII (New England Biolabs, R0543M). For each viewpoint approximately 1 μg of the 4C-seq library was amplified using 16 individual PCR reactions with inverse primers containing Illumina Solexa adapter sequences.
Circular Chromosome Conformation Capture (4C) technique was performed as described (Noordermeer et al, 2011 Science). Cross-linked chromatin was digested with NlaIII, ligated under diluted conditions, cross links were reversed and DNA was further digested by DpnII and again ligated under diluted conditions.

value: Anterior trunk cells


title: 4C_E10.5_Mx_WT_Dlx1


title: 4C_E10.5_FB_InvItga6_HoxDVRVIII_Wt1


title: 4C_E10.5_FB_InvItga6_HoxDVRVIII_Hoxd4


title: 4C_E10.5_FB_InvItga6_HoxDVRVIII_Hoxd13


title: 4C_E10.5_FB_InvItga6_HoxDVRVIII_Sp9


title: 4C_EScells_WT_Hoxc4


title: 4C_E10.5_FB_WT_Hoxc4

extract_protocol:
Illumina adapters (single end reads) were included in the inverse 4C PCR primers used for 4C-seq library preparation
Cells were isolated in PBS supplemented with 10% Fetal Calf Serum. Tissue sample were further made single cell by collagenase treatment and applying a cell-strainer cap (BD Biosciences). Chromatin was crosslinked in the presence of 2% formaldehyde.
PCR amplification using primers with included adapter sequences (for Illumina HiSeq2000)

treatment_protocol:
4C–seq libraries were constructed as described before (Noordermeer et al. 2011, Science). 4C-seq libraries were prepared from 12 E10.5 forebrains. The primary restriction enzyme used was NlaIII (New England Biolabs, R0125L) and the secondary restriction enzyme was DpnII (New England Biolabs, R0543M). For each viewpoint approximately 1 μg of the 4C-seq library was amplified using 16 individual PCR reactions with inverse primers containing Illumina Solexa adapter sequences.
Circular Chromosome Conformation Capture (4C) technique was performed as described (Noordermeer et al, 2011 Science). Cross-linked chromatin was digested with NlaIII, ligated under diluted conditions, cross links were reversed and DNA was further digested by DpnII and again ligated under diluted conditions.


title: 4C_E10.5_FB_WT_Hoxa4


title: 4C_E10.5_FB_InvItga6_HoxDVRVIII_Dlx1


title: 4C_E10.5_FB_WT_Alx4


title: 4C_E10.5_FB_WT_Hoxd4


title: 4C_E10.5_FB_WT_Hoxc9


title: 4C_E10.5_FB_WT_Pax6


title: 4C_E10.5_FB_WT_Hoxa9


title: 4C_E10.5_FB_WT_Hoxb4


title: 4C_E10.5_FB_WT_Pdk1


title: 4C_E10.5_Jct_WT_Dlx1


title: 4C_E10.5_FB_WT_Hoxd13


title: 4C_E10.5_FB_WT_Wt1


title: 4C_E10.5_FB_WT_Dlx1


title: 4C_EScells_WT_Dlx1


title: 4C_E10.5_FB_WT_Cd44


title: ChIPseq_E10.5_FB_InvItga6_HoxDVRVIII_H3K27me3

data_processing:
Mapping and generation of density files was done through HTSstation (David et al, 2014 PLoS ONE and http://htsstation.epfl.ch/).
Reads were mapped to the mm9 (NCBIM37) Mouse genome assembly with bowtie version 0.12.7 (Langmead et al, 2009 Genome Biology) with parameters -n 2 --best -strata -m 5.
If read length was shorter than 100 bp, reads were extended to 100 bp. Reads mapping to top and bottom strand were shifted 50 bp to compensate for average fragment length. Genome coverage densities were calculated for each strand separately, then averaged and normalized by the total number of mapped reads (times 10^-7).
Genome_build: mm9
Supplementary_files_format_and_content: density file (bigWig) per cell type. Density file (bigWig) with inversed genomic fragment for Inv(Itga6_HoxDVRVIII) cells

extract_protocol:
Libraries (single end reads) were prepared according to the manufacturers description
Cells were isolated in PBS supplemented with 10% Fetal Calf Serum. Samples were further made single cell by collagenase treatment and applying a cell-strainer cap (BD Biosciences). Chromatin was crosslinked in the presence of 2% formaldehyde. Nuclei were sonicated to generate fragments with average length around 300 bp.
Immuno-precipitated DNA was prepared using the TruSeq ChIP Sample Preparation Kit (Illumina)

treatment_protocol:
ChIP-seq libraries were constructed as described before (Noordermeer et al. 2011, Science). Libraries were prepared from 8 E10.5 forebrain samples. Nuclei were sonicated to generate fragments with average length around 300 bp. For each ChIP, 10 ug of sonicated nuclei were lysed and histone-DNA complexes were isolated with indicated antibody.
Lysates were clarified from sonicated nuclei and histone-DNA complexes were isolated with H3K27me3 (#17-622, Millipore) antibody.

tag: antibody


title: ChIPseq_E10.5_FB_WT_H3K27me3


title: ChIPseq_E10.5_Jct_WT_H3K27me3

extract_protocol:
Samples were sequenced on the Illumina HiSeq system using 50 bp single-end reads according to the manufacturer’s specifications. ChIP-seq reads were sorted and aligned using the 4C-seq pipeline of the BBCF HTSstation (David et al. 2014, Plos ONE and available at http://htsstation.epfl.ch). Samples were mapped to the ENSEMBL Mouse assembly NCBIM37 (mm9).
Libraries (single end reads) were prepared according to the manufacturers description
Cells were isolated in PBS supplemented with 10% Fetal Calf Serum. Samples were further made single cell by collagenase treatment and applying a cell-strainer cap (BD Biosciences). Chromatin was crosslinked in the presence of 2% formaldehyde. Nuclei were sonicated to generate fragments with average length around 300 bp.
Immuno-precipitated DNA was prepared using the TruSeq ChIP Sample Preparation Kit (Illumina)

treatment_protocol:
ChIP-seq libraries were constructed as described before (Noordermeer et al. 2011, Science). Libraries were prepared from 125 E10.5 junction samples. Nuclei were sonicated to generate fragments with average length around 300 bp. For each ChIP, 10 ug of sonicated nuclei were lysed and histone-DNA complexes were isolated with indicated antibody.
Lysates were clarified from sonicated nuclei and histone-DNA complexes were isolated with H3K27me3 (#17-622, Millipore) antibody.


title: 4C_E10.5_FB_WT_Sp9


title: Trim24-KO rep2

city: HOUSTON

first: Michelle

middle: C.

last: Barton

state: TEXAS

address: 6767 Bertner St. Unit Number: 1000 Room Number: S9.8116B

department: Department of Epigenetics and Molecular Carcinogenesis,

country: USA

institute: The University of Texas MD Anderson Cancer Center

data_processing:
The sequencing of 6 RNA-Seq samples (3 biological replicates of RNA samples from livers of Trim24-/- and Trim24+/+ mice) produced Illumina 75 bp paired-end sequenced reads. These reads were aligned to the mouse genome (mm9), through the use of TopHAT 1.4.1 with –r 150 –G UCSC_genes.gtf (downloaded from UCSC mm9 July 2007) parameters.
EdgeR was applied to determine differentially expressed genes between Trim24-/- and Trim24+/+ samples at a false discovery rate (FDR) adjusted p-value (q-value) of 1%
The number of reads aligning to the exons of each gene was computed using HTSeq (Anders and Huber, 2010)
Genome_build: mm9
Supplementary_files_format_and_content: tab-delimited text files include read counts for each Sample ...

source_name: Liver

extract_protocol:
Total RNA from homogenized mouse liver was extracted with TRIzol reagent (Life Technologies, Invitrogen) according to the manufacturer’s instructions. Complementary DNA was obtained by reverse transcription of 2.5 μg of RNA using the SuperScript system (Invitrogen).
RNA libraries were prepared for sequencing using standard Illumina protocols

value: liver

value: 8 weeks


title: Trim24-KO rep1


title: Trim24-KO rep3


title: WT rep3

value: wild type


title: WT rep2


title: WT rep1


title: JAG1 wildtype 2

city: Los Angeles

first: Anais

last: Briot

state: CA

address: 621 Charles E. Young Drive, South

department: Molecular, Cell and Developmental Biology

institute: University of California, Los Angeles

data_processing:
Basecalls performed by RTA
qseq files demultiplexed using custom script, allowing for one mismatich in the tag
convert to fastq using custom script
align fastq to mus musculus GRCm37/mm9 genome using tophat v 2.0.4, allowing only uniquely mapped reads and up to two mismatches, and using Ensembl 67 annotation
count reads mapped per gene using HTSeq 0.5.4p3, using the non-empty intersection method
gene counts normalized by DESeq 1.16.0 using a robust normalization process
RPKM calculated as normalized counts divided by union exon length per gene and mean library size
p-values calculated using DESeq 1.16.0, pooling replicates together to estimate dispersion
Genome_build: GRCm37/mm9
Supplementary_files_format_and_content: xlsx format spreadsheet. Contains normalized read counts per gene, gene function, location, calculated length (union of all exons), RPKM, log base 2 ratios per comparison, p-value per comparison

source_name: JAG1 lox/lox Cre- SMC

extract_protocol:
RNA was isolated using Rneasy Micro kit (Qiagen);  Illumina TruSeq RNA Sample Prep Kit  was used with 500ng of total RNA for the construction of sequencing libraries.
RNA libraries were prepared for sequencing using standard Illumina protocols

tag: developmental stage

value: descending aorta


title: JAG1 wildtype 1


title: JAG1 heterozygous 2

source_name: JAG1 lox/+ Cre+ SMC


title: JAG1 heterozygous 1


title: Control 3

source_name: ImSMC + DMSO

extract_protocol:
RNA was isolated using Rneasy Mini kit (Qiagen);  Illumina TruSeq RNA Sample Prep Kit  was used with 1ug of total RNA for the construction of sequencing libraries.
RNA libraries were prepared for sequencing using standard Illumina protocols

treatment_protocol: ImSMC were treated for 3 days with g-secretase inhibitor (DAPT, 50uM) or DMSO vehicle control

value: immorto mouse

value: vehicle control


title: Treated 2

source_name: ImSMC + DAPT

value: gamma - secretase


title: ChIPseq_E10.5_Mx_WT_H3K27me3

treatment_protocol:
ChIP-seq libraries were constructed as described before (Noordermeer et al. 2011, Science). Libraries were prepared from 125 E10.5 maxillary arch samples. Nuclei were sonicated to generate fragments with average length around 300 bp. For each ChIP, 10 ug of sonicated nuclei were lysed and histone-DNA complexes were isolated with indicated antibody.
Lysates were clarified from sonicated nuclei and histone-DNA complexes were isolated with H3K27me3 (#17-622, Millipore) antibody.


title: Control 2


title: Treated 3


title: Treated 1


title: JAG1 knockout 1

source_name: JAG1 lox/lox Cre+ SMC


title: Control 1


title: RAG2 RNA Pol II Ser5P ChIP-Seq

city: Marseille

first: Mohamed

last: BELHOCINE

address: 163, Avenue de Luminy

country: France

institute: INSERM

description: RAG2 RNA Pol II Ser5P ChIP-Seq

data_processing:
The Genome Analyzer II System, basecalls were performed using manufacturer’s instructions.
Alignment of reads to the mm9 genome was conducted using CASAVA Eland and BFast 0.7.0 for Illumina sequencing run, according to manufacturer’s instructions. Only reads with a unique best scoring alignment were retained. Clonal artifacts were filtered out retaining the longer reads.
Aligned reads were processed using a previously described in-house ChIP-Seq analysis pipeline (Koch, Fenouil et al 2011).  Using a stepwise tag size extension in 10bp increments, fragment size was estimated in silico.
Tags were then elongated to the estimated mean fragment size and converted to wig format using a binning window of 50bp. Visual inspection of invariant targets (e.g. β-actin) allowed further fine tuning and scaling of tag-counts to reflect potential differences in signal to noise ratios.
Genome_build: mm9
Supplementary_files_format_and_content: Fragments were extended using an R pipeline described in Koch, Fenouil, Gut et al. 2011. Fixed-step 50bp wig files were generated using an R pipeline described in Koch, Fenouil, Gut et al. 2011.

source_name: primary Rag2 -/- thymocytes

growth_protocol: Thymuses were extracted from 5-6-week-old Rag2-/- knockout as well as C57BL/6 wild type mice and stored in RPMI medium. To recover thymocytes, thymuses were subsequently passed twice through a 70µm Falcon cell strainer and stored in PBS. Cells were crosslinked 10 min in 10% formaldehyde followed by 5 min quenching in 250 mM glycine.

extract_protocol:
Phenol-chroloform
The library preparation was carried out as per manufacturer’s instructions (Illumina) using all the ChIPed material. Samples were subsequently size-selected via gel migration and extraction, with average cut sizes at about 156 bp, including adapters. Sequencing was carried out on an Illumina Genome II Analyzer. The quality of libraries was checked with the Bioanalyser DNA High sensitivity and by Q-PCR.

treatment_protocol: For ChIP-Seq, thymocytes were  aliquoted in crosslinked pellets of 6 10+7 cells and lysed by rotation for 10 min at 4°C in 2x 1.25ml LB1 (50mM Hepes pH 7.5, 140mM NaCl, 1mM EDTA, pH 8, 10% glycerol, 0.75% NP-40, 0.25% Triton X-100). Following centrifugation at 1,350g for 5 minutes at 4°C, nuclei were subsequently treated with 2x1.25ml LB2 (200mM NaCl, 1mM EDTA pH 8, 0.5mM EGTA pH 8, 10mM Tris pH 8) on a rotating wheel for 10 minutes at 4°C. Chromatin was resuspended in 1.5ml LB3 (1mM EDTA pH8, 0.5mM EGTA pH 8, 10mM Tris pH 8, 100mM, NaCl, 0.1% Na-deoxycholate, 0.5% N-lauroylsarcosine) in 15ml polypropylene falcon tubes and then sonicated for 10 cycles (30 seconds on, 30 seconds off) at 40W using a VCX130 sonicator (VibraCell) down to an average size of about 250bp. 50µl aliquots were collected as Input controls. All buffers contained final concentrations of 1X protease inhibitors (Roche, France) as well as 0.2mM PMSF and 1μg /ml pepstatin. ChIPs were conducted using protein-G DynaBeads (Invitrogen).Antibody and bead mixes were washed three times with 1ml BSA blocking buffer (0.5% BSA in 1x DPBS), incubated and rotated overnight at 4°C, and subsequently washed another three times  with 1ml BSA blocking buffer. Immunoprecipitation was carried out overnight in 750µl LB3 at 4°C on a rotating wheel. Samples were then washed 8 times in 1ml RIPA buffer (50mM Hepes pH 7.6, 500mM LiCl, 1mM EDTA pH 8, 1% NP-40, 0.7% Na-Deoxycholate) and once in 1ml TE buffer with 50mM NaCL. Two 15-min and 10-min subsequent elutions were carried out in a waterbath at 65°C, vortexing every other minute, for a final volume of 100µl. 100µl 1X elution buffer (50mM Tris pH 8, 10mM EDTA pH 8, 1% SDS) was added to eluates which were reverse-crosslinked overnight in a 65°C waterbath. Equal volumes of TE buffer (100µl) were added to samples as well as 0.2μg/ml RNAse A and placed at 37°C for 2 hours, followed by proteinase K treatment at 0.2μg/ml at 55°C. DNA purification was carried out via phenol-chloroform extraction followed by elution using QiaQuick PCR purification kits. (QiaGen) Samples were pooled per replicate then SpeedVac-ed down to 30µl. DNA quantification of eluates was obtained using Bioanalyzer High Sensitivity chips (Agilent). Library preparation was carried out as per manufacturer’s instructions (Illumina). Samples were subsequently size-selected via gel migration and extraction, with average cut sizes at about 156 bp including adapters.  Sequencing was performed using either Genome Analyzer II (Illumina), whereby samples were sequenced at the CNAG, Barcelona, Spain. The quality of the libraries was checked with the Bioanalyser DNA High sensitivity and by Q-PCR.

tag: strain background

value: 5-6 weeks

tag: genotype/variation

value: primary Rag2 -/- thymocytes

tag: # of cells used

tag: chip antibody

value: Pol II Antibody (N-20)

tag: chip antibody vendor

value: Santa Cruz

tag: chip antibody cat. #

tag: chip antibody quantity

value: 5 µg

tag: magnetic beads

value: Protein G dynabeads

tag: bead volume

tag: estimated fragment size


title: P5424 H3K4me2 (DMSO) ChIP-Seq

data_processing:
Sequencing of ChIP samples were performed according to the Illumina Genome Analyzer ChIP-seq protocol and aligned against the mouse mm9 genome using the integrated Eland software.
As pre-filtering steps, only uniquely mapped tags were used for further processing and all duplicate tags (those with identical coordinates) were filtered out to remove possible sequencing and/or alignment artifacts. Remaining tags were processed using a custom R pipeline, employing the ShortRead library3
Wiggle files for genome-wide scores were generated following a binning step, calculating the average score every 50 bp .
Genome_build: mm9
Supplementary_files_format_and_content: wig

source_name: P5424 H3K4me2 (DMSO)

growth_protocol: Exponentially growing P5424 cells in RPMI medium for 18 hours at 37°C

extract_protocol:
Phenol-chroloform
Library preparation was carried out as per manufacturer’s instructions (Illumina) using 10ng material. Samples were subsequently size-selected via gel migration and extraction, with average cut sizes at about 230 bp, including adapters. Sequencing was carried out on an Illumina Genome II Analyzer. The quality of libraries was checked with the Bioanalyser DNA High sensitivity and by Q-PCR. Sequencing was performed with the

treatment_protocol: 15x106 P5424 cells of were incubated with either 50 µM of KM05283 (Maybridge, UK) or control DMSO (Sigma-Aldrich, USA) in RPMI medium for 18 hours at 37°C. After incubation, cells were washed two times with 1X DPBS and processed for ChIPs as indicated below.

tag: cells used

tag: antibody vendor/catalog#

value: Abcam (ab32356)

tag: antibody quantity

value: 3 µg

tag: cell line


title: RBP2L_ChIPSeq_1

city: Chicago

first: Elizaveta

middle: V

last: Benevolenskaya

state: IL

address: 900 S. Ashland Ave.

department: Biochemistry and Molecular Genetics

institute: University of Illinois at Chicago

description: Chromatin IP against RBP2 Large isoform

data_processing:
Alignment: Sequence reads were obtained using the Illumina Genome Analyzer Pipeline and reads were mapped to the human (March, 2006) genomes (NCBI36/hg18) using BOWTIE aligner program (version 0.11.3) (http://bowtie-bio.sourceforge.net/index.shtml) (Ref: Langmead B, Trapnell C, Pop M, Salzberg SL. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biology 10:R25.) All reads mapping uniquely with two or fewer mismatches were retained.
Peaks: Peak detection was performed with the Model-based Analysis of ChIP-Seq (MACS) algorithm (http://liulab.dfci.harvard.edu/MACS/)

source_name: Chromatin IP against RBP2 Large isoform

growth_protocol: Human U937 diffuse histiocytic lymphoma cells were seeded at the density 2.5x105 cell/ml and grown in RPMI medium (Mediatech) containing 50 nM TPA (Sigma) for 96 hours. The medium was changed on fresh medium 72 hours after the treatment has started. The cells successfully differentiated to macrophages/monocytes; displayed growth arrest and expressed monocytic markers CD11b and CD14.

extract_protocol: ChIP and genomic library preparation was performed as described (Beshiri, M.L, A. Islam A, D.C. DeWaal, W.F. Richter, Love J, N. Lopez-Bigas, and E.V. Benevolenskaya. 2010. Genome-wide Analysis using ChIP to Identify Isoform-specific Gene Targets. JoVE. 41. http://www.jove.com/index/details.stp?id=2101, doi: 10.3791/2101).  After adapter ligation DNA was PCR amplified with Illumina primers for 18 cycles and library fragments of ~300 bp (insert plus adaptor and PCR primer sequences) were band isolated from an agarose gel. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Genome Analyzer following the manufacturer's protocols.

tag: cell type

value: diffuse histiocytic lymphoma cells

tag: cell differentiation protocol

value: differentiated with TPA for 96 hours


title: RBP2L_ChIPSeq_2

description: Chromatin IP against RBP2 Large isoform


title: RBP2All_ChIPSeq_1

description: Chromatin IP against RBP2

source_name: Chromatin IP against RBP2

value: 2469(non-commercial, unpublished)


title: RBP2All_ChIPSeq_2

description: Chromatin IP against RBP2


title: P5424 H3K4me3 (DMSO) ChIP-Seq

source_name: P5424 H3K4me3 (DMSO)

value: Abcam (ab8580)

tag: antibody


title: P5424 H3K4me3 (KM) ChIP-Seq


title: P5424 H3K4me1 (KM) ChIP-Seq

value: Abcam (ab8895)


title: P5424 H3K4me1 (DMSO) ChIP-Seq

source_name: P5424 H3K4me1 (DMSO)


title: U937_input_gDNA

description: input DNA

source_name: input DNA


title: Flag-E912A iCLIP replicate 2

city: Shanghai

first: Li

last: Yang

address: 131 Dong-An Road

department: Institutes of Biological Sciences

country: China

institute: Fudan University

description:
Flag-E912A iCLIP technical replicate 2
processed data file: e912a_cluster.bed

data_processing:
Basecalling using Illumina Casava1.8.2 software.
High-throughput sequencing reads were separated according to 4-nt experimental barcodes.
3-nt barcodes for identifying PCR duplicates were registered and removed, followed by trimming relevant sequencing adapters.
Trimmed reads were aligned against the GRCh37/hg19 human reference genome with the UCSC Genes annotation (hg19 knownGene.txt updated at 2013/6/30) using TopHat2 (Kim et al., 2013) 2.0.9 (parameters: -a 6 --microexon-search -m 2).
Mapped reads with same genomic locations and the same 3-nt barcode were combined.
Peak calling and the false discovery rate (FDR) calculating were determined according previous work(Konig et al., 2010).
Genome_build: hg19 for human samples
Supplementary_files_format_and_content: bed files describing significant crosslink sites for individual treatment, combined samples from technical replicates.

source_name: Flag-E912A iCLIP

growth_protocol: To obtain ADAR1 KD cells, H9 cells grown on Matrigel (BD Biosciences) were digested into single cells with Accutase (Millipore). Cells were incubated with MEF-condition medium (MEF-CM) containing 10 μl concentrated lentivirus, 5 μg/ml polybrene (sigma) and 10 μM Y-27632 (Tocris) at 37°C for 1 hr in a 15 ml-conical tube and were then seeded onto Matrigel coated culture dishes. 24 hr later, cells were fed with pre-warmed MEF-CM containing 5 μg/ml polybrene for 15 min and then infected the second time on the plates. 4 days after 1st infection, cells were split and cultured for 5 additional days. GFP-positive colonies were mechanically detached and cultured individually. To obtain a pure and stable ADAR1 KD H9 cell population, the RNAi cells were further split and selected for more than 3 passages.

extract_protocol:
The UV-crosslinked cells were resuspended in lysis buffer and sonicated for 20s x 4 times on 25% duty. After centrifugation at 14,000rpm at 4℃, the supernatant were incubated with 8μg anti-flag antibody and 100μl DynaBeadsG at 4℃ for 2 hours. Then the protein-RNA complexes were treated with 5 Kunz U/ml of micrococcal nuclease with 5 μg yeast tRNAs for 5 min at 37°C, and RNA 3’ ends were dephosphorylated by PNK at pH6.5. Then pre-adenylated linker L3 (3’ modification with biotin) was linked to 3’ end of RNA at 16°C overnight. 5’ end of RNA was labeled with [γ-32P] ATP and eluted with 1x Nupage loading buffer (Invitrogen) at 70°C for 10 min. Protein-RNA complexes were purified by monomeric avidin beads (Fischer Scientific) and eluted again with 1xNuPAGE loading buffer. Complexes were separated on 4-12% NuPAGE Bis-Tris gel (Invitrogen), transferred to a nitrocellulose membrane and exposure to a Fuji film at -80℃. Protein-RNA complexes were digested with proteinase K, and RNA were reverse transcribed by Superscript III reverse transcriptase (Invitrogen) with Rclip primers. cDNAs were separated on 6% TBE-urea gel, then three bands were cut at 120-200 nt (high), 85-120 nt (medium) and 70-85 nt (low).
cDNAs were circularized by Circligase II (Epicentre). Single-stranded cDNAs were hybridized with cut_oligo, and then digested by BamHI. Linearized cDNAs were PCR amplified and purified on PAGEs to obtain iCLIP libraries, which were subjected to Illumina HiSeq 2000.

treatment_protocol: ADAR1 KD H9 cells infected with Flag-ADAR1 or Flag-ADAR1-E912A were washed with cold 1xPBS, then UV cross-linked at 400mJ/cm2 and collected by scraper.

tag: cell line background

value: ADAR1 KD H9

value: embryonic stem cells

tag: infected with

value: ADAR1 KD H9 with expressing Flag-E912A

value: anti-Flag

tag: antibody vendor

value: Sigma-Aldrich

tag: antibody cat. #

tag: antibody lot #

tag: barcode sequence

value: NNNCCGANN


title: Flag-ADAR1 iCLIP replicate 1

description:
Flag-ADAR1 iCLIP technical replicate 1
processed data file: adar1_cluster.bed

source_name: Flag-ADAR1 iCLIP

tag: genotype/variation

value: ADAR1 KD H9 with expressing Flag-ADAR1

value: NNNGGTCNN


title: Flag-E912A iCLIP replicate 1

description:
Flag-E912A iCLIP technical replicate 1
processed data file: e912a_cluster.bed

value: NNNCGGANN


title: JAG1 knockout 2

tag: developmental stage

tag: tissue

value: descending aorta


title: Flag-ADAR1 iCLIP replicate 2

description:
Flag-ADAR1 iCLIP technical replicate 2
processed data file: adar1_cluster.bed


title: sRNA from WT Day 35 (Forebrain)

data_processing:
Basecalling using Illumina Casava1.8.2 software
Map to human genome hg19 using Tophat(v 2.0.8)
Bam to Bedgraph using genomeCoverageBed
Bedgraph to bigwig using UCSC bedGraphToBigWig
For each gene, we first merged all exons from different isoforms, then counted the mapping reads and normalized as RPKM values.
We use miRDeep2 pipeline to quantify miRNA expression. Briefly, small RNA-seq reads were trimmed to remove 3’ adapters, mapped to the genome (hg19), and piped up for miRNA expression (miRBase 18).
Genome_build: hg19
Supplementary_files_format_and_content: bigwig file (UCSC bigwig in an indexed binary format. http://genome.ucsc.edu/goldenPath/help/bigWig.html)
Supplementary_files_format_and_content: tab-delimited text files include RPKM values for each gene and RPM values for each miRNA

source_name: embryonic stem cells

growth_protocol: H9 hESCs were maintained on irradiated-MEF feeder cells and induced to differentiate into the region-specific neural cells according to two distinct protocols. Briefly, for forebrain neural cells induction, hESCs were detached from MEF cells and suspended in hESCs medium without FGF2 for 4 days in Petri dish. After the aggregates floated in neural induction medium (NIM) consisting of DMEM/F12, N2 supplement, 1xNEAA and heparin for additional 2-3 days, the aggregates were induced to adhere to laminin-coated substrate, and primitive neuroepithelial (NE) would be observed on days 8-10, followed by the definitive NE cells in the center of colonies occurring at days 14-17 of differentiation. The definitive NE were gently blown off (day 17) and suspended in NIM for one week and then replated on laminin-coated substrate for terminal differentiation. For motor neuron generation, primitive NE at day 10 was administrated with RA (0.1 μM) for caudalization. After isolation of definitive NE, RA, purmorpahmine and B27 supplement were added to NIM for ventralization. Samples from day 0 (d0), day 10 (d10), day 17 (d17) and day 35 (d35) were collected for subsequent analyses.

extract_protocol:
Total RNAs from cultured cells (0.5 x 106 - 1 x 107) were extracted with Trizol (Invitrogen) according to the manufacturer’s instruction, followed by DNase I treatment at 37 °C for 30 mins (Ambion, DNA-freeTM Kit). The polyadenylated RNA population was purified by oligo(dT) magnetic beads (Invitrogen) from 5 μg of total RNAs.
Library was prepared by using Illumina TruSeq Stranded Total RNA HT Sample Prep Kit (P/N 15031048) and Illumina TruSeq Small Sample Prep Kit (P/N 15004197).

tag: strain

tag: differential stage

value: day 35 to FB

tag: knockdown

value: wild type


title: sRNA from ADAR1 KD Day 35 (Motor Neuron)

value: day 35 to MN

value: ADAR1 shRNA


title: sRNA from ADAR1 KD Day 17 (Forebrain)

value: day 17 to FB


title: sRNA from WT Day 35 (Motor Neuron)


title: sRNA from ADAR1 KD Day 17 (Motor Neuron)

value: day 17 to MN


title: sRNA from ADAR1 KD Day 0

value: day 0


title: sRNA from ADAR1 KD Day 35 (Forebrain)


title: sRNA from WT Day 17 (Motor Neuron)


title: P5424 H3K4me2 (KM) ChIP-Seq

tag: cells used

tag: antibody vendor/catalog#

value: Abcam (ab32356)

tag: antibody quantity

value: 3 µg

tag: magnetic beads

value: Protein G dynabeads

tag: bead volume

tag: estimated fragment size


title: sRNA from WT Day 17 (Forebrain)


title: sRNA from WT Day 10

value: day 10


title: mRNA from ADAR1 KD Day 35 (Forebrain)


title: P5424 RNA Pol II (KM) ChIP-Seq

source_name: P5424 RNA Pol II (KM)

value: Santa Cruz (sc-899x)

tag: antibody

value: RNA Pol II

value: 10 µg

tag: cell line


title: mRNA from ADAR1 KD Day 35 (Motor Neuron)


title: P5424 RNA Pol II (DMSO) ChIP-Seq

source_name: P5424 RNA Pol II (DMSO)


title: sRNA from WT Day 0


title: mRNA from ADAR1 KD Day 17 (Forebrain)


title: mRNA from ADAR1 KD Day 10


title: mRNA from WT Day 17 (Motor Neuron)


title: mRNA from WT Day 35 (Forebrain)


title: mRNA from WT Day 35 (Motor Neuron)


title: mRNA from WT Day 17 (Forebrain)


title: mRNA from ADAR1 KD Day 17 (Motor Neuron)


title: mRNA from ADAR1 KD Day 0


title: mRNA from WT Day 10


title: bisulfite treated genomic DNA from old sun protected epidermis 3

city: Baltimore

first: Andrew

last: Feinberg

state: Maryland

address: 855 N. Wolfe St

institute: Johns Hopkins University School of Medicine

data_processing:
Alignment performed using BSmooth bisulfite alignment pipeline (version 0.4.5-beta), Bowtie2 version 2.0.1
Filtered measurments with mapping quality <20 or nucleotide base quality <10, removed measurements from 5' most 10 nucleotides of both mates.
Genome_build: Hg19
Supplementary_files_format_and_content: Methylation measurements smoothed using Bsseq (height of 20,000).Columns are labelled by sample, chromosome and location information in "chr", "start", "end" columns.

source_name: old sun protected epidermis 3

extract_protocol:
Epicentre Masterpure Genomic DNA Extraction
Bisulfite converted Illumina TruSeq DNA library

tag: gender

value: Male

tag: age

tag: exposure

value: sun protected

value: dermis


title: bisulfite treated genomic DNA from old sun exposed epidermis 2

source_name: old sun exposed epidermis 2

value: sun exposed


title: sRNA from ADAR1 KD Day 10


title: bisulfite treated genomic DNA from old sun exposed epidermis 3

source_name: old sun exposed epidermis 3


title: bisfulfite treated genomic DNA from old sun exposed epidermis 1

source_name: old sun exposed epidermis 1


title: bisfulfite treated genomic DNA from old sun protected epidermis 1

source_name: old sun protected epidermis 1


title: bisulfite treated genomic DNA from young sun protected epidermis 2

source_name: young sun protected epidermis 2


title: bisfulfite treated genomic DNA from old sun protected epidermis 2

source_name: old sun protected epidermis 2


title: bisfulfite treated genomic DNA from young sun exposed epidermis 2

source_name: young sun exposed epidermis 2


title: bisfulfite treated genomic DNA from young sun protected epidermis 1

source_name: young sun protected epidermis 1


title: bisulfite treated genomic DNA from young sun exposed epidermis 3

source_name: young sun exposed epidermis 3


title: bisulfite treated genomic DNA from young sun protected epidermis 3

source_name: young sun protected epidermis 3


title: bisfulfite treated genomic DNA from young sun exposed epidermis 1

source_name: young sun exposed epidermis 1


title: Input_10ng_BisSeq

city: Bethesda

middle: J

last: Oler

state: MD

address: 31 Center Drive, Room 3B62E

department: Bioinformatics and Computational Biosciences Branch (BCBB)

institute: NIAID/NIH

data_processing:
Library strategy: ChIP-seq protocol with additional bisulfite conversion step (ChIP-Bis-seq)
Basecalls performed using CASAVA version 1.7
Reads were aligned with Novoalign V2.07.09 using paired-end (-FILMFQ -t120 -h120 -b2 -a -i PE 20-600) or single-end settings (-FILMFQ -t120 -h120 -b2 -a).  A Novoalign index was created for the hg18 genome (UCSC), plus the Lambda genome (Genbank accession number J02459.1) and adapter sequences (Illumina PE PCR Primer 2.0: CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCT) with these options: novoindex -k 13 -s 3 -b.  Repeat alignments intersecting at least one uniquely aligned sequence realigned using single-end settings, allowing up to 7 alignments (-r A 7), which includes 85% of the repeat reads.  Reads from 8082X1_110523_SN141_0354_BB0236ABXX_3_1 were aligned as single-end as there were HiSeq issues with the second read; reads in 8082X1_110523_SN141_0354_BB0236ABXX_3_2 were not aligned.
The rates of conversion of methylated cytosines in the reads aligning to Lambda were 99.87% and 99.64% for input and pol III libraries, respectively, as determined by NovoalignBisulfiteParser (USeq package).
Analysis of aligned data was performed with the USeq package of programs (useq.sourceforce.net).  For ChIP-seq analysis, alignments were converted to single-position point data with NovoalignParser (with no quality filter, to include repeat alignments) and peaks were called with ScanSeqs (using peakshift of 116bp, as determined by PeakShiftFinder, and 300bp window) and EnrichedRegionMaker with thresholds of 20 or 70, 13, and 1 for Q-value FDR, Empirical FDR, and log2 ratio, respectively.  To obtain a list of enriched pol III-transcribed genes (including SINEs), pol III-enriched regions (Q-value 20 threshold) were intersected with an annotation file containing all SINEs, tRNA genes and fragments, and other pol III-transcribed genes and elements from the RepeatMasker track of UCSC.  The annotated genes/repeat elements were scored by DefinedRegionScanSeqs using the PointData and Q-value 70 threshold was used.   The number of enriched genes by class are 1472 Alu, 242 tRNA, 83 MIR, 7 U6, 4 hY, 3 7SL/SRP, 3 HVG, 2 rRNA, 2 miRNA, one RNaseP, one MRP, one BC200, and one 7SK to a total of 1822 genes.
For methylation analysis, alignments were converted to per-base methylated and unmethylated cytosines with NovoalignBisulfiteParser (with no quality filter to include repeat alignments).  Regions statistically enriched or reduced for methylation were determined with BisSeq (using settings -w 10 -m 10 -f 13 -l 0).  Percent methylation scores for enriched pol III target genes (including SINEs) was obtained using ScoreMethylatedRegions.
Genome_build: hg18
Supplementary_files_format_and_content: .xls file contains output of ChIP-seq and bisulfite sequencing analysis.

growth_protocol: All samples were from the same batch of cross-linked HeLa cells, grown to 75% confluence in T75 flasks with DMEM +10% (v/v) FBS, 10 mM glutamine in standard growth conditions.  Cells were cross-linked in 1% formaldehyde for 30 minutes.

extract_protocol: Lysates were clarified from sonicated nuclei and protein-DNA complexes were isolated with antibody. About 20-40 x 10^6 cells were used per immunoprecipitation reaction. DNA was purified from protein-DNA complexes (10 ChIP reactions pooled) and ChIP-seq library was prepared using Illumina ChIP-seq library protocol with the addition of bisulfite conversion after addition of adapters but before amplification.  In parallel, DNA was purified from nuclear sonicate and two input libraries were prepared: 1) starting with 10ng, using ChIP-seq library protocol with the addition of bisulfite conversion after addition of adapters but before amplification, and 2) standard Illumina Bisulfite DNA library protocol. All libraries included addition of 2% sheared Lambda DNA to control for bisulfite conversion.  The Qiagen EpiTect kit was used for bisulfite conversion, with four periods of incubation at 60°C (for 25, 85, 175, and 120 min), each preceded by denaturation for 10 min at 98°C.  Size selection was not performed for the amplified libraries (except the standard Input library) due to the small insert size, and the small  amount of total library after amplification.

value: HeLa cervical carcinoma cells

tag: chip antibody

tag: starting library amount


title: Rat non-pineal mixed tissue - ZT7 PolyA

first: Steven

last: Coon

address: 49 Convent Drive Room 6A83

department: NICHD/PDEGEN

institute: NIH

description: Total RNA was extracted from each tissue, and then equal amounts of each of the 15 tissues were combined for the final pooled sample.

data_processing:
Image analysis and base calling was done by Illumina Genome Analyzer Pipeline software (GAPipeline) with default parameters.
RNA-Seq reads were aligned to the rn4 genome assembly using Tophat-1.3.1.Linux_x86_64
FPKM values were calculated using Cufflinks-1.2.0.Linux_x86_64
Genome_build: rn4
Supplementary_files_format_and_content: Files include FPKM data

organism: Rattus norvegicus

source_name: mixed tissues

extract_protocol:
Total RNA was extracted with Trizol, followed by clean-up using RNeasy Micro kit with on-column DNase treatment. Quantitation and quality assuance were performed using a NanoDrop and Bioanalyzer, respectively.
The Illumina standard kit was used whereby 10 µg of total RNA was polyA mRNA enriched using streptavidin-coated magnetic beads, followed by chemical mRNA fragmentation. Fragmented mRNA was converted to cDNA using reverse transcriptase (SuperScript II) and random primers. The cDNA was further converted into double stranded cDNA and, after an end repair process (Klenow fragment, T4 polynucleotide kinase and T4 polymerase), was finally ligated to Illumina paired end (PE) adaptors. Size selection was performed using a 2% agarose gel. Libraries were enriched using 15 cycles of PCR and purified by the QIAquick PCR purification kit (Qiagen). The enriched libraries were diluted with Elution Buffer to a final concentration of 10 nM. Each library was run at a concentration of 7 pM on one Illumina Genome Analyzer II (GAii) lane using paired-end 51 base-pair sequencing. Image analysis and base calling was done by Illumina Genome Analyzer Pipeline software (GAPipeline) with default parameters.

treatment_protocol: Rats were housed in 14:10 lighting cycle

value: Sprague-Dawley

value: Adult

tag: tissue

value: cortex, cerebellum, midbrain, hypothalamus, hindbrain, spinal cord, retina, pituitary, heart, liver, lung, kidney, skeletal muscle, small intestine, adrenal gland

tag: sampling time

value: ZT7 (mid-day)


title: Input_standard_Illumina_BisSeq

data_processing:
standard Illumina Bisulfite DNA library protocol
Basecalls performed using CASAVA version 1.7
Reads were aligned with Novoalign V2.07.09 using paired-end (-FILMFQ -t120 -h120 -b2 -a -i PE 20-600) or single-end settings (-FILMFQ -t120 -h120 -b2 -a).  A Novoalign index was created for the hg18 genome (UCSC), plus the Lambda genome (Genbank accession number J02459.1) and adapter sequences (Illumina PE PCR Primer 2.0: CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCT) with these options: novoindex -k 13 -s 3 -b.  Repeat alignments intersecting at least one uniquely aligned sequence realigned using single-end settings, allowing up to 7 alignments (-r A 7), which includes 85% of the repeat reads.  Reads from 8082X1_110523_SN141_0354_BB0236ABXX_3_1 were aligned as single-end as there were HiSeq issues with the second read; reads in 8082X1_110523_SN141_0354_BB0236ABXX_3_2 were not aligned.
The rates of conversion of methylated cytosines in the reads aligning to Lambda were 99.87% and 99.64% for input and pol III libraries, respectively, as determined by NovoalignBisulfiteParser (USeq package).
Analysis of aligned data was performed with the USeq package of programs (useq.sourceforce.net).  For ChIP-seq analysis, alignments were converted to single-position point data with NovoalignParser (with no quality filter, to include repeat alignments) and peaks were called with ScanSeqs (using peakshift of 116bp, as determined by PeakShiftFinder, and 300bp window) and EnrichedRegionMaker with thresholds of 20 or 70, 13, and 1 for Q-value FDR, Empirical FDR, and log2 ratio, respectively.  To obtain a list of enriched pol III-transcribed genes (including SINEs), pol III-enriched regions (Q-value 20 threshold) were intersected with an annotation file containing all SINEs, tRNA genes and fragments, and other pol III-transcribed genes and elements from the RepeatMasker track of UCSC.  The annotated genes/repeat elements were scored by DefinedRegionScanSeqs using the PointData and Q-value 70 threshold was used.   The number of enriched genes by class are 1472 Alu, 242 tRNA, 83 MIR, 7 U6, 4 hY, 3 7SL/SRP, 3 HVG, 2 rRNA, 2 miRNA, one RNaseP, one MRP, one BC200, and one 7SK to a total of 1822 genes.
For methylation analysis, alignments were converted to per-base methylated and unmethylated cytosines with NovoalignBisulfiteParser (with no quality filter to include repeat alignments).  Regions statistically enriched or reduced for methylation were determined with BisSeq (using settings -w 10 -m 10 -f 13 -l 0).  Percent methylation scores for enriched pol III target genes (including SINEs) was obtained using ScoreMethylatedRegions.
Genome_build: hg18
Supplementary_files_format_and_content: .xls file contains output of ChIP-seq and bisulfite sequencing analysis.

value: standard Illumina protocol (1ug)


title: Rat pineal gland– ZT7 – PolyA

source_name: Pineal gland

tag: strain

value: pineal gland


title: Rat pineal gland– ZT19 – PolyA

value: ZT19 (midnight)


title: dim-3_min_Ncrassa_BSseq

status: Public on Mar 22 2015

city: Colorado Springs

middle: David

last: Klocko

state: Colorado

address: 278 Centennial Hall, 1420 Austin Bluffs Pkwy

department: Chemistry and Biochemistry

institute: University of Colorado Colorado Springs

data_processing:
The BRAT-BW software package (compbio.cs.ucr.edu/brat/; Harris et al, 2012) was used to prepare and map the reads to the N. crassa OR74A (annotation NC12) genome, which was converted to a four stranded reference genome to permit bisulfite mapping.
BRAT-BW acgt-count “-B” option cytosine-only files produced for the forward and reverse strand reads were merged.
A python script (bidensity) was written to calculate the average 5mC level over a 500bp sliding window across the genome, producing a .wig file
.wig files were displayed on IGV (Integrative Genomics Viewer; Broad Institute, Robinson et al, 2011)
Genome_build: Ncrassa version12
Supplementary_files_format_and_content: .wig files show the ratio (0-1.0; 0 is DNA that is not cytosine methylated, 1.0 is DNA where all of the cytosines are methylated) of methylated cytosines to unmethylated cytosines as peaks of cytosine methylation enrichment

organism: Neurospora crassa

source_name: N2108 dim-3 Ncrassa (Selker)

growth_protocol: grown under standard dark cycle in 1xVogels medium + 1.5% sucrose (minimum medium) or minimum medium supplemented with histidine (0.5mg/mL [final]) for 48 hours on rocking shaker at 32oC

extract_protocol:
Genomic DNA was isolated using a protocol modified from (Oakley et al, 1987).  Genomic DNA (50ng) was treated with bisulfite using the EZ DNA Methylation LightningTM kit (ZYMO Research) per the manufacturer's protocol.
Libraries were prepared for sequencing with the EpiGnomeTM Methyl-Seq Kit (Epicentre cat #EGMK91324) and EpiGnomeTM Index PCR Primers (Epicentre cat#  EGIDX81312) per the manufacturer's protocol, purified with Agencourt AMPure XP beads (Beckman Coulter) per the manufacturer's protocol, Qubit HS Assay quantified (Life Technologies) per the manufacturer's protocol, visualized on the Fragment AnalyzerTM (Advanced Analytical) per the manufacturer's protocol, and sequenced on a Illumina HiSeq 2000

tag: time point in light cycle

tag: growth medium

value: 1xVogels, 1.5% sucrose (minimum)

tag: bisulfite treated

value: bisulfite


title: WT H3K9me3 ChIPseq

data_processing:
raw data .fastq files were mapped to the Ncrassa10 genome using Bowtie2, generating .sam files
using samtools, .sam files were converted to .bam files, duplicates were removed (rmdup), and the .bam file was sorted
the sorted .bam file was indexed, and then used for building the .tdf file on igvtools (below)
Genome_build: Ncrassa version10
Supplementary_files_format_and_content: .tdf files were generated using the "count" function in igvtools (Integrative Genomics Viewer; Broad Institute, Robinson et al, 2011), using a window size of 200bp, showing peaks of H3K9me3 enrichment

source_name: N3752 (WT) Selker lab

growth_protocol: grown under standard dark cycle in 1xVogels medium + 1.5% sucrose (minimum medium) for 16 hours on rocking shaker at 32oC

extract_protocol:
Lysates were generated from sonicated wet tissue, and histone-DNA complexes were isolated with H3K9me3 antibody
Libraries were prepared according to Illumina's standard protocol (TruSeq kit, ref# IP-202-1012)

value: wildtype

tag: time point in light

value: 1x Vogels, 1.5% sucrose

value: Active Motif Cat# 39162 (H3K9me3)


title: RPC155_ChIP_BisSeq

value: RPC155 (serum 1900, Bob White lab)


title: MISO_0014

status: Public on Mar 21 2015

city: Toronto

first: Shirley

last: Tam

state: Ontario

address: 661 University Avenue, Suite 510, MaRS Building, West Tower

department: Genome Technologies

country: Canada

institute: Ontario Institute for Cancer Research

description: Raw sequence reads were aligned using multiple alignment algorithms and parameter settings.  The processed data file supplied represents the miRNA count data derived from alignment using BWA with no mismatches allowed in the seed region.  Other alignment outputs can be generated from the raw fastq files using the specified parameters listed in the data processing section.

data_processing:
Base-calling was performed using CASAVA v1.1.  Short read sequences were output in FASTQ format with corresponding base quality scores.  The raw data was initially filtered for reads containing ambiguous base calls, which did not meet the Illumina chastity filter based on quality measures.  Quality control of the remaining sequences from each sequenced library was investigated using FastQC (v0.11.2) to check for homopolymers, adapters, and distribution of base quality.  The reads were then filtered for low quality reads, contaminating 5’ adapters, homopolymers, and trimmed for 3’ adapters.  The pre-processed reads were aligned in a sequential manner: first to ribosomal RNA/repeats, miRBase v20, RefSeq, and finally to a genomic reference (hg19).
Several general-purpose aligners were evaluated, including BWA, Bowtie, Bowtie 2 and Novoalign.   Sequence alignment was performed with the following parameters for each aligner, respectively:
1.   BWA 0.7.4 : bwa aln -n 1 -o 0 -e 0 -k 1 -t 4
2.   BWA 0.7.4 (0 mismatch in seed): bwa aln -n 1 -o 0 -e 0 -l 8 -k 0 -t 4
3.   Bowtie 0.12.9 : bowtie -n 1 -l 8 -a --best --strata --phred33-quals
4.   Bowtie 0.12.9 (0 mismatch in seed): bowtie -n 0 -l 8 -a --best --strata --phred33-quals
5.   Bowtie2 2.1.0 : bowtie2 --local -p 8 -q --phred33 -D 20 -R 3 -N 0 -L 8 -i S,1,0.50
6.   Novoalign 3.00.05: novoalign -a TGGAATTCTCGGGTGCCAAGG -l 15 -t 30 -r A
Only reads uniquely aligned to miRNAs were retained and counted.  The miRNAs were filtered for targets with a minimum of 5 counts in at least 25% of the samples.
Genome_build: hg19
Supplementary_files_format_and_content: Tab-delimited text file contains raw counts for each aligned miRNA.

source_name: Universal Human Reference RNA

extract_protocol:
Universal Human Reference RNA was obtained from Agilent Technologies.  Twelve miRNAs from the Arabidopsis Thaliana genome were spiked into 1μg of this reference RNA at 12 different concentrations.  The samples were subjected to all experimental steps, including small RNA purification (PureLink miRNA Isolation kit, Life Technologies).
Libraries were prepared from fractionated RNA samples as per Illumina's TruSeq Small RNA protocol (version D) (Illumina, San Diego, CA, USA).

tag: sample type

value: reference


title: MISO_0013


title: MISO_0017


title: MISO_0016


title: dim-3 H3K9me3 ChIPseq

source_name: N2108 (dim-3) Selker lab


title: mRNA from WT Day 0

source_name: embryonic stem cells

tag: differential stage

value: day 0

tag: knockdown

value: wild type


title: MISO_0015


title: MISO_0011


title: MISO_0009


title: MISO_0008


title: MISO_0007


title: MISO_0006


title: Control tumor 1

status: Public on Mar 20 2015

first: Nienke

last: de Vries

institute: NKI

data_processing:
Sequence reads were mapped to mm9 using tophat version 1.4.0 with parameters --library-type fr-unstranded, -M, --transcriptome-index, Ensembl GTF version 66
Readcounts were obtained by counting the number of reads per gene using HTSeq-count.
Genome_build: mm9
Supplementary_files_format_and_content: The file gene_exp.txt contains the absolute gene expressions.

source_name: Control tumor

growth_protocol: high-grade gliomas developed in mice after intracranial injections of GICs

extract_protocol:
Total RNA was extracted using TRIzol Reagent (Invitrogen) and submitted to next generation sequencing (Illumina HiSeq2000).
RNA libraries were prepared for sequencing using standard Illumina TruSeq RNA Sample Prep protocols.

treatment_protocol: doxycycline hyclate (Sigma) administered at 2mg/ml supplemented with 1% sucrose in the drinking water

tag: strain source

value: p53;Ink4a/Arf;Krasv12;LucR compound conditional mice

tag: source cell type

value: SVZ derived neural stem cells (NSCs)

tag: cell type

value: glioma initiating cells (GICs)

tag: infected with

value: a tet-inducible, doxycycline-responsive short hairpin construct (FH1-tUTG-shEzh2)

tag: host strain

value: NOD-SCID mice

value: control tumor without Ezh2 knockdown


title: Control tumor 2


title: Ezh2 KD tumor 7

source_name: Ezh2 KD tumor

value: tumor with prolonged Ezh2-knockdown


title: Ezh2 KD tumor 8


title: Ezh2 KD tumor 9


title: Ezh2 KD tumor 6


title: dim-3_+his_Ncrassa_BSseq

value: 1xVogels, 1.5% sucrose+Histidine (0.5mg/mL final)


title: Ezh2 KD tumor 5


title: Ezh2 KD tumor 4


title: Ezh2 KD tumor 3


title: Ezh2 KD tumor 2


title: Ezh2 KD tumor 1


title: MISO_0012


title: MISO_0010


title: d2PGCLC_H3_1

city: Kyoto

first: Yukihiro

last: Yabuta

state: Kyoto

address: Yoshida-Konoe-cho, Sakyo-ku

department: Anatomy and Cell Biology

country: Japan

institute: Kyoto University, Graduate school of medicine

data_processing:
Read data were mapped on mouse mm9 genome by bowtie v0.12.9 with "-n 3 -m 1" option.
Mapped reads were sorted and format converted using samtools v0.1.18 and picard-tools v1.85.
Tdf files are made using igvtools v2.3.5.
Peak calling for EGFP-BLIMP1, T, H3K4me3, and H3K27ac were performed using MACS v1.4.2 with default setting.
To enable comparison of peaks in different samples, we scanned all peaks throughout the genome, and considered peaks detected in proximities (within 1 Kb) as a single peak.
Genome_build: mm9
Supplementary_files_format_and_content: Tdf files are made using igvtools v2.3.5 with "-w 25 -e 250" setting
Supplementary_files_format_and_content: Bed files of H3K27me3 and H3K9me2 were made by counting fpkm in 2kb-bin at every 1kb on the genome. Fpkm values were divided by that of input value and subsequently normalized by QPCR data.
Supplementary_files_format_and_content: Bed files for H3K27ac_all_peaks and H3K4me3_all_peaks were made as follows: we scanned all peaks in all samples throughout the genome, and considered peaks detected in proximities (within 1 Kb) as a single peak to be able to compare read  intensity among samples.

source_name: PGCLC 2days

growth_protocol: BVSC and EGFP-Blimp1 knock-in ESC lines were induced into EpiLCs as described for 48 hrs (BVSC) or 36 hrs (EGFP-Blimp1 knock-in).  The EpiLCs were then cultured under a floating condition by plating 2-3×103 cells per well of lipidure-coated U-bottom 96-well plate (Thermo Scientific) in the GK15 medium containing LIF (1000 U/mL) for induction of d2 LIF aggregate (d2 LIF Ag), LIF and BMP4 (500 ng/mL) for d2 PGCLCs, and LIF, BMP4, BMP8a (500ng /mL), SCF (100 ng/mL), and EGF (50 ng/mL) for d6 PGCLCs, respectively.

extract_protocol:
Cells were suspended in 1 mL of phosphate buffered saline (PBS) and fixed by adding 27 mL of 36.5% formalin (Sigma) for 10 min at room temperature.  Fixation was terminated by adding 110 mL of 1.5 mM glycine.  The fixed cells were washed twice in PBS containing 0.1% bovine serum albmin (BSA), and were lysed in 400 mL SDS-lysis buffer [50 mM Tris-HCl (pH 8.0) containing 1% SDS, 10 uM EDTA, and 1 mM PMSF] on ice for 10 min.  Chromatins were then solubilized using Bioruptor UCD250 with ice-chilled water, with 15 cycles of 30 sec sonication at the high power and 60 sec interval.  The sonication products were centrifuged at 16,000 g for 5 min.  The supernatants were diluted in 1 mL ChIP dilution buffer [16.7 mM Tris-HCl (pH 8.0), 0.01% SDS, 1.1% Triton X100, 1.2 mM EDTA, 167 mM NaCl, 1 mM SDS, 200 mg/mL tRNA (Roche)], and were divided into 0.1 mL as input DNA and 1.3 mL as ChIP samples.  The ChIP samples were mixed with the Dynabeads-antibody complexes and rotated at 4 ºC overnight.  The ChIP-ed Dynabeads were recovered using the DynaMag-2 magnet (Life Technologies), and washed with 200 mL of low salt buffer [20mM Tris-Cl (pH 8.0), containing 0.1% SDS, 1% Triton X100, 2 mM EDTA, and 150 mM NaCl], high salt buffer [20mM Tris-Cl (pH 8.0), containing 0.1% SDS, 1% Triton X100, 2 mM EDTA, and 500 mM NaCl], RIPA buffer [10mM Tris Cl (pH8.0), containing 1% Sodium deoxycholate, 1% NP40, 250 mM LiCl, and 1 mM EDTA], and TE buffer [10 mM Tris Cl (pH8.0), containing 1 mM EDTA].  The ChIP-ed Dynabeads were then incubated in new tubes containing 50 mL of Elution buffer (0.1 mM NaHCO3, 1% SDS, 10 mM DTT, and 0.06 mM tRNA) for 15 min at room temperature.  The supernatants were collected using DynaMag-2, and were reverse-crosslinked by adding 8 mL of 2.5 M NaCl and incubating at 65 ºC overnight.  Then, 2 mL of 0.5 M EDTA and 4 mL of 0.645 mg/mL proteinase K solution were added to reaction mixture, and further incubated at 45 ºC for 1 hr.  The ChIP-ed DNAs were then purified with Qiaquick PCR purification columns, using the buffer EB supplemented with 4 ng/mL tRNA (Qiagen).
The ChIP-ed and Input DNAs were sheared to an average size of about 150 bp by ultra-sonication (Covaris, Woburn, MA).  ChIP-ed DNAs obtained from 1×106 cells were then subjected to the procedures for SOLiD Fragment Library Preparation (Life Technologies) according to the manufacture’s instruction except for the ligation reaction that was performed overnight.  The libraries were amplified with 11-13 cycles of PCR for H3K4me3 and H3K27ac or 8 cycles for H3K9me2 and H3K27me3.
ChIP-ed DNA for histone modifications from 1×105 cells were subjected to the procedures for SOLiD Fragment Library Preparation with a modification where the size fractionation step using AMPure XP beads (Agencourt) was omitted and ligation reaction was performed overnight.  The libraries were amplified with 12-16 cycles of PCR for H3K27ac or 8 cycles for H3K9me2 and H3K27me3.
We developed a new method to prepare and precisely amplify libraries of ChIPed DNA from a small number of cells, and applied this method to H3K4me3 from 1 x 105 cells and transcription factors from 3×105 ~ 1×106 cells.  A modified adaptor for SOLiD library was prepared by annealing 50 mM each of P1-T Adaptor/F (CCACTACGCCTCCGCTTTCCTCTCTATGGGCAGTCGGTGAT) and Barcode-Internal+12mer/R (TCACCGACTGCCACGCCTTGGCCGTACAGCAGCCTCTTACACAGAG, with Phosphorylated 5'-end) in 1×Linker buffer [50 mM Tris-HCl (pH 8.0) containing 100 mM NaCl and 1 mM EDTA] with a step-wise cooling procedure (95 ºC, 70 ºC, 50 ºC, 40 ºC and 25 ºC for 15 min, respectively).  The sheared DNAs were end-repaired using End-It DNA End-Repair Kit (Epicentre) in a 120 mL scale according to the manufacture’s instruction, and purified using MinElute PCR purification kit (Qiagen) with 540 mL of binding buffer PB3 (5.6 M Gu HCl, 33.3% isopropanol) supplemented with 10 ng/mL tRNA and 10 mL of elution buffer EB (Qiagen) with 4 ng/mL tRNA.  The end-repaired DNAs were incubated in 20 mL of dA tailing mixture [0.2 mM dATP (GE Healthcare), 1×High Yield buffer (Greiner), 4 ng/mL tRNA 2.5 unit of Taq DNA polymerase (Greiner)], for 10 min at 70 ºC, and purified using MinElute PCR purification kit with buffers PB (Qiagen) and EB (11 mL) supplemented with tRNA.  The dA-tailed DNAs were incubated in 15 mL ligation mixture [1×DNA Ligase Reaction Buffer (Life Technologies), 0.1 mM the modified adaptor, and 2.5 unit ExpressLink T4 DNA Ligase (Life Technologies)] at 16 ºC for more than 22 hr.  The adaptor-ligated DNAs were then amplified using the Platinum Taq DNA Polymerase in a 50 mL reaction scale (Life Technologies) with 1 mM each of Library PCR primer 1 (Life Technologies) and Library PCR primer Barcode001+Internal adaptor (CTCTGTGTAAGAGGCTGCTGTACGGCC).  PCR was performed with 8 cycles for ChIP-ed DNAs of H3K4me3 and with 10 cycles for those of EGFP-BLIMP1.  PCR products were purified using 50 mL AMPure XP beads supplemented with 3.33 mL of 25% PEG8000 and 13.7 mL of 2.5 M NaCl, and eluted in 30 mL of buffer EB supplemented with tRNA.  The PCR products were then subjected to another 5-cycle PCR using 1 mM each of Library PCR Primer 1 and P2-Barcode-N-Internal primer [CTGCCCCGGGTTCCTCATTCTCT (N10) CTGCTGTACGGCCAAGGCGT, where N10 corresponds to SOLiD barcode sequences other than barcode 1; e.g., AGGGAGTGGT for barcode 2], and purified twice with AMPure XP beads supplemented with PEG8000 and NaCl.

treatment_protocol: d2 and d6 PGCLCs of the BVSC line were purified with Fluorescence-activated cell sorter (FACS) (ARIA III; BD Biosciences) by fluorescence of BV.  For the EGFP-BLIMP1 knock-in line, d2 PGCLCs were purified with FACS using anti-Kit antibody and d6 PGCLCs using anti-SSEA1 and anti-Integrin b3 antibodies.

tag: chip antibody

value: anti H3 (Abcam, ab1791)

tag: genotype

value: Blimp1-mVenus reporter (BV),Prdm14-mVenus reporter (PV)


title: d6PGCLC_H3K9me2_2

source_name: PGCLC 6days

value: anti H3K9me2, MsIgG, CMA317


title: d6PGCLC_H3_1


title: d2PGCLC_H3_2


title: d2PGCLC_H3K9me2_2


title: ESC_H3_1

source_name: ESC


title: EpiLC_H3_2

source_name: EpiLC


title: EpiLC_H3_1


title: d6PGCLC_H3K9me2_1


title: d6PGCLC_H3_2


title: EpiLC_H3K9me2_2


title: ESC_H3_2


title: EpiLC_H3K9me2_1


title: d2PGCLC_H3K9me2_1


title: MS04T61

description: SC3seq amplified from single cell of E4.5 blastocysts

data_processing:
All the reads were surveyed and the adaptor or the poly-A sequences were trimmed by cutadapt-1.3 with options "-c -m 30 -a CTCGAGGGCGCGCCGGATCCATATACGCCTTGGCCGTACAGCAG -g CTCGAGGGCGCGCCGGATCCATATACGCCTTGGCCGTACAGCAG -a AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA". The trimmed reads with less than 30 bp were discarded.
Untrimmed and trimmed reads of 30 bp or longer were mapped onto the mouse genome mm10 and the ERCC spike-in RNA sequences with tophat-1.4.1/bowtie1.0.1 with the “—no-coverage-search” option.
Mapped reads on the genome and the ERCC were separated, and the reads on the genome were converted into the expression levels by cufflinks-2.2.0 using the “—compatible-hits-norm”, “—no-length-correction” and “—library-type fr-secondstrand” options and mm10 reference gene annotations with extended TTSs. We also set the cufflinks option “—max-mle-iterations” to 50,000, because default iterations (5,000) resulted in “FAILED” when estimating the expression levels of some genes. For the reference gene annotations used in cufflinks, we extended the TTSs of the reference genes up to 10 kb downstream to correctly estimate the expression levels of genes whose transcripts are longer than the reference toward the 3 prime.
Genome_build: mm10
Supplementary_files_format_and_content: Tab-delimited text files with abundance measurements

source_name: Blastocyst

growth_protocol:
The mouse embryonic stem cell (mESC) line BVSC R8 was cultured in a 2i+LIF, feeder-free culture condition.
For the analysis of human iPSCs (hiPSCs), the two iPSC lines, 585A1 and 585B1, were cultured either under a conventional culture condition [DMEM/F12 [Life Technologies (11330-32), Carlsbad, CA] supplemented with 20% (vol/vol) Knockout Serum Replacement [KSR; Life Technologies (10828-028)], 1% (vol/vol) GlutaMax [Life Technologies (35050-061)], 0.1 mM nonessential amino acids [Life Technologies (11140-050)], 4 ng/ml recombinant human bFGF [Wako Pure Chemical Industries (064-04541), Osaka, Japan] and 0.1 mM 2-mercaptoethanol [Sigma-Aldrich (M3148)]] on the SNL feeder cells [Watanabe, K., et al. (2007). Nature Biotechnology, 25(6), 681.] or under a feeder-free condition as reported previously [Nakagawa, M., et al. (2014). Scientific Reports, 4, 3594.][Miyazaki, T., et al. (2012). Nature Communications, 3, 1236.].

extract_protocol:
Total RNAs from the mouse embryonic stem cell (mESC) line BVSC R8 were extracted using an RNeasy mini kit [Qiagen (74104), Hilden, Germany] according to the manufacturer’s instructions. The isolated RNAs were serially diluted by double-distilled water (DDW) to concentrations of 250 ng/μl, 25 ng/μl, 2.5 ng/μl, 250 pg/μl, and 25 pg/μl for use in evaluation of the quantitative performance of the SC3-seq.
For isolating mouse blastocysts, C57BL/6 mice were mated and noon of the day when a copulation plug was identified was designated as embryonic day (E) 0.5. At E4.5, peri-implantation blastocysts were flushed from the uteri by KSOM [Merck Millipore (MR-020P-5D), Darmstadt, Germany], and then they were bisected into a polar part containing an inner cell mass (ICM) and polar trophectoderm (pTE) and a mural part containing mural TE (mTE) by a glass needle under a dissection microscope [Leica Microsystems (M80), Wetzlar, Germany]. Each fragment was incubated with 0.25% trypsin/PBS [Sigma-Aldrich (T4799), St. Louis, MO] for around 10 min at 37°C, then dissociated into single cells by repeated pipetting, and dispersed in 0.1 mg/ml of PVA/PBS [Sigma-Aldrich (P8136)] in preparation for the SC3-seq analysis.
For the isolation of single hiPSCs cultured with the feeders, the culture was first treated with CTK solutions [0.25% Trypsin [Life Technologies (15090-046)], 0.1 mg/mL Collagenase IV [Life Technologies (17104-019)], 1 mM CaCl2 [Nacalai Tesque (06729-55)] (Fujioka, T., et al. (2004). Int.J.Dev.Biol., 48(10), 1149.)] for the removal of the feeder cells, then dissociated into single cells using Accutase [Innovative Cell Technologies, San Diego, CA]. For the preparation of single cells from a feeder-free system, the cells were dissociated into single cells with 0.5 × TrypLE Select [TrypLE Select [Life Technologies (12563011)] diluted 1:1 with 0.5 mM EDTA/PBS] [Nakagawa, M., et al. (2014). Scientific Reports, 4, 3594.]. Dissociated single hiPSCs were transferred several times into a pick-up medium consisting of 1% KSR/PBS containing 10 μM of the ROCK inhibitor Y-27632 [Wako Pure Chemical Industries (257-00511)] [Watanabe, K., et al. (2007). Nature Biotechnology, 25(6), 681–686.] in preparation for the SC3-seq analysis.
cDNA synthesis and amplification from isolated RNAs/single cells were perfomed essentially as reported in [Kurimoto, K.et.al., Nucleic Acids Research, 34(5), e42.] except that the spike-in RNAs developed by the External RNA Control consortium [ERCC; Life Technologies (4456740)] were used and different numbers of PCR cycles were employed for amplification depending on the amounts of starting total RNAs (total RNA 100 ng: 7 cycles; 10 ng: 11 cycles; 1 ng: 14 cycles; 100 pg: 17 cycles; 10 pg: 20 cycles). 5 ng of amplified and quality-checked cDNAs were added to the pre-amplification buffer [1 × ExTaq buffer [Takara Bio (RR006), Shiga, Japan], 0.2 μM of each dNTP [Takara Bio (RR006)], 0.01 μg/μl of the N-V3 (dT)24 primer (HPLC-purified, attachment of amine at the 5-prime end), 0.01 μg/μl of the V1(dT)24 primer (HPLC-purified), and 0.025 U/μl of ExTaqHS [Takara Bio (RR006)]], and were amplified by four cycles of PCR. The surplus primers and PCR byproducts such as primer dimers were removed by size selection through three rounds of purification using a 0.6 × volume of AMPureXP beads for each  round [Beckman Coulter (A63881), Tokyo, Japan] according to the manufacturer’s instructions. The purified cDNAs were diluted to 130 μl by double-distilled water (DDW) and fragmented by shearing with Covaris S2 or E210 (duty cycle: 10%; intensity: 5; cycles per burst: 200; duration: 50 seconds; an intensifier was installed in the case of E210) [Covaris, Woburn, MA], and then end-polished in the End-polish buffer [1 × NEBnext End Repair Reaction buffer [NEB (B6052S), Ipswich, MA], 0.01 U/μl of T4 DNA polymerase [NEB (M0203)], and 0.033 U/μl of T4 polynucleotide kinase [NEB (M0201)]] for 30 min at 20oC. After incubation, a 0.8 × volume of the AMPureXP was immediately added, the solution was mixed for more than 20 min, and then the supernatant was transferred to a 1.2 × volume of the AMPureXP reagent and the cDNAs were purified. Next, to provide the purified cDNAs with an Int-adaptor sequence, the cDNAs were incubated in 30 μl of the Internal adaptor extension buffer [1 × ExTaq Buffer, 0.23 mM of each dNTP, 0.67 μM of the IntV1 (dT)24 primer (HPLC-purified), 0.033 U/μl of ExTaqHS] using the following thermal cycler program: 95oC for 3 min, 67oC for 2 min, and 72oC for 2 min. The reactions were terminated by chilling in an ice-block, and after the addition of 20 μl of the P1-adaptor ligation buffer [a mixture of 10 μl of 5 × NEBNext Quick Ligation Reaction Buffer [NEB (B6058S)], 0.6 μl of 5 μM of the P1-T adaptor [Life Technologies (4464411)], and 1 μl of T4 ligase [NEB (M0202M)]], the solution was incubated for 15 min at 20oC and for 20 min at 72oC. After two rounds of cDNA purification by adding a 1.2 × volume of AMPure XP, the cDNAs were added into the Final amplification buffer [1 × ExTaq buffer, 0.2 mM of each dNTP, 1 μM of the P1 primer, 1 μM of the BarT0XX_IntV1 primer (HPLC-purified), 0.025 U/μl of ExTaqHS] and amplified by PCR using the following thermal cycler program: 95oC for 3 min; followed by 9 cycles of 95oC for 30 sec, 67oC for 1 min and 72oC for 1 min; with a final extension of 72oC for 3 min. Finally, the cDNA libraries were purified two times by using a 1.2 × volume of AMPureXP and dissolved in 20 μl of TE buffer. The quality and quantity of the constructed libraries were evaluated by LabChip GX or Bioanalyzer 2100, a Qubit dsDNA HS assay kit [Life Technologies (Q32851)], and a SOLiD Library TaqMan Quantitation kit [Life Technologies (4449639)]. The cDNA libraries were sequenced with an ABI SOLiD 5500XL system (Applied Biosystems) for 50-bp single-end reads according to the instructions of the manufacturer.

tag: tissue

value: Blastocyst

tag: developmental stage


title: ESC_H3K9me2_2

tag: strain


title: ESC_H3K9me2_1


title: MS04T60


title: MS04T59


title: MS04T58


title: MS04T63


title: MS04T57


title: MS04T53


title: MS04T56


title: MS04T55


title: MS04T52


title: MS04T48


title: MS04T50


title: MS04T64

description: SC3seq amplified from single cell of hiPSC on SNL

source_name: hiPSC on SNL

tag: cell line

value: hiPSC on SNL


title: MS04T43


title: MS04T47


title: MS04T38


title: MS04T40


title: MS04T39


title: MS04T41


title: MS04T42


title: MS04T36


title: MS04T37


title: MS04T34


title: MS04T49


title: MS04T30


title: MS04T62


title: MS04T35


title: MS04T31


title: MS04T28


title: MS04T29


title: MS04T23


title: MS04T27


title: MS04T22


title: MS04T24


title: MS04T21


title: MS01T32

description: SC3seq amiplified from 10 pg total RNA

value: BVSC R8

value: ESC


title: MS01T31

description: SC3seq amiplified from 10 pg total RNA


title: MS01T29

description: SC3seq amiplified from 10 pg total RNA


title: MS01T28

description: SC3seq amiplified from 10 pg total RNA


title: MS01T30

description: SC3seq amiplified from 10 pg total RNA


title: MS01T22

description: SC3seq amiplified from 10 pg total RNA


title: MS04T19


title: MS04T18


title: MS01T26

description: SC3seq amiplified from 10 pg total RNA


title: MS01T24

description: SC3seq amiplified from 10 pg total RNA


title: MS01T25

description: SC3seq amiplified from 10 pg total RNA


title: MS01T27

description: SC3seq amiplified from 10 pg total RNA


title: MS01T20

description: SC3seq amiplified from 10 pg total RNA


title: MS04T20


title: MS01T18

description: SC3seq amiplified from 10 pg total RNA


title: MS01T16

description: SC3seq amiplified from 100 pg total RNA


title: MS01T15

description: SC3seq amiplified from 100 pg total RNA


title: MS01T14

description: SC3seq amiplified from 100 pg total RNA


title: MS01T19

description: SC3seq amiplified from 10 pg total RNA


title: MS01T13

description: SC3seq amiplified from 100 pg total RNA


title: MS01T17

description: SC3seq amiplified from 10 pg total RNA


title: MS01T11

description: SC3seq amiplified from 100 pg total RNA


title: MS01T12

description: SC3seq amiplified from 100 pg total RNA


title: MS01T10

description: SC3seq amiplified from 100 pg total RNA


title: MS01T09

description: SC3seq amiplified from 100 pg total RNA


title: MS01T08

description: SC3seq amiplified from 1 ng total RNA


title: MS01T23

description: SC3seq amiplified from 10 pg total RNA


title: MS01T05

description: SC3seq amiplified from 1 ng total RNA


title: MS01T06

description: SC3seq amiplified from 1 ng total RNA


title: MS01T07

description: SC3seq amiplified from 1 ng total RNA


title: MS01T21

description: SC3seq amiplified from 10 pg total RNA


title: MS01T01

description: SC3seq amiplified from 100 ng total RNA


title: MS01T03

description: SC3seq amiplified from 10 ng total RNA


title: BVSC_input_d6PGCLC_1

description: BVSC_input_ESC, BVSC_input_d6PGCLC_1, BVSC_input_d6PGCLC_2 and BVSC_input_EpiLC(GSM1260395) are combined to make one BVSC_input control.

tag: genotype/variation


title: EGFP-BLIMP1_input

value: EGFP-Blimp1 knock-in


title: BVSC_input_d6PGCLC_2


title: d2PGCLC_EGFP-BLIMP1_1

value: antiGFP, RbIgG, Abcam ab290


title: BVSC_input_ESC


title: d6PGCLC_EGFP-BLIMP1_2


title: d2PGCLC_H3K27me3_2

value: anti H3K27ame3, MsIgG, CMA327


title: d6PGCLC_H3K27me3_1


title: d2PGCLC_H3K27me3_1


title: d2LIF_Ag_H3K27me3_1

source_name: EpiLC aggregated only with LIF, 2days


title: EpiLC_H3K27me3_1


title: d2LIF_Ag_H3K27me3_2


title: ESC_H3K27me3_2


title: d6PGCLC_H3K27ac_2

value: anti H3K27ac, MsIgG, CMA309


title: MS01T04

description: SC3seq amiplified from 10 ng total RNA


title: MS01T02

description: SC3seq amiplified from 100 ng total RNA


title: d6PGCLC_H3K27ac_1


title: d2PGCLC_H3K27ac_2


title: d2PGCLC_H3K27ac_1


title: d6PGCLC_EGFP-BLIMP1_1


title: d2PGCLC_EGFP-BLIMP1_2


title: d6PGCLC_H3K27me3_2


title: d2LIF_Ag_H3K27ac_1


title: EpiLC_H3K27ac_1


title: ESC_H3K27ac_2


title: d6PGCLC_H3K4me3_2

value: anti H3K4me3, MsIgG, CMA304


title: EpiLC_H3K27me3_2


title: d6PGCLC_H3K4me3_1


title: d2LIF_Ag_H3K4me3_1


title: d2PGCLC_H3K4me3_1


title: ESC_H3K27ac_1


title: d2PGCLC_H3K4me3_2


title: EpiLC_H3K4me3_2


title: MS04T95


title: MS04T92


title: ESC_H3K27me3_1


title: MS04T90


title: MS04T89


title: ESC_H3K4me3_1


title: ESC_H3K4me3_2


title: MS04T91


title: MS04T94


title: MS04T84

description: SC3seq amplified from single cell of hIPSC, feeder free

source_name: hIPSC, feeder free

value: hIPSC, feeder free


title: MS04T82


title: d2LIF_Ag_H3K27ac_2


title: d2LIF_Ag_H3K4me3_2


title: MS04T83


title: MS04T86


title: MS04T85


title: MS04T81


title: MS04T88


title: MS04T80


title: MS04T79


title: MS04T78


title: MS04T76


title: EpiLC_H3K4me3_1


title: MS04T75


title: MS04T70


title: MS04T73


title: MS04T69


title: MS04T71


title: MS04T72


title: MS04T67


title: MS04T68


title: MS04T66