title: BMDM miRNA seq KO4

city: New Haven

first: Yi-Chien

last: Lu

state: Connecticut

address: 10 Amistad Street

department: Laboratory Medicine

institute: Yale Medical School

description:
Small RNA
miRNAseq

data_processing:
Illumina Casava1.4 software used for basecalling on miRNA and mRNAseq data, CASAVA-1.8.2 software used for PARCLIP data.
Reads Per Kilobase of exon per Megabase of library size (RPKM) were calculated by using GobyWeb project.
MicroRNA sequencing results were trimmed and mapped to miRBase 855 mouse stem-loop sequences (version 19) using the Bowtie alignment program (version 0.12.7) without any mismatch.
PARalyzer program was used to analyze PAR-CLIP dataset for idntifiying RBP binding clusters.
Genome_build: mm9
Supplementary_files_format_and_content: tab-delimited text files include RPKM values for mRNA seq samples, raw reads values for miRNA and PAR-CLIP samples.

source_name: Bone marrow derived macrophage

growth_protocol: Bone marrow cells from mouse femur and tibia were collected by flushing through PBS with a 23-guage needle.  Bone marrow cells were cultured in one 150mm Petri dishes with complete DMEM and 20% of L929 cell culture medium for 6 days

extract_protocol:
Total RNA and small RNAs were isolated as detailed in the manufacturer’s protocols (Clonetech NucleoSpin miRNA) and RNA quality was checked by an Agilent 2100 bioanalyzer (Agilent technologies)
RNA libraries were prepared for sequencing using standard Illumina protocols, PAR-CLIP sample libraries were prepared with PAR-CLIP + TruSeq Small RNA prep.

value: Bone marrow derived macrophage

value: ELAVL1f/f, LysM-Cre

tag: par-clip antibody


title: BMDM miRNA seq KO3

status: Public on Apr 23 2015

type: SRA

country: USA

description:
Small RNA
miRNAseq

library_source: transcriptomic

molecule: total RNA

organism: Mus musculus

tag: strain

tag: cell type

tag: genotype


title: BMDM miRNA seq WT2

description:
Small RNA
miRNAseq


title: HB (Sample 24)

city: Lund

first: Malin

last: Parmar

address: BMC A11

department: Wallenberg Neuroscience Center

country: Sweden

institute: Lund University

data_processing:
Mature and hairpin microRNAs were downladed from mirbase.org 2013-06-22
Preprocessing with fastx, http://hannonlab.cshl.edu/fastx_toolkit/index.html
The adapter TGGAATTCTCGGGTGCCAAGGAACTCCAGTCAC was removed
Sequences shorter than 15 nt were removed.
A fasta file with read counts was produced for each sample
Bowtie2 was used for alignment. Reference : Langmead B, Trapnell C, Pop M, Salzberg SL. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol 10:R25.
Bowtie indices were built using the mature and hairpin files from mirbase.
Alignment was performed for the reads of each sample. Default bowtie2 scores were used
For each sample and micro-RNA, the total count was found. The counts were multiplicatively normalized such that each sample had the same total count. The counts were logarithm tansformed by adding a pseudocount of 1 to all counts and taking the base 2 logarithm.
Genome_build: Mirbase release 19
Supplementary_files_format_and_content: csv file with normalized counts for the microRNAs for each sample

organism: Homo sapiens

source_name: Differentiated hindbrain cells from ES cells d14

growth_protocol: Kirkeby A, Nelander J, Parmar M. Generating regionalized neuronal cells from pluripotency, a step-by-step protocol.  Front Cell Neurosci. 2013 Jan 3;6:64. doi: 10.3389/fncel.2012.00064

extract_protocol: RNA was exracted using the miRNeasy mini kit form Qiagen

treatment_protocol: Kirkeby A, Grealish S, Wolf DA, Nelander J, Wood J, Lundblad M, Lindvall O, Parmar M. Generation of regionally specified neural progenitors and functional neurons from human embryonic stem cells under defined conditions. Cell Rep. 2012 Jun 28;1(6):703-14. doi: 10.1016/j.celrep.2012.04.009. Epub 2012 May 26.

tag: protocol

value: H9 cells were differentiated towards a  hindbrain fate according to the protocol in Kirkeby et al 2012, Frontiers in Cellular Neuroscience.

tag: cell line


title: P493 shTerc

city: Singapore

first: Julius

last: Müller

address: 61 Biopolis Drive

department: IMCB

country: Singapore

institute: A*Star Singapore

description: Sample 3_P493

library_source: genomic

data_processing:
Illumina Casava v1.8 used for basecalling.
In order to make the downstream analysis more comparable to the P493 samples, all mouse Eu Myc samples were trimmed to 35Bp long reads using trimmomatic.
Sequences were mapped to hg19 and mm10 using bowtie2 v2.2.3 with parameters -N 0 --sensitive --no-unal -q -U
MACS v2.0.10.20131216 was used for peak calling with parameters --llocal=10000 --bw 200 --shiftsize 100 -q 0.05 --slocal=1000  --keep-dup=3  --nomodel
All MACS output files contain only peaks with a pileup greater than 4, a q-value smaller or equal to 0.05 and a minimum enrichment of 2.
Genome_build: mm10 (Primary lymphoma cells) and hg19 (P493)
Supplementary_files_format_and_content: MACS v2.0.10.20131216 peaks file

molecule: genomic DNA

source_name: P493 cells

growth_protocol: P493 cells were maintained in RPMI medium 1640 supplemented with 10% FBS, penicillin, penicillin and streptomycin.

extract_protocol:
Cells were treated with 1% formaldehyde for 10 minutes at room temperature to crosslink protein-DNA complexes, and reactions were then quenched using 125 mM glycine for 5 minutes. The cells were then lysed with sodium dodecyl sulfate (SDS) lysis buffer and sonicated for 10 minutes using the Diagenode Bioruptor. The fragmented chromatin was pre-cleared with bovine serum albumin (BSA), protein A Sepharose beads (Millipore) at 4 ˚C for 2 hours before immunoprecipitation with c-MYC or rabbit control IgG antibodies overnight at 4 ˚C. Sepharose beads were washed as described previously and eluted with SDS elution buffer before subjecting to reverse cross-linking at 65 ˚C overnight. Finally samples were purified using the QIAquick PCR Purification Kit (Qiagen) according to the manufacturer’s protocol.
Single read - Illumina

treatment_protocol: P493 cells were stably infected with the following viruses: pLKO shControl, pLKO shTERT and pCMV-GFP-NheI shTerc.

value: P493 cells

tag: treatment

value: shTerc

tag: chip antibody

value: c-Myc Antibody (N-262) (Santa Cruz, catalog# sc-764, lot# D0413)


title: FB (Sample 13)

source_name: Differentiated forebrain cells from ES cells d14

value: H9 cells were differentiated towards a forebrain fate according to the protocol in Kirkeby et al 2012, Frontiers in Cellular Neuroscience.


title: Corinpos vMB (Sample 33)

source_name: FACS Corin+ cells from ventral midbrain cells from ES cells d14

value: H9 cells were differentiated towards a ventral midbrain fate according to the protocol in Kirkeby et al 2012, Frontiers in Cellular Neuroscience.In addition, cells were further ventralized by adding Purmophamine to the media (StemGent, 5uM) during day0-11 of differentiation.  On day 14 the cells were stained for Corin (R&D 1:200) and FACS sorted.


title: FB (Sample 14)

value: Differentiated forebrain cells from ES cells d14


title: FB (Sample 12)


title: BMDM miRNA seq WT1

description:
Small RNA
miRNAseq


title: ES cells (Sample 11)

source_name: Embryonic stem cells

value: H9 cells were expanded and maintained to gamma-irradiated mouse fibroblasts in DMEM/F12, 20%KSR, 0.05 mM 2-merkaptoethanom, 0.5% Pen/Strep, 0.5% glutamine and 10ng/ml bFGF. Cells were passaged once weeksly with 0.5mM EDTA.

value: Embryonic stem cells


title: ES cells (Sample 9)


title: BMDM mRNA seq KO1

description:
Poly A tailed mRNA
mRNAseq


title: BMDM mRNA seq KO4

description:
Poly A tailed mRNA
mRNAseq


title: BMDM mRNA seq KO3

description:
Poly A tailed mRNA
mRNAseq


title: ES cells (Sample 10)


title: ES cells (Sample 8)


title: BMDM mRNA seq KO2

description:
Poly A tailed mRNA
mRNAseq


title: BMDM mRNA seq WT4

description:
Poly A tailed mRNA
mRNAseq


title: BMDM mRNA seq WT3

description:
Poly A tailed mRNA
mRNAseq


title: HLF (Sample 7)

source_name: Lung fibroblast

value: Human Fetal Lung Fibroblasts (hFL1s) were purchased from American Type Culture Collection (ATCC). Passage 12 – 16 hFL1s were expanded in DMEM supplemented with 10% FBS, 0.5% Pen/Strep, 0.5% glutamine.

value: Lung fibroblast


title: HLF (Sample 6)


title: BMDM mRNA seq WT1

description:
Poly A tailed mRNA
mRNAseq


title: HLF (Sample 5)


title: HLF (Sample 4)


title: NS cells (Sample 3)

source_name: Neural stem cells

value: Subdissected human fetal ventral midbrai was plated at 12,500cells/cm2 onto plastic coated with polyornithine, fibornectin and laminin. Neurobasal medium was supplemented with B27, Glutamine, BDNF, GDNF and ascorbic acid and was exchanged every 2-3days.

value: Neural stem cells

tag: origin

value: Subdissection from embryonic human tissue


title: NS cells (Sample 2)


title: NET-seq HeLa S3 Rep1

city: Boston

first: Stirling

last: Churchman

state: Massachusetts

address: 77 Avenue Louis Pasteur

department: Genetics

institute: Harvard Medical School

description:
nascent RNA
library strategy: NET-seq

data_processing:
For NET-seq and HEK 293T cytoplasmic RNA-seq libraries, the six 5’ end nucleotides corresponding to the molecular barcode, are trimmed from the reads, but remain associated with the read using a custom python script.
For all samples, reads are aligned using the STAR aligner (v2.4.0) (Dobin et al., 2013) with the following parameters : --clip3pAdapterSeq ATCTCGTATGCCGTCTTCTGCTTG --clip3pAdapterMMp 0.21 --clip3pAfterAdapterNbases 1 –outFilterMultimapNmax 101 –outSJfilterOverhangMin 3 1 1 1 –outSJfilterDistToOtherSJmin 0 0 0 0 –alignIntronMin 11 –alignEndsType EndToEnd. The reference genome used consisted in the human reference genome (hg19), with additional sequences containing the rDNA sequences (genBank U13369.1, genBank U67616.1), and the sequence of processed tRNA (with the additional CCA at their end, and intron removed, when present).
For NET-seq data, to avoid any bias towards favoring annotated regions the alignment was performed without providing transcriptome information, while for RNA-seq data, the GENCODE annotation v16 was used as the transcriptome.
Only uniquely aligned reads are kept to generate the processed data files.
For NET-seq data, only the position corresponding to the 5’ end of the sequencing read (after removal of the barcode), which corresponds to the 3’ end of the nascent RNA fragment, is recorded with a custom python script using HTSeq package (Anders et al., 2014), while the whole read coverage is recorded for RNA-seq data.
For NET-seq and HEK 293T cytoplasmic RNA-seq libraries, reverse transcription mispriming events are identified where barcode sequences correspond exactly to the genomic sequence adjacent to the aligned read. These reads are removed from further analysis.
For NET-seq and HEK 293T cytoplasmic RNA-seq libraries, the few reads that align to the same genomic position and contain identical barcode are considered PCR duplication events and are filtered out.
For NET-seq libraries, evidence of splicing intermediates (Figure S1F) revealed that at sites where intermediates map (exact single bp 3’ ends of introns and exons) it is unclear which reads are due to splicing intermediate contamination and which are due to 3’ ends of nascent RNA. Thus, reads that map precisely at the exact single bp ends of introns and exons are discarded and the single 1 bp genomic positions are removed from subsequent analysis.
Genome_build: hg19
Supplementary_files_format_and_content: BedGraph files (http://genome.ucsc.edu/goldenPath/help/bedgraph.html); Column1=chromosome; Column2=start (left most coordinate; 0 based; position included); Column3=end (right most coordinate; 0 based; position not included); Column4=Coverage (3'end nt for NET-seq and whole read for RNA-seq).

source_name: HeLa S3 cells

growth_protocol: HeLa S3 cells (ATCC, CCL-2.2) were grown in DMEM containing 10% FBS, 100 U/ml penicillin and 100 µg/ml streptomycin until 90% confluent.

extract_protocol:
Cell fractionation was performed as described in (Pandya-Jones & Black, RNA, 2009), (Bhatt et al., Cell, 2012) and based on (Wuarin & Schibler, MCB, 1994) with modifications. All subsequent steps have been conducted on ice or at 4°C and in the presence of 25 µM α-amanitin (Sigma, A2263), 50 Units SUPERaseIN (Life Technologies, AM2696) and Protease inhibitors cOmplete (Roche, 11873580001) according to manufacturer’s instructions using RNAse free equipment. All buffers have been pre-chilled on ice before use. The cell pellet corresponding to 1x107 cells was gently resuspended in 200 µl cytoplasmic lysis buffer (0.15% NP-40, 10 mM Tris-HCl pH 7.0, 150 mM NaCl). The cell lysate was incubated for 5 min on ice, layered onto 500 µl sucrose buffer (10 mM Tris-HCl pH 7.0, 150 mM NaCl, 25% sucrose) and centrifuged at 16,000 g for 10 min. The supernatant corresponding to the cytoplasmic fraction was carefully removed. For subcellular RNA-seq (described below) the cytoplasmic fraction was mixed with 3.5x volumes of RLT buffer (74104, QIAGEN). RNA purification from the cytoplasmic and following fractions is described below.
Sequencing library preparation was performed as described in (Churchman & Weissman, 2012) with modifications. 3 µg of purified chromatin associated RNA was used to prepare one DNA sequencing library. 3’ ligation of RNA with a DNA linker was performed essentially as originally described, except that a DNA linker with a mixed random hexameric sequence at its 5’ end was used (new DNA linker: 5’-AppNNNNNNCTGTAGGCACCATCAAT/3ddC-3’). The ligation efficiency was monitored by ligating the new DNA linker to a 28 nt long RNA control oligo (oGAB11: 5’-AGUCACUUAGCGAUGUACACUGACUGUG-3’OH). The ligation efficiency as determined by polyacrylamide gel electrophoresis was ≥95%. After ligation the RNA was fragmented by partial alkaline hydrolysis essentially as originally described. The fragmentation time was adjusted so that most RNA molecules were fragmented into the range of 35 to 100 nt, size-selected and converted into cDNA. Reverse transcription was performed with the following modifications to the original protocol. First, for reverse transcription a new RT primer was used (oLSC007: 5’-Phos/ATCTCGTATGCCGTCTTCTGCTTG/iSp18/CACTCA/iSP18/ TCCGACGATCATTGATGGTGCCTACAG-3’). Second, the RT primer was applied at a lower final concentration (1.9 µM instead of 10.9 µM). Next, cDNA was circularized and vectors that contained cDNA corresponding to original mature snRNAs, snoRNAs, rRNAs and mitochondrial tRNAs that were heavily sequenced in first NET-seq experiments were specifically depleted. For specific depletion, biotinylated oligos were designed that were complementary to the 3’ ends of the 19 most heavily sequenced mature RNAs of HeLa S3 cells (see Supplemental Table S1). Depletion was performed similar to the rRNA depletion approach described by (Ingolia et al., Nature Protocols, 2012). Per depletion 5.0 µl circularization reaction was used. 5.0 µl of circularization reaction was combined with 1.0 µl depletion oligo pool (10 µM of each oligo, prepared in 10 mM Tris-HCl, pH 8.0; see Supplemental Table S1), 1.0 µl 20x SSC (Life Technologies, AM9763) and 3.0 µl DNase-free H2O. Tubes were placed in a thermal cycler, denatured for 90 sec at 99°C and then annealed at 0.1°C s-1 to 37°C. Tubes were incubated for 15 min at 37°C. 37.5 µl of MyOne Streptavidin C1 DynaBeads (10 mg/ml, Life Technologies, 65001) were used per depletion reaction. Magnetic beads were washed 3x with 1x bind/wash buffer (1M NaCl, 0.5 mM EDTA, 2.5 mM Tris-HCl pH 7.0, 0.1% (vol/vol) Triton-X-100). After the last wash, magnetic beads were resuspended in 15 µl 2x bind/wash buffer (2 M NaCl, 1 mM EDTA, 5 mM Tris-HCl pH 7.0, 0.2% (vol/vol) Triton-X-100) and incubated at 37°C. 10 µl of the depletion reaction were transferred directly to the bead aliquot, immediately mixed by pipetting and incubated for 15 min at 37°C with mixing at 1000 rpm. Finally, tubes were transferred to a magnetic rack and 20 µl of supernatant were recovered and isopropanol precipitated. After depletion, the circularized DNA served as template for PCR amplification. PCR amplification was performed as originally described (Churchman & Weissman, 2012), using minimal amplification cycles. The concentration, the quality and the size distribution of final DNA libraries were assessed by Qubit 2.0 Fluorometer (Invitrogen) and 2100 Bioanalyzer (Agilent) measurements. 3’ end sequencing was performed on NextSeq 500 (SE 75 nt) and HiSeq 2000 (SE 50 nt) Illumina sequencing platforms using the following custom sequencing primer: oLSC006: 5’-TCCGACGATCATTGATGGTGCCTACAG-3’.

value: HeLa S3

tag: library type

value: 3'end RNA fragment sequencing


title: NS cells (Sample 1)


title: TH17 Effector_2960

city: Nashville

first: Charles

middle: F

last: Spurlock III

state: Tennessee

address: 1161 21st Avenue South

department: Medicine

institute: Vanderbilt University School of Medicine

data_processing:
Illumina Casava1.7 software used for basecalling.
Quality control to filter out bad samples and reads
Alignment to human genome (hg19) using Tophat 2
Read count quantification using HTSeq
Genome_build: hg19
Supplementary_files_format_and_content: Read Count Table

source_name: PBMC

growth_protocol: Human PBMC were isolated from healthy volunteers and stimulated with soluble mouse anti-human CD28 (1 mg/ml; 555725; BD Biosciences) and plate-bound anti-CD3 (10 mg/ml; OKT3 clone American Type Culture Collection), 1 x 10^6 cells/ml, under TH1: IL-12 (10 ng/ml), TH2: IL-4 (10 ng/ml), or TH17: IL-1b (20 ng/ml), IL-6 (50 ng/ml), IL-23 (20 ng/ml), IL-21 (100 ng/mL), TGF-b1 (5 ng/ml) and mouse anti-human IFN-g (10 mg/ml; 554698; BD Biosciences), polarizing conditions.  TH1 and TH2 cultures were harvested after 5 days to obtain RNA from primary cultures.  TH17 cultures were harvested after 7 days to obtain RNA from primary cultures. After 5 days (TH1 and TH2) or 7 days (TH17), cultures were restimulated with plate-bound anti-CD3 and harvested 2 days later to obtain RNA from effector cultures.

extract_protocol:
Total RNA was isolated with TRI Reagent (Molecular Research Center) and purified with the RNeasy MinElute Cleanup kit (Qiagen) using an on-column DNAse treatment to ensure absence of genomic DNA contamination according to the manufacturer’s supplied protocol.
"2695" libraries were constructed using the Illumina TruSeq Stranded mRNA kit (Cat. No. RS-122-2101) following the manufacturer's supplied protocol. "2960" libraries were constructed using the Illumina TruSeq Stranded Total RNA kit (Catalog No. RS-122-2201) following the manufacturer's supplied protocol.

treatment_protocol: Cells were treated with cytokine and antibody cocktails detailed above to generate TH1, TH2, and TH17 in vitro cultures from healthy human donors.

value: peripheral blood mononuclear

tag: polarizing conditions

tag: rna fraction

value: Total RNA


title: TH1 Effector_2695

value: polyA+ RNA


title: TH1 Primary_2960


title: TH17 Effector_2695


title: TH17 Primary_2695


title: TH1 Effector_2960


title: TH2 Effector_2695


title: TH2 Effector_2960


title: TH17 Primary_2960


title: TH2 Primary_2960


title: TH1 Primary_2695


title: TH2 Primary_2695


title: mNET_8WG16_siLuc

city: Lisboa

first: Tomás

last: Gomes

address: Av. Professor Egas Moniz

country: Portugal

institute: Instituto de Medicina Molecular

description: nascent RNA from unphosphorylated Pol II

data_processing:
For mNET-seq data, adaptor trimming was preformed using Cutadapt (v1.1), only keeping the reads with more than 10 bases.
mNET-seq data was then aligned to the human genome (build hg19) using TopHat (v2.0.9), with a minimum anchor length of 5 bases and allowing only for unique alignments.
For ChrRNA-seq data, alignment was preformed using TopHat (v2.0.9) with a minimum anchor length of 5 bases, allowing only for unique alignments and allowing read pairs to be separated by up to 3kb.
To aquire the last nucleotide incorporated by the polymerase in the aligned mNET-seq reads, a combination of SAMtools and inhouse python scripts was used
Then, both ChrRNA-seq and mNET-seq singe-nucleotide aligned reads were separated by strand using SAMtools, selecting the strands according to the read flag.
To make bedgraphs of the reads aligning in different strands, genomeCoverageBed (v2.17.0) was used.
After that, the values in the bedgraphs were normalized to reads per 108 sequences using an inhouse python script.
Finally, to convert the bedgraphs to bigWig, the program bedGraphToBigWig from UCSC was used.
Genome_build: hg19
Supplementary_files_format_and_content: processed data files are in bigWig format; the scores in the files represent the number of reads per 108 sequences

source_name: HeLa cells

growth_protocol: HeLa cells were maintained in DMEM with 10% fetal bovine serum

extract_protocol:
Approximately 1.6x108 cells were used to generate nuclear and chromatin fractions. Isolated chromatin was washed in 1 ml of 1x Micrococcal Nuclease (MNase) buffer (NEB) and then incubated with MNase (40 u/mL) on Thermomixer (eppendorf, 1,400 rpm) at 37oC for 90 sec. MNase was inactivated by EGTA (25 mM) addition immediately after the reaction and soluble digested chromatin was collected by 13,000 rpm centrifuge for 5 min. The supernatant was diluted with 9 ml of NET-2 buffer and add Pol II antibody-conjugated beads. 40 mg of Pol II antibody was used for each mNET-seq experiment. Immuno-precipitation was performed at 4oC for one hour. The beads were washed with 1 ml of NET-2 buffer six times and with 500 ml of 1xPNKT (1xPNK buffer and 0.05 % Triton X-100) buffer once in the cold room. The washed beads were incubated in 100 ml of PNK reaction mix (1xPNKT, 1 mM ATP and 0.05 U/ml T4 PNK 3'phosphatase minus (NEB) ) on Thermomixer (1,400 rpm) at 37oC for 6 min. After the reaction the beads were washed with 1 ml of NET-2 buffer once and RNA was extracted with Trizol reagent. RNA was resolved on 8 % denaturing acrylamide 7 M urea gels for size purification. 35-100 nt fragments were eluted from the gel using RNA elution buffer (1 M NaOAc and 1 mM EDTA) and RNA was precipitated in 75 % Ethanol.
RNA libraries were prepared according to the manual of Truseq small RNA library prep kit (Illumina). Deep sequencing was conducted by WTCHG in Oxford.

treatment_protocol: Control siRNA against Luciferase ORF (final conc. 30nM) were transfected into HeLa cell using Lipofectamine RNAiMAX reagent (Life technologies) according to the manual and incubated for 72 hours.

value: HeLa

tag: mnet-seq antibody

value: Luciferase siRNA


title: mNET_CMA603_siLuc

description: nascent RNA from Ser5 phosphorylated Pol II


title: BMDM mRNA seq WT2

description:
Poly A tailed mRNA
mRNAseq

value: Bone marrow derived macrophage


title: mNET_8WG16_siCPSF73

treatment_protocol: SMARTpool siRNA against human CPSF73 (CPSF3)  (final conc. 30nM) were transfected into HeLa cell using Lipofectamine RNAiMAX reagent (Life technologies) according to the manual and incubated for 72 hours.

value: CPSF73 siRNA


title: mNET_CMA603_PlaB

treatment_protocol: HeLa cell was treated with pladienolides B (Pla-B, final conc. 1mM) for 4 hours.

value: Pla-B


title: mNET_CMA603_siCPSF73


title: ChrRNA_siCstF64tau

description: chromatin-bound RNA

extract_protocol:
Chromatin RNA fraction was prepared from ~80% confluent HeLa cells in 100mm Dishes. Approximately 7x106 cells were washed with ice-cold PBS twice. The cells were lysed with ice-cold 4 ml of HLB/NP40 buffer (10 mM Tris-Hcl pH 7.5, 10 mM NaCl, 0.5% NP40 and 2.5 mM MgCl2) and incubated on ice for 5 min. After the incubation, 1 ml of ice-cold HLB/NP40/Sucrose buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 0.5% NP40, 2.5 mM MgCl2 and 10 % Sucrose) was under-laid and then the nuclei were collected under 1,400 rpm centrifuge at 40C for 5 min. Isolated nuclei were resuspended in 1.25 ml of NUN1 solution (20 mM Tri-HCl pH 8.0, 75mM NaCl, 0.5 mM EDTA, 50% Glycerol and proteinase inhibitor 1xComplete (Roche)) and added 1.2 ml NUN2 buffer (20 mM HEPES-KOH pH 7.6, 7.5 mM MgCl2, 0.2 mM EDTA, 300 mM NaCl, 1 M Urea, 1% NP40, proteinase inhibitor 1xComplete and phosphatase inhibitor 1xPhosStop (Roche)). 15 min incubation was carried out on ice with mixing by max speed vortex for 5 sec every ~4 min and then chromatin pellets were precipitated under 13,000 rpm centrifuge at 4oC for 10min. Chromatin pellet was resuspended in 200 ml HSB (10mM Tris-HCl pH 7.5, 500 mM NaCl and 10 mM MgCl2) with 0.25 U/ml TURBO DNase (Life technologies) at 37oC for 10 min and then treated with Proteinase K for 10 min. RNA was extracted by Trizol reagent (Life technologies). This extraction steps were repeated three times.
In prior to RNA library preparations, rRNAs were depleted using Ribo-Zero rRNA removal kits (Epicentre) from 5 mg of Chromatin RNA. RNA was also fragmented 150-200 nt by heat treatment (94oC) for 15 min in 1xNEB first strand synthesis buffer. 200 ng of chromatin RNA was used for RNA library preparations. These were carried out according to NEBNext Ultra Directional RNA Library Prep kit for Illumina (NEB). Deep sequencing Hiseq2500 were performed by the Wellcome Trust Centre for Human Genetics (WTCHG) Oxford UK.

treatment_protocol: Silencer select siRNA against CstF64 tau (CSTF2T) was designed by Life technologies as following sequence, Sense: CCAUUAUUGACUCACCCUAtt, Antisense: UAGGGUGAGUCAAUAAUGGgc. The siRNA (final conc. 30nM) were transfected into HeLa cell using Lipofectamine RNAiMAX reagent (Life technologies) according to the manual and incubated for 72 hours.

value: CstF64tau siRNA


title: NucleoplasmRNA_DMSO

description: nucleoplasm fraction RNA

extract_protocol:
Nucleoplasm RNA was prepared from ~80% confluent HeLa cells in 100mm Dishes. Approximately 7x106 cells were washed with ice-cold PBS twice. The cells were lysed with ice-cold 4 ml of HLB/NP40 buffer (10 mM Tris-Hcl pH 7.5, 10 mM NaCl, 0.5% NP40 and 2.5 mM MgCl2) and incubated on ice for 5 min. After the incubation, 1 ml of ice-cold HLB/NP40/Sucrose buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 0.5% NP40, 2.5 mM MgCl2 and 10 % Sucrose) was under-laid and then the nuclei were collected under 1,400 rpm centrifuge at 40C for 5 min. Isolated nuclei were resuspended in 1.25 ml of NUN1 solution (20 mM Tri-HCl pH 8.0, 75mM NaCl, 0.5 mM EDTA, 50% Glycerol and proteinase inhibitor 1xComplete (Roche)) and added 1.2 ml NUN2 buffer (20 mM HEPES-KOH pH 7.6, 7.5 mM MgCl2, 0.2 mM EDTA, 300 mM NaCl, 1 M Urea, 1% NP40, proteinase inhibitor 1xComplete and phosphatase inhibitor 1xPhosStop (Roche)). 15 min incubation was carried out on ice with mixing by max speed vortex for 5 sec every ~4 min and then chromatin pellets were removed under 13,000 rpm centrifuge at 4oC for 10min. Nucleoplasm supernatant was treated with 0.25 U/ml TURBO DNase (Life technologies) at 37oC for 10 min. RNA was extracted by Trizol reagent (Life technologies). This extraction steps were repeated three times.
In prior to RNA library preparations, rRNAs were depleted using Ribo-Zero rRNA removal kits (Epicentre) from 5 mg of nucleoplasm RNA. RNA was also fragmented 150-200 nt by heat treatment (94oC) for 15 min in 1xNEB first strand synthesis buffer. 200 ng of nucleoplasm RNA was used for RNA library preparations. These were carried out according to NEBNext Ultra Directional RNA Library Prep kit for Illumina (NEB). Deep sequencing using Hiseq2500 were performed by the Wellcome Trust Centre for Human Genetics (WTCHG) Oxford UK.

treatment_protocol: HeLa cell was treated with DMSO for 4 hours.

value: DMSO


title: NucleoplasmRNA_PlaB

description: nucleoplasm fraction RNA

extract_protocol:
Nucleoplasm RNA was prepared from ~80% confluent HeLa cells in 100mm Dishes. Approximately 7x106 cells were washed with ice-cold PBS twice. The cells were lysed with ice-cold 4 ml of HLB/NP40 buffer (10 mM Tris-Hcl pH 7.5, 10 mM NaCl, 0.5% NP40 and 2.5 mM MgCl2) and incubated on ice for 5 min. After the incubation, 1 ml of ice-cold HLB/NP40/Sucrose buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 0.5% NP40, 2.5 mM MgCl2 and 10 % Sucrose) was under-laid and then the nuclei were collected under 1,400 rpm centrifuge at 40C for 5 min. Isolated nuclei were resuspended in 1.25 ml of NUN1 solution (20 mM Tri-HCl pH 8.0, 75mM NaCl, 0.5 mM EDTA, 50% Glycerol and proteinase inhibitor 1xComplete (Roche)) and added 1.2 ml NUN2 buffer (20 mM HEPES-KOH pH 7.6, 7.5 mM MgCl2, 0.2 mM EDTA, 300 mM NaCl, 1 M Urea, 1% NP40, proteinase inhibitor 1xComplete and phosphatase inhibitor 1xPhosStop (Roche)). 15 min incubation was carried out on ice with mixing by max speed vortex for 5 sec every ~4 min and then chromatin pellets were removed under 13,000 rpm centrifuge at 4oC for 10min. Nucleoplasm supernatant was treated with 0.25 U/ml TURBO DNase (Life technologies) at 37oC for 10 min. RNA was extracted by Trizol reagent (Life technologies). This extraction steps were repeated three times.
In prior to RNA library preparations, rRNAs were depleted using Ribo-Zero rRNA removal kits (Epicentre) from 5 mg of nucleoplasm RNA. RNA was also fragmented 150-200 nt by heat treatment (94oC) for 15 min in 1xNEB first strand synthesis buffer. 1200 ng of nucleoplasm RNA was used for RNA library preparations. These were carried out according to NEBNext Ultra Directional RNA Library Prep kit for Illumina (NEB). Deep sequencing using Hiseq2500 were performed by the Wellcome Trust Centre for Human Genetics (WTCHG) Oxford UK.


title: mNET_CMA603_DMSO


title: ChrRNA_siCstF64

description: chromatin-bound RNA

extract_protocol:
Chromatin RNA fraction was prepared from ~80% confluent HeLa cells in 100mm Dishes. Approximately 7x106 cells were washed with ice-cold PBS twice. The cells were lysed with ice-cold 4 ml of HLB/NP40 buffer (10 mM Tris-Hcl pH 7.5, 10 mM NaCl, 0.5% NP40 and 2.5 mM MgCl2) and incubated on ice for 5 min. After the incubation, 1 ml of ice-cold HLB/NP40/Sucrose buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 0.5% NP40, 2.5 mM MgCl2 and 10 % Sucrose) was under-laid and then the nuclei were collected under 1,400 rpm centrifuge at 40C for 5 min. Isolated nuclei were resuspended in 1.25 ml of NUN1 solution (20 mM Tri-HCl pH 8.0, 75mM NaCl, 0.5 mM EDTA, 50% Glycerol and proteinase inhibitor 1xComplete (Roche)) and added 1.2 ml NUN2 buffer (20 mM HEPES-KOH pH 7.6, 7.5 mM MgCl2, 0.2 mM EDTA, 300 mM NaCl, 1 M Urea, 1% NP40, proteinase inhibitor 1xComplete and phosphatase inhibitor 1xPhosStop (Roche)). 15 min incubation was carried out on ice with mixing by max speed vortex for 5 sec every ~4 min and then chromatin pellets were precipitated under 13,000 rpm centrifuge at 4oC for 10min. Chromatin pellet was resuspended in 200 ml HSB (10mM Tris-HCl pH 7.5, 500 mM NaCl and 10 mM MgCl2) with 0.25 U/ml TURBO DNase (Life technologies) at 37oC for 10 min and then treated with Proteinase K for 10 min. RNA was extracted by Trizol reagent (Life technologies). This extraction steps were repeated three times.
In prior to RNA library preparations, rRNAs were depleted using Ribo-Zero rRNA removal kits (Epicentre) from 5 mg of Chromatin RNA. RNA was also fragmented 150-200 nt by heat treatment (94oC) for 15 min in 1xNEB first strand synthesis buffer. 200 ng of chromatin RNA was used for RNA library preparations. These were carried out according to NEBNext Ultra Directional RNA Library Prep kit for Illumina (NEB). Deep sequencing using Hiseq2500 were performed by the Wellcome Trust Centre for Human Genetics (WTCHG) Oxford UK.

treatment_protocol: SMARTpool siRNA against CstF64 (CSTF2) (final conc. 30nM) were transfected into HeLa cell using Lipofectamine RNAiMAX reagent (Life technologies) according to the manual and incubated for 72 hours.

value: CstF64 siRNA


title: ChrRNA_rep2

description: chromatin-bound RNA


title: PB2, Template-switching (4h)

city: Cambridge

first: David

last: Koppstein

state: MA

address: 9 Cambridge Center

institute: MIT

description:
Sample name: PB2_TS
A/WSN/33 Influenza (H1N1)
PB2, Template-switching (4h)
Template-switching 5′-RACE RNA-Seq

data_processing:
Randomized nucleotides in the TSO and all subsequent contiguous Gs were used as unique molecular identifiers to collapse PCR duplicates. Cutadapt was used to trim sequences that perfectly matched the complement of positions 0–9 in the vRNA (NS1, HA, NP, NA: GCAAAAGCAG; MP, PA, PB1, PB2: GCGAAAGCAG). Reads without a perfect match and reads containing Ns within the remaining sequence were not considered, as were reads in which the remaining heterogeneous sequence was <9 or >15 nt. To account for prime-and-realign, any sequences with ≥2 3′-terminal nucleotides matching the 5′ end of the complement of positions 0–9 in the vRNA were iteratively trimmed up to four times from heterogeneous sequences. The resulting reads were designated trimmed host leaders. Trimmed host leaders < 9 nt were again discarded. The number of read counts and nucleotides trimmed at the 5′ and 3′ end were retained in the FASTQ header as meta-information. Annotated TSSs were obtained from Gencode 17 and iteratively trimmed at each 5′ terminus until the first nucleotide was not a G. A 51 nt window around each adjusted TSS was extracted using BEDTools and converted to a Bowtie index. Cellular fragments were mapped to the custom Bowtie index, requiring a perfect, sense match (Bowtie parameters: -S –norc -p 24 –all –tryhard -v 0), converted to hg19 human genome coordinates in BAM format, and intersected with the Gencode annotations using BEDTools. If a read mapped to multiple paralogs, one paralog was chosen at random and the rest were removed from the dataset by collapsing on the Gencode 17 gene_name attribute. Reads mapping to multiple snRNA/snoRNA paralogs were also removed, accounting for somewhat variable gene_name attributes sometimes assigned to these paralogs.
Genome_build: hg19
Supplementary_files_format_and_content: Tab-delimited file with headers on the first line. Header descriptions: READ_CHROM, READ_START, READ_END, READ_STRAND: Zero-based, half-open coordinates of the mapped read. READ_NAME: Read FASTQ header. READ_CODE: Same as SAM format specifications. TSS_CHROM, TSS_START, TSS_END, TSS_STRAND: Zero-based, half-open coordinates of the Gencode 17 TSS the read maps to. TSS_NAME, TSS_CODE, TSS_SOURCE, TSS_ATTRIBUTE: Correspond to TSS GTF format attributes of the same names. UNKNOWN_DOT: Placeholder for dot in GTF file. TSS_META: verbatim list of GTF attributes for that TSS; parsed in other headers as well (see below). ORIGINAL_SEQUENCE: Sequence of the read. ORIGINAL_SEQUENCE_LENGTH: length of the original read. OFFSET_FROM_START: strand-oriented offset from the annotated TSS start. 5PTRIMMED: Sequences trimmed from the 5' end of the read 3PTRIMMED: Sequences trimmed from the 3' end of the transcript (after trimming viral sequence, positions 0-9). GENOMIC_SEQUENCE: Sequence of the stranded TSS window the read maps to. COUNT: number of PCR-deduplicated reads with this sequence that have the same 3PTRIMMED and 5PTRIMMED attributes. SEQUENCE_COUNT: number of PCR-deduplicated reads with this sequence, regardless of the 3PTRIMMED and 5PTRIMMED attributes. ZEROS: Number of times this sequence maps to a TSS precisely. ZEROS_TO_TOTAL: ratio of ZEROS to total number of mapping events for this transcript. THREEPRIME_OF_CLEAVAGE: Genomic sequence 3' of the mapped read. GENE_STATUS, EXON_NUMBER, LEVEL, TAG, GENE_ID, TRANSCRIPT_ID, HAVANA_TRANSCRIPT, HAVANA_GENE, TRANSCRIPT_NAME, GENE_TYPE, TRANSCRIPT_TYPE, GENE_NAME: correspond to the same fields in the GTF format.

organism: Influenza A virus (A/WSN/1933(H1N1))

source_name: A549 cells

growth_protocol: standard cell culture protocol (DMEM + FBS/pen/strep)

extract_protocol:
TRI Reagent, according to manufacturer's protocol (Life Technologies)
TEMPLATE-SWITCHING: 5-10 μg RNA was diluted in 50 mM NaCl and 3.3 μM gene-specific outer primer and adjusted to a volume of 6 μl. The mixture was heated to 65°C for 5 minutes, followed by a cooling ramp (0.1°C/s) to 4°C. In parallel, 2 μl of 100 μM template-switching oligonucleotide (TSO, IDT, Supplemental Table 2) was denatured at 65°C for 5 minutes and snap-cooled on ice. These were combined in a SuperScript II mixture (Life Technologies) with sorbitol/trehalose and betaine as described previously (42, 43). After 60 minutes at 42°C, RNA was hydrolyzed by adding NaOH to a final concentration of 130 mM at 90°C for 10 minutes, then quenched with five-fold molar excess of prechilled 1 M HEPES pH 7, extracted with phenol:chloroform:isoamyl alcohol 25:24:1 pH 8 (Sigma Aldrich) and chloroform. cDNA was precipitated with Pellet Paint NF co-precipitant (EMD Millipore) in ethanol at -20°C, resuspended in 1x Gel Loading Buffer II (Life Technologies) and gel-purified on a thin denaturing 6% polyacrylamide gel, excising the slice corresponding 200–1000 nt, eluting in 800 μl 0.3 M NaCl at room temperature overnight, and precipitating as before. cDNA was then PCR amplified with one common primer and one gene-specific primer (Supplemental Table 2), using Phusion HF according to the manufacturer’s protocol (New England Biolabs) with an extension time of 30 seconds, for 21–27 cycles. The resulting PCR product was precipitated and gel-purified as before, this time excising the corresponding PCR product by SYBR gold staining (Life Technologies). Following precipitation, libraries were resuspended in 10 μl Tris pH 8.0, and sequenced on an Illumina HiSeq 2000.
CIP-TAP: RNA was isolated from influenza-infected A549 cells as described in the Methods. 10 μg RNA was treated with Calf Intestinal Phosphatase (NEB) for 1 hour at 37°C in a 20 µl reaction containing 40 units Superase-In (Life Technologies), according to manufacturers’ protocols. The reaction was then brought to 250 μl in 0.3 M NaCl, sequentially extracted with equal volumes of phenol:chloroform:isoamyl alcohol 25:24:1 pH 8 (Sigma Aldrich) and chloroform, and precipitated with ethanol and glycoblue (Life Technologies) overnight. The precipitate was pelleted, resuspended in water, then treated with Tobacco Acid Pyrophosphatase (Epicentre) according to manufacturer’s protocol in a 10 μl reaction. RNA was extracted, precipitated, resuspended in an 8 μl mixture of 12.5% PEG 8000 (NEB), 10 μM 5′ adapter (Supplemental Table 2), 80 mM ATP, T4 RNA Ligase Buffer (NEB), 4 units Superase-In, and 8 units T4 RNA Ligase 1 (NEB), and incubated at room temperature overnight. The ligated product was boiled in 1x Gel Loading Buffer II (Life Technologies) and gel-purified on a thin denaturing 4% polyacrylamide gel, excising the slice corresponding to >750 nt but excluding the well. The eluate was precipitated, reverse transcribed, and amplified as described in the Methods section.

treatment_protocol: A day before infection, confluent cells in a T-75 flask were diluted four-fold and split into 6-well plates. Three wells were washed twice with 2 mL of PBS supplemented with Ca2+ and Mg2+ (PBS+). 500 μl infection media, consisting of PBS+ w/ 0.2% BSA, pen/strep, and A/WSN/33 (H1N1) influenza at a final MOI of 1-3, was added to each well after vortexing briefly. RNA from three wells was harvested with TRI reagent (Life Technologies) according to protocol after the indicated duration of infection.

tag: infected host cell line

tag: viral mrna

tag: hpi

tag: method

value: template-switching


title: ChrRNA_rep1

description: chromatin-bound RNA


title: PB1, Template-switching (4h)

description:
Sample name: PB1_TS
A/WSN/33 Influenza (H1N1)
PB1, Template-switching (4h)
Template-switching 5′-RACE RNA-Seq


title: PA, Template-switching (4h)

description:
Sample name: PA_TS
A/WSN/33 Influenza (H1N1)
PA, Template-switching (4h)
Template-switching 5′-RACE RNA-Seq

value: PA


title: SNS-Seq (pool) from Bg3 cells, replicate 2

city: Basel

first: Federico

last: Comoglio

state: Basel

address: Mattenstrasse, 26

department: Department of Biosystems Science and Engineering

country: Switzerland

institute: ETH Zurich

data_processing:
S2 and Bg3 SNS-Seq reads were quality filtered and aligned to the dm3 Drosophila reference genome using Bowtie2 version 2.2.0 (Langmead et al., 2012) allowing one mismatch in seed alignment (-N 1 -L 22).
The output was converted from SAM format to BAM using samtools version 0.1.19 (Li et al., 2009), and BAM files were sorted and indexed.
Origin peaks were called using MACS version 1.4.1 (Zhang et al., 2008) with parameters -g dm -m 5,30 for each replicate. Only peaks supported by unique alignments were considered further. For single fraction SNS-Seq data, technical replicates for each fraction were pooled prior to peak calling.
Genome_build: dm3
Supplementary_files_format_and_content: All processed data files are tab-separated TXT files containing genomic coordinates of the origin peaks. In addition, S2 and Bg3 origins also contain a column with the estimated origin efficiency.
Supplementary_files_format_and_content: S2origins.txt: Origin peaks in S2 cells
Supplementary_files_format_and_content: Bg3origins.txt: Origin peaks in Bg3 cells
Supplementary_files_format_and_content: F4origins.txt: Origin peaks in S2 cells, from short SNS
Supplementary_files_format_and_content: F5origins.txt: Origin peaks in S2 cells, from intermediate SNS
Supplementary_files_format_and_content: F6origins.txt: Origin peaks in S2 cells, from long SNS

organism: Drosophila melanogaster

growth_protocol: Drosophila S2-DRSC cells and ML-DmBG3-c2 cells were cultured at 25 °C in 145 mm plates (Greiner) at a density of 1.5 x 10^6 cells/ml in Schneider’s Insect Cell Medium (Sigma S0146) and Shields and Sang M3 insect medium (Sigma S3652) with 10 μg/ml insulin (Sigma), respectively, supplemented with 10 % FBS (Pansera ES).

extract_protocol:
SNS fragments were isolated essentially as described in (Cayrou et al. 2011). Adjustments were made according to SW-41 Ti rotor specifications (Beckman-Coulter) and centrifugation was carried out at 4 °C and 26700 rpm for 21 hours. DNA fragments of 0.5-2.5 kb were collected, purified and subjected to up to five rounds of T4 PNK (Fermentas) phosphorylation and lambda Exonuclease (Fermentas) digestion. SNS were then prepared for sequencing by digesting the RNA-primer, second strand synthesis (NEBNext mRNA Second Strand Synthesis) and purified with AMPure XP beads (Beckman-Coulter).
DNA libraries were prepared with the Nextera XT DNA Sample Preparation Kit (Illumina) following manufacturer’s instructions.

tag: material

value: Short nascent leading strands

tag: celll line


title: NS1, Template-switching (90 min)

description:
Sample name: NS1_90
A/WSN/33 Influenza (H1N1)
NS1, Template-switching (90 min)
Template-switching 5′-RACE RNA-Seq


title: SNS-Seq (single fraction) from S2 cells, fraction 4 (short SNS fragments)


title: NS1, Template-switching (60 min)

description:
Sample name: NS1_60
A/WSN/33 Influenza (H1N1)
NS1, Template-switching (60 min)
Template-switching 5′-RACE RNA-Seq


title: NS1, Template-switching (4h)

description:
Sample name: NS1_TS
A/WSN/33 Influenza (H1N1)
NS1, Template-switching (4h)
Template-switching 5′-RACE RNA-Seq


title: SNS-Seq (pool) from Bg3 cells, pooling replicates 1 and 2


title: SNS-Seq (pool) from S2 cells, replicate 2


title: mNET_CMA603_siPTBP1

treatment_protocol: SMARTpool siRNA against human PTBP1  (final conc. 30nM) were transfected into HeLa cell using Lipofectamine RNAiMAX reagent (Life technologies) according to the manual and incubated for 60 hours.

value: PTBP1 siRNA


title: NS1, Template-switching (45 min)

description:
Sample name: NS1_45
A/WSN/33 Influenza (H1N1)
NS1, Template-switching (45 min)
Template-switching 5′-RACE RNA-Seq


title: SNS-Seq (single fraction) from S2 cells, fraction 5 (middle-sized SNS fragments)


title: GRdim LPS+Dex 3

city: Neuherberg

first: Henriette

last: Uhlenhaut

state: Munich

address: Ingolstaedter Landstr 1

department: HDC

country: Germany

institute: Helmholtz

data_processing:
Software: HCS 1.5.15 / RTA 1.13.48/ Recipe Fragment: 1.3.26 CASAVA 182
TopHat 1.4.1 alignment + cufflinks 2.0.2
files NHU017-019 are triplicates for the condition "LPS" and files NHU020-022 are triplicates for the condition "LPS+Dex"
Genome_build: mm9
Supplementary_files_format_and_content: Excel file of all "expressed" transcripts, with FPKM values for combined replicates (one per condition) and statistical test for differential expression between the two conditions

growth_protocol: primary macrophages were differentiated from mouse bone marrow (legs, arms) in the presence of L-929 conditioned medium on bacterial plates, after alkaline lysis and a Ficoll gradient. After 5days, macrophages were plated in Macrophage serum free medium (Gibco) and treated with Dex 1uM overnight. The next day, cells were treated with LPS for 6hrs.

extract_protocol:
RNeasy Qiagen with Dnase treatment
Illumina, following manufacturer's instructions (Illumina TruSeq RNA sample prep kit). stranded RNA-Seq Illumina.
PE TruSeq Cluster Kit v3 TruSeq 200 cycle kit v3 Multiplexed PE 2x101 cycle +7 Index

treatment_protocol: See Uhlenhaut et al., Mol Cell 2013

value: GRdim genotype (homozygous mut.)

tag: cell type

value: primary bone marrow derived macrophages


title: GRdim LPS+Dex 1

tag: genotype


title: NS1, CIP-TAP (4h)

description:
Sample name: NS1_5R
A/WSN/33 Influenza (H1N1)
NS1, CIP-TAP (4h)

value: CIP-TAP


title: GRdim LPS+Dex 2


title: NS1, Template-switching (30 min)

description:
Sample name: NS1_30
A/WSN/33 Influenza (H1N1)
NS1, Template-switching (30 min)
Template-switching 5′-RACE RNA-Seq


title: GRdim LPS 3


title: SNS-Seq (pool) from S2 cells, replicate 1


title: NS1, Template-switching (240 min)

description:
Sample name: NS1_240
A/WSN/33 Influenza (H1N1)
NS1, Template-switching (240 min)
Template-switching 5′-RACE RNA-Seq


title: GRdim LPS 2


title: SNS-Seq (pool) from S2 cells, pooling replicates 1 and 2


title: NP, Template-switching (4h)

description:
Sample name: NP_TS
A/WSN/33 Influenza (H1N1)
NP, Template-switching (4h)
Template-switching 5′-RACE RNA-Seq

value: NP


title: PB2, CIP-TAP (4h)

description:
Sample name: PB2_5R
A/WSN/33 Influenza (H1N1)
PB2, CIP-TAP (4h)


title: NS1, Template-switching (120 min)

description:
Sample name: NS1_120
A/WSN/33 Influenza (H1N1)
NS1, Template-switching (120 min)
Template-switching 5′-RACE RNA-Seq


title: GRdim LPS 1


title: MP, Template-switching (4h)

description:
Sample name: MP_TS
A/WSN/33 Influenza (H1N1)
MP, Template-switching (4h)
Template-switching 5′-RACE RNA-Seq

value: MP


title: HA, Template-switching (4h)

description:
Sample name: HA_TS
A/WSN/33 Influenza (H1N1)
HA, Template-switching (4h)
Template-switching 5′-RACE RNA-Seq

value: HA


title: BAT_d3_siRNA

first: Juan

middle: R

last: Alvarez-Dominguez

address: 7 Divinity Ave

department: Stem Cell and Regenerative Biology

institute: Harvard University

data_processing:
low quality reads were removed using the fastq_quality_trimmer (“-t 20 -l 25” parameters) from the FASTX-Toolkit (http://hannonlab.cshl.edu/fastx_toolkit/index.html). Only reads in proper pairs were mapped to mm9 using TopHat v.2.0.4.12 (Trapnell et al., 2009) with default parameters and “--min-anchor 5”. The resulting junction files from the BAT, iWAT and eWAT samples were then concatenated and used for another run of TopHat using the --no-novel-juncs parameter.
Genome_build: mm9
Supplementary_files_format_and_content: bigwig

source_name: brown adipocyte_siRNA

growth_protocol: Primary fat tissues were isolated from 8-week-old B/C mice, and primary brown pre-adipocytes were isolated from 3~4-week-old mice and differentiated in culture as described (Sun et al., 2011)

extract_protocol:
Total RNA was isolated using a Qiagen kit as described (Sun et al., 2011)
RNA libraries were prepared for sequencing using standard Illumina protocols

treatment_protocol: RNAi in pre-adipocytes followed by induction of differentiation was performed as described (Sun et al., 2013)

tag: strain

value: B/C

tag: age

tag: tissue

value: brown adipocyte

tag: treated with

value: BATE1-targetting siRNA


title: BAT_d3_NC

source_name: brown adipocyte_NC

value: control siRNA


title: BAT_d5_NC


title: wt_spb75_37C_7days

first: Tim

last: Roloff

address: Maulbeerstrasse 66

department: Functional Genomics

institute: FMI

data_processing:
All samples were sequenced (together with other libraries) on an HiSeq 2000 instrument. Individual reads were assigned to their sample based on the TruSeq barcode. Illumina software Casava v 1.8 was used.
The 3' adapter was removed by aligning it to the read allowing one or two mismatches in prefix alignments of at least 7 or 10 bases respectively. Low complexity reads were filtered out based on their dinucleotide entropy (removing <1% of the reads). All the reads that were shorter than 14 nucleotides were removed.
Alignments to the S. pombe genome (May 8, 2009, http://www.sanger.ac.uk/Projects/S_pombe/) were performed by the software bowtie (version 0.9.9.1) ( Langmead et al., 2009) with parameters -v 2 -a -m 100, tracking up to 100 best alignment positions per query and allowing at most two mismatches. Alignments were normalized for the size of each library. sRNA enriched regions were identified by peak calling with MACS algorithm.
Genome_build: S. pombe genome (May 8, 2009, http://www.sanger.ac.uk/Projects/S_pombe/)
Supplementary_files_format_and_content: tabular file with small RNA enrichment over MACS peaks, normalized for the length of the feature

organism: Schizosaccharomyces pombe

growth_protocol: Strains grown in YES medium to OD600=0.5 at 30°C or 37°C

extract_protocol:
Total RNA was isolated from exponentially growing cells using the hot phenol method (Leeds, 1991). The RNA was fractionated using RNeasy Midi columns (Qiagen) following the „RNA cleanup protocol“ provided by the manufacturer. The flow-through fraction was precipitated („small RNA“ fraction). 25 μg of the "small RNA" fraction were separated on a 17.5% PAGE and the 18 – 28 nt population was purified.
The isolated small RNA populations were prepared for sequencing using the Illumina® TruSeqTM small RNA preparation protocol (Cat.# RS-930-1012).

treatment_protocol: no specific treatment

tag: genotype/variation

value: wt

tag: temperature

tag: time


title: eWAT

source_name: epididymal adipose tissue

value: epididymal adipose tissue


title: SNS-Seq (pool) from Bg3 cells, replicate 1


title: SNS-Seq (single fraction) from S2 cells, fraction 6 (long SNS fragments)


title: wt_spb75_37C_8hours


title: wt_spb75_30C_1


title: dcr_delta_spb309

value: dcr delta


title: BAT_d5_siRNA


title: BAT_rep1

source_name: brown adipose tissue

value: brown adipose tissue


title: wt_spb80_30C


title: S355_GFPplus_3 paired-end

city: Dresden

last: Calegari

address: Fetscherstr. 105

department: Center for Molecular and Cellular Bioengineering (CMCB)

institute: TU Dresden

data_processing:
basecalling: bcl2fastq 1.8.2
data processing: 34 bases of the R2 reads in the paired end were truncated
alignment: alignment of paired-end data against mm9 with tophat version 2.0.10 (--b2-sensitive --library-type fr-firststrand) and Bowtie 2 version 2.2.1; per cell type
alignment: alignment of single-end data against mm9 with tophat version 2.0.10 (-j paired-end_cellType_junctions --b2-sensitive --library-type fr-firststrand) and Bowtie 2 version 2.2.1; per cell type
transcriptome construction: transcriptome construction with Cufflinks version 2.1.1 (-g EnsemblGene-67.TR.gtf --library-type fr-firststrand --max-bundle-frags 2000000); per cell type and within cell type per sequencing data (single-end and paired-end separately)
transcriptome construction: merging of Cufflinks' transcript files with Cuffmerge (-g EnsemblGene-67.TR.gtf --ref-sequence mm9.fa)
filter steps: Cuffmerge transcript file was filtered with Bedtools version 2.16.2 for transcripts which overlap known transcript or its 2 kb up- and downstream flanks on the same strand; overlapping single exon transcripts; transcripts which had a lower estimated abundance than their confidence interval in one of the cell types; transcripts which showed protein-coding conservation
alignment: for differential gene expression analysis; se and pe data was aligned with bwa version 0.5.9-r16 against mm9 with an exon-exon junction library, which is based on Ensembl version 67
read counting: counting was done with featureCounts version 1.4.4 (single-end settings: -s 2 -Q 1 -O; paired-end settings: -s 2 -Q 1 -O -p -B) and the filtered Cuffmerge transcript gtf file on the bwa bam files
gene expression: DESeq2 version 1.4.5 and the count files from featureCounts
Genome_build: mm9
Supplementary_files_format_and_content: tab delimited files from featurecounts (genecount.se.ensembl67.txt, genecount.pe.ensembl67.txt); columns are in the following order: geneid, chromosom, start, end, length, strand, bam files
Supplementary_files_format_and_content: tab delimited files from deseq (deseq.rminus_rplus.ensembl.csv, deseq.rplus_gplus.ensembl.csv); first seven columns are standard output from deseq; columns 8-13 normalised counts for libraries; column 14-15 normalised counts for condition; 16-22 gene information from ensembl (chromosome name, start position, end position, strand, biotype, description)
Supplementary_files_format_and_content: tab delimited files from deseq (deseq.rminus_rplus.ensembl.csv, deseq.rplus_gplus.ensembl.csv); first seven columns are standard output from deseq; columns 8-13 normalised counts for libraries; column 14-15 normalised counts for condition; 16-22 gene information from ensembl (chromosome name, start position, end position, strand, transcript ids)

source_name: Lateral cortex of the brain

extract_protocol:
E14.5 Btg2GFP/Tubb3GFP cortices were dissociated using the papain-based neural dissociation kit (Miltenyi Biotec) after removal of meninges and ganglionic eminences. FACS was performed at 4˚C in the 4-way purity mode with a flow rate of 20 μl/min using side and forward scatter light to eliminate debris and aggregates and gating established for green (488 nm) and red (561 nm) fluorescence. About 1x106 sorted cells from >3 embryos from different litters were immediately lysed in μMACS™ mRNA Isolation Kit and lysates cleaned on LysateClear Colums (Miltenyi) resulting in ca. 1 µg of poly-A RNAs with a integrity number >9.2
Library preparation and enrichment was performed using 15 µl Sera-Mag Oligo(dT) beads (Thermo Scientific) in 50 µl 10 mM Tris-HCl. Samples were treated with 1U Turbo DNAse (Ambion) and purified with Agencourt RNAclean XP beads. Eluted mRNA (18 µl) was chemically fragmented with NEBNext-Mg RNA Fragmentation Module (New England Biolabs), re-purified with RNAclean XP beads and eluted in 13,5 µl nuclease-free water. First strand cDNA synthesis was performed using 0.15 µg/µl Random Primers (New England Biolabs), 1x First Strand Synthesis Reaction Buffer (New England Biolabs), 10 U/uL Superscript II (Invitrogen) with an initial hybridization for 5 min at 65°C with mRNA and primers followed by incubation at 25°C for 10 min, 42°C for 50 min, and 70°C for 15 min. After purification with Agencourt Ampure XP-beads (Beckman Coulter), second strand synthesis was performed using the Second Strand Synthesis module (New England Biolabs) replacing the 2nd strand synthesis buffer with a NTP-free buffer and adding equimolar 2.5 mM of d-nucleotides. Incubation for 2.5 h at 16°C was followed by Ampure XP beads purification as described above. End-Repair was done with the NEBnext End Repair Module (New England Biolabs) followed by XP beads purification and A-Tailing using the NEBnext dA-Tailing Module. Adaptors were ligated  (Adaptor-Oligo 1: 5'-ACA-CTC-TTT-CCC-TAC-ACG-ACG-CTC-TTC-CGA-TCT-3', Adaptor-Oligo 2: 5'-P-GAT-CGG-AAG-AGC-ACA-CGT-CTG-AAC-TCC-AGT-CAC-3') using 1x NEBnext Quick Ligation Buffer (New England Biolabs), 0.3 uM DNA Adaptors, 1 uL Quick T4 DNA Ligase (New England Biolabs) in 50 µl. XP beads purification was followed by dUTP cleavage with 1 U USER enzyme mix (New England Biolabs) per sample and direct enrichment using the PCR Enrich Adaptor Ligated cDNA Library module (New England Biolabs) with indexed primers.

tag: developmental stage

value: Embryonic day 14.5

value: Neurons Tubb3 positive


title: BAT_rep2


title: S355_GFPplus_3 single


title: S353_RFPminus_3 paired-end

value: Proliferating Progenitors Btg2 negative


title: S354_RFPplus_3 paired-end

value: Differentiating Progenitors Btg2 positive


title: NA, Template-switching (4h)

description:
Sample name: NA_TS
A/WSN/33 Influenza (H1N1)
NA, Template-switching (4h)
Template-switching 5′-RACE RNA-Seq

value: template-switching


title: S336_GFPplus_2 paired-end


title: iWAT

source_name: inguinal adipose tissue

value: inguinal adipose tissue


title: wt_spb75_30C

data_processing:
All samples were sequenced (together with other libraries) on an HiSeq 2000 instrument. Individual reads were assigned to their sample based on the TruSeq barcode. Illumina software Casava v 1.8 was used.
The 3' adapter was removed by aligning it to the read allowing one or two mismatches in prefix alignments of at least 7 or 10 bases respectively. Low complexity reads were filtered out based on their dinucleotide entropy (removing <1% of the reads). All the reads that were shorter than 14 nucleotides were removed.
Alignments to the S. pombe genome (May 8, 2009, http://www.sanger.ac.uk/Projects/S_pombe/) were performed by the software bowtie (version 0.9.9.1) ( Langmead et al., 2009) with parameters -v 2 -a -m 100, tracking up to 100 best alignment positions per query and allowing at most two mismatches.
Wiggle files were generated with window size 100, normalized to the library size and transformed into log2 scale.
Genome_build: S. pombe genome (May 8, 2009, http://www.sanger.ac.uk/Projects/S_pombe/)
Supplementary_files_format_and_content: wig file for sequencing coverage with fixed step 100bp windows


title: wt_spb75_30C_2


title: S354_RFPplus_3 single


title: S353_RFPminus_3 single


title: S336_GFPplus_2 single


title: Yeast Core Promoter Library-Targeted Sequencing

city: Rehovot

first: Eran

last: Segal

address: 234 Herzl St.

country: Israel

institute: Weizmann Institute

description: plasmid DNA

data_processing:
Library strategy: Targeted sequencing
Library sequences were padded by N's and concatenated to form a single library ref. sequence.
PE sequencing reads were aligned to the library ref. sequence using the novoalign software (downloaded on 2/Dec/2011) with the options -i PE 180,5 -n 101
Supplementary_files_format_and_content: Tab delimited text; col #1 - sequence name, col #2 - 118bp core promoter sequence, col #3 - measured core promoter activity (expression) in [a.u.]

organism: Saccharomyces cerevisiae

source_name: Yeast cells

extract_protocol: The library was prepared for sequencing using standard Illumina protocols

tag: source

value: yeast cells


title: S335_RFPplus_2 single


title: S334_RFPminus_2 paired-end


title: S334_RFPminus_2 single


title: S168_GFPplus_1 single


title: S167_RFPplus_1  single


title: S167_RFPplus_1 paired-end


title: wt_spb75_37C_8h


title: wt_spb75_37C_7d


title: BMDM PAR-CLIP KO

description:
Total RNA
PAR-CLIP

source_name: Bone marrow derived macrophage

extract_protocol:
Total RNA and small RNAs were isolated as detailed in the manufacturer’s protocols (Clonetech NucleoSpin miRNA) and RNA quality was checked by an Agilent 2100 bioanalyzer (Agilent technologies)
RNA libraries were prepared for sequencing using standard Illumina protocols, PAR-CLIP sample libraries were prepared with PAR-CLIP + TruSeq Small RNA prep.

value: Bone marrow derived macrophage

value: ELAVL1f/f, LysM-Cre

tag: par-clip antibody

value: Anti mouse Ago2 (WAKO 2D4, 041-22023)


title: BMDM PAR-CLIP WT

description:
Total RNA
PAR-CLIP


title: S166_RFPminus_1 single


title: Eu Myc Tert wt Input

description: Sample 1_Eµ-Myc

source_name: Primary lymphoma cells

value: Primary lymphoma cells

tag: treatment

tag: chip antibody

value: none (input)

value: Tert +/+


title: Eu Myc Tert ko Input

description: Sample 3_Eµ-Myc

value: Tert -/-


title: Eu Myc Tert ko ChIP

description: Sample 4_Eµ-Myc

value: c-Myc Antibody (N-262) (Santa Cruz, catalog# sc-764, lot# D0413)


title: S. pombe_20M_20min

status: Public on Apr 22 2015

city: Stanford

first: William

middle: J

last: Greenleaf

state: CA

address: 279 Campus Drive West

institute: Stanford University

description:
S. pombe ATAC-seq sample
processed data files: Pombe.nucpos.bed, Pombe.nucsig.bw, Pombe.occ.bw, pombe.Scores.bw, pombe.mappable.bed

data_processing:
Library strategy: ATAC-seq

Adapters were trimmed from reads before alignment using bowtie 2 with the -X 2000 flag.  Duplicate fragments for each sample were discarded, as were reads mapping to chrM, reads with mapping quality less than 30, and reads that were not properly paired.

Nucleosome calling was performed in broad open chromatin regions determined using MACS2 and filtered for high mappability.  For the osmotic stress time-course, nucleosome calling was performed in regions around TSS that overlapped with broad open chromatin peaks in at least one time point.

genome build: sacCer3 for S. cerevisiae, ASM294v2.21 for S. pombe, and hg19 for human GM12878

processed data files format and content: *.nucpos.bed.gz files contain nucleosome positions.  Columns are as follows:  1) chromosome 2) dyad position (0-based) 2) dyad position (1-based) 3)

processed data files format and content: *.nucleoatac_signal.bw are BigWig files containing the normalized NucleoATAC signal

processed data files format and content: *.occ.bw are BigWig files contining the nucleosome occupancy

processed data files format and content: *.peaks.final.bed are highly mappable open chromatin regions for which nucleosome calling was performed. For the osmotic stress time course, analyis was performed on regions around tss that overlapped with peaks (osmotic.tss_peak_regions.bed).

processed data files format and content: *.Scores.bw  are BigWig files that contain transposase preference scores used in the sequence bias normalization for nucleosome calling.  These scores represent the log of the relative probability of insertion.

source_name: Schizosaccharomyces pombe cells

growth_protocol: Cells were grown to mid-log phase in YES

extract_protocol:
20 M Cells were washed twice in Sorbitol Buffer  (1.4 M Sorbitol, 40 mM HEPES-KOH pH 7.5, 0.5 mM MgCl2)  + 10 mm BME, incubated in Sorbitol Buffer + 2mm BME for 30 minutes, and the incubated in Sorbitol buffer with 0.25 mg/mL 100T Zymolyase for 23 minutes, and then washed twice in Sorbitol Buffer before incubated with 2.5 μl of Nextera Transposase in 47.5 μl of 1x TD buffer at 37 degrees for 30 minutes.
Sequencing libraries were constructed using a modified version of the Illumina Nextera DNA Sample prep kit, as per Buenrostro et al. (2013).

tag: growth stage

value: mid-log


title: S. pombe_5M_20min

extract_protocol:
5 M Cells were washed twice in Sorbitol Buffer  (1.4 M Sorbitol, 40 mM HEPES-KOH pH 7.5, 0.5 mM MgCl2)  + 10 mm BME, incubated in Sorbitol Buffer + 2mm BME for 30 minutes, and the incubated in Sorbitol buffer with 0.25 mg/mL 100T Zymolyase for 23 minutes, and then washed twice in Sorbitol Buffer before incubated with 2.5 μl of Nextera Transposase in 47.5 μl of 1x TD buffer at 37 degrees for 30 minutes.
Sequencing libraries were constructed using a modified version of the Illumina Nextera DNA Sample prep kit, as per Buenrostro et al. (2013).


title: S. pombe_5M_5min

extract_protocol:
5 M Cells were washed twice in Sorbitol Buffer  (1.4 M Sorbitol, 40 mM HEPES-KOH pH 7.5, 0.5 mM MgCl2)  + 10 mm BME, incubated in Sorbitol Buffer + 2mm BME for 30 minutes, and the incubated in Sorbitol buffer with 0.25 mg/mL 100T Zymolyase for 5 minutes, and then washed twice in Sorbitol Buffer before incubated with 2.5 μl of Nextera Transposase in 47.5 μl of 1x TD buffer at 37 degrees for 30 minutes.
Sequencing libraries were constructed using a modified version of the Illumina Nextera DNA Sample prep kit, as per Buenrostro et al. (2013).


title: S. pombe_1M_5min

extract_protocol:
1 M Cells were washed twice in Sorbitol Buffer  (1.4 M Sorbitol, 40 mM HEPES-KOH pH 7.5, 0.5 mM MgCl2)  + 10 mm BME, incubated in Sorbitol Buffer + 2mm BME for 30 minutes, and the incubated in Sorbitol buffer with 0.25 mg/mL 100T Zymolyase for 5 minutes, and then washed twice in Sorbitol Buffer before incubated with 2.5 μl of Nextera Transposase in 47.5 μl of 1x TD buffer at 37 degrees for 30 minutes.
Sequencing libraries were constructed using a modified version of the Illumina Nextera DNA Sample prep kit, as per Buenrostro et al. (2013).


title: Input_5hmC_DIP

city: Dundee

first: Kasper

middle: Dindler

last: Rasmussen

address: Dow Street

department: Division of Molecular, Cellular, and Developmental Biology (MCDB)

country: United Kingdom

institute: School of Life Sciences, University of Dundee

data_processing:
Image capture, analysis and base calling were performed using Illumina’s CASAVA 1.8. Raw sequencing reads obtained from 5hmC-DIP-seq experiments were split according to the sample index, and 3'-adaptor sequences as well as low quality nucleotides were trimmed prior to mapping to the GRCm38 (mm10) mouse genome using bowtie with parameters “-m1 --strata --best”. Potential PCR duplicates were removed by only keeping a single read if multiple reads mapped to the exact same genomic sequence. Finally, peak calling was performed using MACS 1.4 with the IgG sample as control and a p-value cut-off of 1E-5. Pre-processing, mapping, de-duplication and peak calling were performed on the public server of the Galaxy Project.
Genome_build: mm10
Supplementary_files_format_and_content: BED files: Tab-delimited text files containing genomic intervals representing 5hmC-DIP-seq peaks. BIGWIG files: Binary files generated from the pile up track of MACS

source_name: Kit-enriched hematopoietic bone marrow cells retrovirally transduced with AML1-ETO

growth_protocol: Primary Kit-enriched bone marrow cells from Tet2fl/fl;CreER mice were freshly transduced with MigR1-AE-IRES-GFP, sorted for GFP and plated in methylcellulose medium (M3534, StemCell Technologies). After three rounds of replating, the cells were shifted to grow in suspension in non-tissue culture treated plasticware in media containing 1× StemPro®-34 SFM (Invitrogen), 1x L-Glutamine, with SCF (CHO producer cell line), 10ng/ml IL-3 (Peprotech), 10ng/ml IL-6 (Peprotech) and 0.1mM 2-mercapto-ethanol.

extract_protocol:
Immature myeloid cells (GFP+, CD11b-, Gr1-, CD16/32+) were sorted from AML1-ETO cultures. Genomic DNA was isolated using the AllPrep DNA/RNA micro kit (Qiagen).
5hmC DNA Immunoprecipitation and sequencing (5hmC-DIP-seq) of genomic DNA was performed as previously described (Williams et al., 2011, Nature 473, 343–348.). In brief, genomic DNA was sonicated using the Bioruptor (Diagenode) to obtain fragments of approximately 200-500bp. Next, adaptors were ligated using the NEBNext® DNA sample Prep Master Mix according to manufacturer’s instructions (New England Biolabs) and resuspended in 1× IP buffer (10mM NaPhosphate pH 7.0, 0.14M NaCl, 0.5% Triton X-100). 1ug of adaptor ligated DNA was briefly denatured and incubated 4h using an IgG control antibody or a polyclonal rabbit antibody directed against 5hmC. Anti-Rabbit dynabeads (Invitrogen) were added and incubated for additional 2h before immunoprecipitation, washing and elution by proteinase K digestion. The eluted DNA was PCR-amplified using indexed multiplex primers for illumina sequencing (New England Biolabs), pooled and sequenced on HiSeq 2000.

treatment_protocol: To induce the CreER-mediated excision of Tet2, the cells were treated with 200 nM 4-OHT (Experimental) or EtOH (Control) for 2 passages (4 days). The cells were subsequently washed to remove residual 4-OHT and passaged every 2 days for a total of 10 to 20 passages.

tag: passage number

tag: dip antibody


title: Tet2_flox_MLL_AF9_p20_Rep3

description: MspI digested, bisulfite-treated

data_processing:
Image capture, analysis and base calling were performed using Illumina’s CASAVA 1.8. Reads were filtered from the adaptor sequence using TrimGalore 0.3.1 (--rrbs option). The processed reads were aligned to a bisulfite-converted mm10 reference genome with Bismark 0.10.1 (Krueger, F., and Andrews, S.R. (2011), Bioinformatics 27, 1571–1572) and Bowtie 2 using default settings. Finally, the CpG-context methylation state were extracted using Bismark methylation extractor to produce the final tab-separated *CpG_context.txt files for each sample. Column headers in *CpG_context.txt files are as follows: 1) Seq-ID, 2) Methylation state, 3) Chromosome, 4) Start/End, 5) Methylation call. Methylated cytosines will receive a '+' orientation, unmethylated cytosines will receive a '-' orientation.
Genome_build: mm10
Supplementary_files_format_and_content: Tab-delimited text files containing methylation calls for cytosines in CpG-context

source_name: Kit-enriched hematopoietic bone marrow cells retrovirally transduced with MLL-AF9

growth_protocol: Primary Kit-enriched bone marrow cells from Tet2fl/fl;CreER mice were freshly transduced with MigR1-AE-IRES-GFP, sorted for GFP and plated in methylcellulose medium (M3534, StemCell Technologies). After three rounds of replating, the cells were shifted to grow in suspension in non-tissue culture treated plasticware in media containing 1× StemPro®-34 SFM (Invitrogen), 1x L-Glutamine, with SCF (CHO producer cell line), 10ng/ml IL-3 (Peprotech), 10ng/ml IL-6 (Peprotech) and 0.1mM 2-mercapto-ethanol. Primary hematopoietic cell lines expressing MLL-AF9 in the Tet2fl/fl;CreER background was generated and maintained in suspension media as previously described (Somervaille, T.C.P., and Cleary, M.L. (2006), Cancer Cell 10, 257–268)

extract_protocol:
Immature myeloid cells (GFP+, CD11b-, Gr1-, CD16/32+) were sorted from AML1-ETO cultures. Genomic DNA was isolated using the AllPrep DNA/RNA micro kit (Qiagen).
Enhanced Reduced Representation Bisulfite Sequencing (eRRBS) libraries were generated as previously described (Akalin, A., et al (2012), PLoS Genet. 8, e1002781) with the exception that end-repair and adaptor ligation were performed using the NEBNext® DNA library Sample Prep Master kit (New England Biolabs). In brief, ~1ug of genomic DNA was digested with MspI enzyme overnight followed by phenol/chloroform extraction. After library generation using pre-annealed methylcytosine-containing illumina adaptors (TruSeq® DNA sample prep kit, Illumina) and size-selection (150-250bp and 250-400bp), the ligated fragments were treated with bisulfite (EZ DNA methylation kit, Zymo) to convert unmethylated cytosines. Finally, the converted fragments were PCR-amplified (15x PCR cycles), pooled and sequenced on an Illumina Hiseq2000.

tag: experiment

tag: lane

tag: index

value: ACAGTG

tag: pcr cycles


title: Tet2_ko_MLL_AF9_p20_Rep2

description: MspI digested, bisulfite-treated

tag: strain

value: CAGATC

tag: genotype


title: Tet2_ko_MLL_AF9_p20_Rep1

description: MspI digested, bisulfite-treated

value: CTTGTA


title: Tet2_flox_MLL_AF9_p20_Rep1

description: MspI digested, bisulfite-treated

value: GCCAAT


title: Tet2_ko_MLL_AF9_p20_Rep3

description: MspI digested, bisulfite-treated

value: CGATGT


title: Tet2_ko_AML1_ETO_p10

description: MspI digested, bisulfite-treated


title: S168_GFPplus_1 paired-end

value: Embryonic day 14.5

tag: cell type

value: Neurons Tubb3 positive


title: Tet2_ko_AML1_ETO_p2

description: MspI digested, bisulfite-treated


title: Tet2_flox_AML1_ETO_p2

description: MspI digested, bisulfite-treated


title: S166_RFPminus_1 paired-end

value: Proliferating Progenitors Btg2 negative


title: S. pombe_20M_5min

extract_protocol:
20 M Cells were washed twice in Sorbitol Buffer  (1.4 M Sorbitol, 40 mM HEPES-KOH pH 7.5, 0.5 mM MgCl2)  + 10 mm BME, incubated in Sorbitol Buffer + 2mm BME for 30 minutes, and the incubated in Sorbitol buffer with 0.25 mg/mL 100T Zymolyase for 5 minutes, and then washed twice in Sorbitol Buffer before incubated with 2.5 μl of Nextera Transposase in 47.5 μl of 1x TD buffer at 37 degrees for 30 minutes.
Sequencing libraries were constructed using a modified version of the Illumina Nextera DNA Sample prep kit, as per Buenrostro et al. (2013).


title: KO_Rep2_5hmC_DIP

value: Polyclonal rabbit Anti-5hmC


title: Flox_Rep2_5hmC_DIP

value: TGACCA


title: Flox_Rep1_5hmC_DIP


title: KO_Rep1_5hmC_DIP


title: Eu Myc Tert wt ChIP

description: Sample 2_Eµ-Myc

extract_protocol:
Cells were treated with 1% formaldehyde for 10 minutes at room temperature to crosslink protein-DNA complexes, and reactions were then quenched using 125 mM glycine for 5 minutes. The cells were then lysed with sodium dodecyl sulfate (SDS) lysis buffer and sonicated for 10 minutes using the Diagenode Bioruptor. The fragmented chromatin was pre-cleared with bovine serum albumin (BSA), protein A Sepharose beads (Millipore) at 4 ˚C for 2 hours before immunoprecipitation with c-MYC or rabbit control IgG antibodies overnight at 4 ˚C. Sepharose beads were washed as described previously and eluted with SDS elution buffer before subjecting to reverse cross-linking at 65 ˚C overnight. Finally samples were purified using the QIAquick PCR Purification Kit (Qiagen) according to the manufacturer’s protocol.
Single read - Illumina


title: IgG control_5hmC_DIP

value: ACTTGA


title: gon_smg2_jr02

city: Santa Cruz

first: James

middle: Matthew

last: Ragle

address: 1165 High Street Sinsheimer Labs 419

department: MCD Biology

institute: University of California-Santa Cruz

description: rRNA depleted RNA

data_processing:
50x50bp paired-end reads were mapped with bowtie2 to the set of RepeatMasker elements for C.elegans, and reads with both ends mapped to such elements were removed from further processing.
Samples were mapped with Tophat2 using Bowtie2 as the underlying alignment tool. The target assemby was ce6. Tophat was supplied with a gene model using the --GTF option. The gene model was from wormBase.
For multiply mapped reads, only the best scoring mapping per bowtie2 were kept. Only mappings with both of the paired-end reads mapped were kept.
Potential PCR duplicates were removed with samtool rmdup.
Splice junctions were extracted from the bam files by examining the CIGAR string, resulting in a non-redundant set of introns spanned by these splice junctions.
Analysis 1:  Tissue-specific alternative splicing events were identified with Splicetrap using a minimum delta IR of 0.3 between gon_n2wt_jr01 and sgo_glp-4_sb26
Analysis 2:  The read-spanned introns were filtered for those with common 5prime position, and multiple 3prime positions within 18nt in gon_smg-2_jr02
Analysis 2:  These introns were then further filtered for those with a minimum splicing change of 0.3 between gon_n2wt_jr01 and sgo_glp-4_sb26
Analysis 3:  The Table Browser from the UCSC Genome Browser was used to filter all introns that terminate in an AG dinucleotide
Analysis 3:  These introns were further filtered to identify those with a terminal AG dinucleotide as well as an AG dinucleotide 6nt upstream
Analysis 3:  These introns were finally filtered for those with a minimum splicing change of 0.3 between gon_n2wt_jr01 and sgo_glp-4_sb26
Genome_build: May 2008 (WS190/ce6)
Supplementary_files_format_and_content: text file of identified tissue-specific splice sites

organism: Caenorhabditis elegans

growth_protocol: Strains: glp-4(bn2ts), smg-2(e2008) and wildtype (Bristol N2 strain) worms were obtained from the Caenorhabditis Genetics Center. Synchronized worm populations were obtained by axenization of a mixed population of worms cultured on liquid media at 20°C to isolate embryos. After synchronization, all worms were cultured at 25°C, the restrictive temperature for glp-4(bn2ts), and the developmental stage was confirmed by microscopy of vulva development (white-crescent stage).

extract_protocol:
Tissues were placed into 300µl TRIzol (Invitrogen) on ice.  60µl chloroform was added to the TRIzol/tissue samples and phases were separated using prespun Phase Lock Gel-Heavy tubes (5 Prime).  The aqueous phase was transferred to a new tube and mixed with 125-175µl isopropanol and 1µl GlycoBlue (Ambion).  RNA was precipitated overnight at  -20°C and pelleted at 13,000xg at 4°C for 30 minutes.  The pellet was washed with 75% ethanol, dried, and resuspended in 10-20µl ddH2O.
Gonad samples were depleted of rRNA with a RiboZero kit (Epicentre) and whole worm samples were poly-A selected.  mRNA sequencing libraries were sequenced according to manufacturer recommendations with Truseq Stranded mRNA Sample kit (Illumina) at the Centre for Genomic Regulation sequencing facility in Barcelona, Spain.

tag: tissue type

value: dissected gonad

tag: rrna status

value: Ribo-zero depleted


title: sgo_glp4_sb26

description: poly-A RNA

value: whole worm (depleted germline)

value: present


title: Tet2_flox_MLL_AF9_p20_Rep2

description: MspI digested, bisulfite-treated


title: sgo_n2wt_sb25

description: poly-A RNA

source_name: N2 wildtype

value: wildtype

value: Bristol N2

value: whole worm


title: gon_n2wt_jr01

description: rRNA depleted RNA


title: sgo_n2wt_sb24

description: poly-A RNA


title: Ch17.B50-PM-3hrs-bio-replicate1

city: Tallahassee

middle: M.

last: Gilbert

state: Florida

address: 319 Stadium Drive

institute: Florida State University

description:
bar code: TGAAACACAGCCATCAAAGC
4C-seq library constructed using HindIII.

data_processing:
Library strategy: 4C-seq
50 bp-long single-end reads were first de-multiplexed using their barcodes and assigned to the corresponding time point and 4C bait. Only the reads that contained one of the valid barcodes was retained for mapping.
Each read was mapped to the mouse genome (UCSC mm9) using the short read alignment mode of BWA (v0.5.9) with default parameter settings. Mapping results were post-processed, and only the reads that mapped uniquely with an alignment quality score of at least 30 and an edit distance of at most 1 were qualified for further analysis.
Each qualified read was assigned to the nearest HindIII cleavage site which represents a restriction fragment.
50 kb upstream and downstream of the bait position was removed from this raw data.
Contact counts at HindIII sites for all chromosomes were windowed (10 kb windows), normalized for sequencing depth and smoothed using a running mean with a span of 30 windows. The resulting smoothed profiles (contact count) were plotted as a function of distance along the chromosome.
Genome_build: mm9
Supplementary_files_format_and_content: rawContactCounts.txt file lists the starting coordinates of all HindIII fragments (defined as the 6-bp recognition site of AAGCTT) and for each such fragment the number of contact counts assigned to that fragment by mapping the library listed in each column.

source_name: C127 cells

growth_protocol: C127 cells were grown in DMEM with 10% fetal calf serum as decribed in (Lu J et.al, 2010)

extract_protocol:
Cell cycle synchronized cultures were crosslinked for 10 minutes at RT in 1% formaldehyde. The reaction was quenched for 5 min at RT. We then applied 4C-seq method. Briefly, nuceli were isolated from the cross-linked cells and digested using the restriction enzyme HindIII. Fragments were ligated under dilute conditions and reversed cross-linked. The samples were then digested with DpnII and circularized. Subsequently bait specific primers were used to amplify the interaction partners and sequenced using the Illumina HiSeq 2500 platform, generating 50 bp reads.
Libraries were generated by amplifying the 4C template using bait specific primers with added illumina adaptors. Bait is the genomic loci of interest whose genome-wide interactions are being measured by 4C-seq. We denote each bait by its chromosome followed by letter B and the approximate megabase-scale coordinate of the bait (e.g. Ch8.B26 means bait with a coordinate on 26th megabase of chromosome 8). Twelve baits were chosen based on their replication timing regulation and were named Ch8.B7,Ch8.B26,Ch8.B44,Ch8.B53,Ch8.B87,Ch8.B118,Ch16.B30,Ch16.B41,Ch16.B46,Ch16.B48,Ch17.B50,Ch17.B50 Coordinates are in mm9.

treatment_protocol: Cultures were synchronized at mitosis using a 4 hr nocodazole block and then released into G1 for 0.5, 1, 2, 3, 4 hr. G2 syncheonization was obtained by 15 hr release. Cells were synchronized in G0 by 5 day serum starvation.

tag: cell cycle phase

value: 3 hrs post-mitosis

tag: cell line


title: Ch17.B76-PM-3hrs-bio-replicate1

description:
bar code: AGATGGCAAACCACATTAAGC
4C-seq library constructed using HindIII.


title: Ch17.B50-PM-30mins-bio-replicate1

value: 30 mins post-mitosis


title: Ch17.B50-PM-2hrs-bio-replicate1

description:
bar code: AGCTGAAACACAGCCATCAAAGC
4C-seq library constructed using HindIII.

value: 2 hrs post-mitosis


title: Ch17.B50-PM-1hr-bio-replicate1

value: 1 hr post-mitosis


title: Tet2_flox_AML1_ETO_p10

description: MspI digested, bisulfite-treated

value: GCCAAT


title: S335_RFPplus_2 paired-end

tag: developmental stage

value: Embryonic day 14.5

value: Differentiating Progenitors Btg2 positive


title: Ch16.B48-PM-4hrs-bio-replicate1

description:
bar code: GGCTGTAGCTTAGGGAAGC
4C-seq library constructed using HindIII.

value: 4 hrs post-mitosis


title: Ch17.B76-PM-2hrs-bio-replicate1

description:
bar code: AGGAGATGGCAAACCACATTAAGC
4C-seq library constructed using HindIII.


title: Ch16.B48-PM-2hrs-bio-replicate2

description:
bar code: GATGGCTGTAGCTTAGGGAAGC
4C-seq library constructed using HindIII.


title: Ch17.B76-PM-30mins-bio-replicate1


title: Ch16.B48-PM-2hrs-bio-replicate1


title: Ch16.B48-PM-1.5hrs-bio-replicate1

value: 1.5 hrs post-mitosis


title: Ch16.B48-PM-30mins-bio-replicate1


title: Ch16.B46-PM-3hrs-bio-replicate1

description:
bar code: GCTACCTAACTGGGCAAAGC
4C-seq library constructed using HindIII.


title: Ch16.B46-PM-1hr-bio-replicate1

description:
bar code: CCTGCTACCTAACTGGGCAAAGC
4C-seq library constructed using HindIII.


title: Ch16.B46-PM-1.5hrs-bio-replicate1


title: Ch16.B46-PM-2hrs-bio-replicate1


title: Ch16.B48-PM-1hr-bio-replicate1


title: Ch16.B41-PM-4hrs-bio-replicate1

description:
bar code: AAGGCACAGGTAGCCAAGC
4C-seq library constructed using HindIII.


title: Ch16.B46-PM-2hrs-bio-replicate2


title: Ch16.B41-PM-2hrs-bio-replicate1

description:
bar code: AACAAGGCACAGGTAGCCAAGC
4C-seq library constructed using HindIII.


title: Ch16.B41-PM-1.5hrs-bio-replicate1


title: Ch16.B41-PM-1hr-bio-replicate1


title: Ch16.B41-PM-30mins-bio-replicate1


title: Ch16.B41-PM-2hrs-bio-replicate2


title: Ch17.B76-PM-1hr-bio-replicate1


title: Ch16.B30-PM-2hrs-bio-replicate1

description:
bar code: GCACGGAAACTCAGCGGTAAAGC
4C-seq library constructed using HindIII.


title: Ch16.B30-PM-4hrs-bio-replicate1

description:
bar code: CGGAAACTCAGCGGTAAAGC
4C-seq library constructed using HindIII.


title: Ch8.B118-PM-1.5hrs-bio-replicate1

description:
bar code: CCTCATACAGCTGGAAGCTT
4C-seq library constructed using HindIII.


title: Ch16.B48-PM-3hrs-bio-replicate1


title: Ch16.B30-PM-1.5hrs-bio-replicate1


title: Ch16.B30-PM-30mins-bio-replicate1


title: Ch16.B30-PM-1hr-bio-replicate1


title: Ch16.B30-PM-2hrs-bio-replicate2


title: Ch8.B87-PM-1.5hrs-bio-replicate1

description:
bar code: GGGCGGAGATGGAAGCTT
4C-seq library constructed using HindIII.


title: Ch8.B53-PM-1.5hrs-bio-replicate1

description:
bar code: AAACATGTTTGGCCAAGCTT
4C-seq library constructed using HindIII.


title: Ch16.B46-PM-4hrs-bio-replicate1


title: Ch8.B118-PM-4hrs-bio-replicate1


title: Ch16.B46-PM-30mins-bio-replicate1


title: Ch8.B118-PM-30mins-bio-replicate1


title: Ch8.B118-PM-3hrs-bio-replicate1


title: Ch8.B118-PM-1hr-bio-replicate2

description:
bar code: CCGACCTCATACAGCTGGAAGCTT
4C-seq library constructed using HindIII.


title: Ch16.B41-PM-3hrs-bio-replicate1


title: Ch8.B118-PM-2hrs-tech-replicate2


title: Ch8.B118-PM-2hrs-tech-replicate1


title: Ch8.B118-PM-1hr-bio-replicate1


title: Ch8.B118-G0-bio-replicate1


title: Ch8.B118-G2-bio-replicate1


title: Ch16.B30-PM-3hrs-bio-replicate1


title: Ch8.B118-G0-bio-replicate2


title: Ch8.B87-PM-4hrs-bio-replicate1


title: Ch8.B87-PM-3hrs-bio-replicate1


title: Ch8.B118-PM-30mins-bio-replicate2


title: Ch8.B87-PM-30mins-bio-replicate2

description:
bar code: CGCAGGGCGGAGATGGAAGCTT
4C-seq library constructed using HindIII.


title: Ch8.B87-PM-2hrs-tech-replicate1


title: Ch8.B87-PM-2hrs-tech-replicate2


title: Ch8.B87-PM-1hr-bio-replicate2


title: Ch8.B87-PM-30mins-bio-replicate1


title: Ch8.B87-PM-1hr-bio-replicate1


title: Ch8.B87-G0-bio-replicate2


title: Ch8.B53-PM-4hrs-bio-replicate1


title: Ch8.B87-G0-bio-replicate1


title: Ch8.B87-G2-bio-replicate1


title: Ch8.B53-PM-30mins-bio-replicate1


title: Ch8.B53-PM-2hrs-tech-replicate1


title: Ch8.B53-PM-3hrs-bio-replicate1


title: Ch8.B53-PM-2hrs-tech-replicate2


title: Ch8.B53-PM-1hr-bio-replicate2

description:
bar code: GTAGAAACATGTTTGGCCAAGCTT
4C-seq library constructed using HindIII.


title: Ch8.B53-PM-1hr-bio-replicate1


title: Ch8.B53-M-0hr-bio-replicate1

value: Mitosis


title: Ch8.B53-G2-bio-replicate1


title: Ch8.B53-G0-bio-replicate2


title: Ch8.B44-PM-3hrs-bio-replicate1

description:
bar code: GGTCATCCCTTTAGAAGCTT
4C-seq library constructed using HindIII.


title: Ch8.B44-PM-4hrs-bio-replicate1


title: Ch8.B53-G0-bio-replicate1


title: Ch8.B44-PM-30mins-bio-replicate1


title: Ch8.B44-PM-30mins-bio-replicate2

description:
bar code: ATCGGTCATCCCTTTAGAAGCTT
4C-seq library constructed using HindIII.


title: Ch8.B44-PM-2hrs-tech-replicate2


title: Ch8.B44-G2-bio-replicate1


title: Ch8.B44-PM-1hr-bio-replicate1


title: Ch8.B44-M-0hr-bio-replicate1


title: Ch8.B26-PM-4hrs-bio-replicate1

description:
bar code: TTGAGTTTGACGCCAAGCTT
4C-seq library constructed using HindIII.


title: Ch8.B44-G0-bio-replicate1


title: Ch8.B44-G0-bio-replicate2


title: Ch8.B53-PM-30mins-bio-replicate2


title: Ch8.B26-PM-3hrs-bio-replicate1


title: Ch8.B26-PM-30mins-bio-replicate2

description:
bar code: TAATTTGAGTTTGACGCCAAGCTT
4C-seq library constructed using HindIII.


title: Ch8.B26-PM-2hrs-tech-replicate1


title: Ch8.B26-PM-30mins-bio-replicate1


title: Ch8.B26-PM-2hrs-tech-replicate2


title: Ch8.B26-PM-1hr-bio-replicate2


title: Ch8.B26-M-0hr-bio-replicate1


title: Ch8.B26-PM-1hr-bio-replicate1


title: Ch8.B26-G2-bio-replicate1


title: Ch8.B7-PM-3hrs-bio-replicate1

description:
bar code: TATTAGGACAGACAGAAGCTT
4C-seq library constructed using HindIII.


title: Ch8.B7-PM-4hrs-bio-replicate1


title: Ch8.B7-PM-30mins-bio-replicate2

description:
bar code: TACTTATTAGGACAGACAGAAGCTT
4C-seq library constructed using HindIII.


title: Ch8.B7-PM-30mins-bio-replicate1


title: Ch8.B44-PM-2hrs-tech-replicate1


title: Ch8.B7-PM-1hr-bio-replicate2


title: Ch8.B7-PM-2hrs-tech-replicate1


title: Ch8.B7-M-0hr-bio-replicate1


title: Ch8.B7-PM-2hrs-tech-replicate2


title: Ch8.B26-G0-bio-replicate1


title: Ch8.B7-G2-bio-replicate1


title: Ch8.B7-PM-1hr-bio-replicate1


title: Ch8.B7-G0-bio-replicate2


title: iOSE4_INPUT_NA_rep2_1

city: Los Angeles

first: Simon

middle: A

last: Gayther

address: 1441 Eastlake Ave.

institute: University of Southern California

data_processing:
Illumina analysis pipeline and aligned to the Human Reference Genome (assembly hg19) using BWA version 0.6.1-r104
Filtered reads with a quality score lower than 20 with samtools
Enriched regions of the genome were identified by comparing ChIP and FAIRE samples using the MACS2 peak caller version 2.0.10 with default settings (this filters duplicate reads)
Genome_build: hg19
Supplementary_files_format_and_content: narrowPeaks generated with MACS2 2.0.10

source_name: ovarian epithelial cell line

growth_protocol: Immortalised OSEC lines (IOE4, IOE11), derived from normal ovaries and immortalized using ectopic expression of TERT, have been previously described and were cultured in medium 199:MCDB105 (mixed in a 1:1 ratio, both Sigma) supplemented with L-glutamine (Lonza) and 15% fetal bovine serum (Hyclone). FTSEC lines (FT33, FT246) were immortalised using TERT, a mutant CDK4 allele, and a shRNA targeting TP53. FTSECs were cultured in DMEM/F12 (Lonza) supplemented with 2% Ultroser G (Crescent Chemicals). An EEC cell line (EEC16), derived from primary endometriosis epithelial culture, has been previously described and was grown in normal ovarian epithelial cell complete medium.

extract_protocol: For each cell line, four 15 cm culture dishes of approximately 90% confluent cells were cross-linked in 1% formaldehyde for 10 min at room temperature before lysing cells in SDS buffer and sonicating to shear genomic DNA to approximately 100-500 bp fragments. Insoluble cell debris was discarded, and the supernatant from pairs of dishes were combined to represent two biological replicates per cell line. The material was divided into aliquots for input, FAIRE and ChIP. Immunoprecipitations were performed overnight at 4°C using antibodies against H3K27ac (Abcam ab4729) and H3K4me1 (Abcam ab8895) followed by incubation with protein A/G agarose beads (Pierce).  Following washing and elution from beads, the DNA was ethanol precipitated, and the pellet washed with 70% ethanol.  Recovered DNA was re-suspended in water for sequencing. Purification and recovery of FAIRE material was performed as previously described in parallel with ChIP to ensure that annotation of open chromatin/nucleosome depletion was performed from the same cell lysate sample as annotation of histone acetylation and methylation marks.

tag: tissue

value: ovarian epithelial cells

tag: antibody


title: iOSE4_INPUT_NA_rep1_1


title: iOSE4_H3K27ac_NA_rep2_1

value: H3K27ac (Abcam ab4729)


title: iOSE4_H3K27ac_NA_rep1_1


title: iOSE4_H3K4me1_NA_rep1_1

value: H3K4me1 (Abcam ab8895)


title: iOSE4_H3K4me1_NA_rep2_1


title: Ch8.B44-PM-1hr-bio-replicate2


title: Ch8.B26-G0-bio-replicate2


title: iOSE4_FAIRE_NA_rep1_1


title: iOSE4_FAIRE_NA_rep2_1


title: iOSE4_FAIRE_NA_rep3_1


title: iOSE11_INPUT


title: iOSE4_FAIRE_NA_INPUT


title: iOSE11_H3K4me1_NA_rep1_1


title: iOSE11_H3K4me1_NA_rep2_1


title: iOSE11_H3K27ac_NA_rep1_1


title: iOSE11_H3K27ac_NA_rep2_1


title: iOSE11_FAIRE_NA_rep1_1


title: iOSE11_FAIRE_NA_rep2_1


title: iOSE11_FAIRE_INPUT


title: iFTSEC33_INPUT_NA_rep1_1

source_name: fallopian tube secretory epithelial cell line

value: fallopian tube secretory epithelial cells


title: Ch8.B7-G0-bio-replicate1


title: iFTSEC33_H3K4me1_NA_rep2_1


title: iFTSEC33_INPUT_NA_rep2_1


title: iFTSEC33_H3K27ac_NA_rep1_1


title: iFTSEC33_H3K27ac_NA_rep2_1


title: iFTSEC33_FAIRE_NA_rep2_1


title: iFTSEC246_INPUT_rep1_1


title: iFTSEC246_INPUT_NA_rep2_1


title: iOSE4_FAIRE_NA_rep4_1


title: iFTSEC33_FAIRE_NA_rep1_1


title: iFTSEC246_H3K4me1_NA_rep2_1


title: Osmotic Stress Time 60 F rep2

description:
S. cerevisiae osmotic stress ATAC-seq time course sample
processed data files: F.nucpos.bed, F.nucsig.bw, F.occ.bw, sacCer3.scores.bw, sacCer3.mappable.bed

source_name: Saccharomyces cerevisiae cells

growth_protocol: Cells were grown to mid-log phase in YPD

extract_protocol:
5 million cells were washed once in Sorbitol buffer (1.4 M Sorbitol, 40 mM HEPES-KOH pH 7.5, 0.5 mM MgCl2) + 10 mm DTT + 0.6 M NaCl before being incubated for 5 min at 30 degrees and shaking at 300 rpm with 0.5 mg/mL 100T zymolyase.  The cells were then washed once in Sorbitol Buffer + 0.6 M NaCl before being incubated with 2.5 ml of Nextera Transposase in 47.5 ml of 1x TD buffer at 37 degrees shaking at 300 rpm for 15 minutes.
Sequencing libraries were constructed using a modified version of the Illumina Nextera DNA Sample prep kit, as per Buenrostro et al. (2013).

treatment_protocol: 0.6 M NaCl was added 60 minutes prior to cells being harvested

tag: growth stage

value: mid-log

tag: treatment

value: 0.6 M NaCl 60 minutes


title: iFTSEC246_H3K27ac_NA_rep2_1


title: iFTSEC246_H3K4me1_NA_rep1_1


title: iFTSEC246_FAIRE_NA_rep2_1


title: Osmotic Stress Time 60 F rep1


title: iFTSEC246_FAIRE_NA_rep1_1


title: iFTSEC246_H3K27ac_NA_rep1_1


title: Osmotic Stress Time 45 E rep1

description:
S. cerevisiae osmotic stress ATAC-seq time course sample
processed data files: E.nucpos.bed, E.nucsig.bw, E.occ.bw, sacCer3.scores.bw, sacCer3.mappable.bed

source_name: Saccharomyces cerevisiae cell

treatment_protocol: 0.6 M NaCl was added 45 minutes prior to cells being harvested

value: 0.6 M NaCl 45 minutes


title: Osmotic Stress Time 45 E rep2


title: Osmotic Stress Time 30 D rep2

description:
S. cerevisiae osmotic stress ATAC-seq time course sample
processed data files: D.nucpos.bed, D.nucsig.bw, D.occ.bw, sacCer3.scores.bw, sacCer3.mappable.bed

treatment_protocol: 0.6 M NaCl was added 30 minutes prior to cells being harvested

value: 0.6 M NaCl 30 minutes


title: Osmotic Stress Time 30 D rep1


title: iEEC16_INPUT_NA_rep2_1

source_name: endometriosis epithelial cell line

value: endometriosis epithelial cells


title: Osmotic Stress Time 15 C rep2

description:
S. cerevisiae osmotic stress ATAC-seq time course sample
processed data files: C.nucpos.bed, C.nucsig.bw, C.occ.bw, sacCer3.scores.bw, sacCer3.mappable.bed

treatment_protocol: 0.6 M NaCl was added 15 minutes prior to cells being harvested

value: 0.6 M NaCl 15 minutes


title: iEEC16_H3K4me1_NA_rep1_1


title: iFTSEC33_H3K4me1_NA_rep1_1


title: Osmotic Stress Time 15 C rep1


title: Osmotic Stress Time 0 B rep2

description:
S. cerevisiae osmotic stress ATAC-seq time course sample
processed data files: B.nucpos.bed, B.nucsig.bw, B.occ.bw, sacCer3.scores.bw, sacCer3.mappable.bed


title: Osmotic Stress Time 0 B rep1


title: iEEC16_H3K27ac_NA_rep2_1


title: Osmotic Stress Time 0 A rep2

description:
S. cerevisiae osmotic stress ATAC-seq time course sample
processed data files: A.nucpos.bed, A.nucsig.bw, A.occ.bw, sacCer3.scores.bw, sacCer3.mappable.bed

extract_protocol:
5 million cells were washed once in Sorbitol buffer (1.4 M Sorbitol, 40 mM HEPES-KOH pH 7.5, 0.5 mM MgCl2) + 10 mm DTT before being incubated for 5 min at 30 degrees and shaking at 300 rpm with 0.5 mg/mL 100T zymolyase.  The cells were then washed once in Sorbitol Buffer before being incubated with 2.5 ml of Nextera Transposase in 47.5 ml of 1x TD buffer at 37 degrees shaking at 300 rpm for 15 minutes.
Sequencing libraries were constructed using a modified version of the Illumina Nextera DNA Sample prep kit, as per Buenrostro et al. (2013).


title: iEEC16_H3K27ac_NA_rep1_1


title: iEEC16_FAIRE_NA_rep1_1


title: GM12878_GSM1155960

description:
Nucleosome calling for merged dataset from GSM1155957, GSM1155958, GSM1155959, GSM1155960 ATAC-seq samples; processed data available on the series level
processed data files: GM.nucpos.bed, GM.nucsig.bw, GM.occ.bw, hg19.Scores.bw, GM.peaks.final.bed

source_name: GM12878 cells

growth_protocol: GM12878 cells were grown in suspension using RPMI 1640 with 15% FBS in T-75 flasks, cells were maintained between 200,000-800,000 cells/ml.

extract_protocol:
GM12878 cells were harvested at a concentration of 500,000 cells/ml, CD4+ T cells were isolated from 5 ml of blood using negative selection. For both cell types, 50,000 cells were used for each transposition reaction. For the GM12878 cells, an additional experiment was performed using 500 cells. Nuclei were prepared prior to transposition.
Sequencing libraries were constructed using a modified version of the Illumina Nextera DNA Sample prep kit, as per Buenrostro et al. (2013).


title: S. cerevisiae lin6c

description:
S. cerevisiae ATAC-seq sample. Four populations of S. cerevisiae strain GSY14744 were used, with three of the four populations (lin2, lin3, and lin6) derived from the first (lin0) and differing only in that they were grown on plates for 200 generations in conditions to minimize selective pressures.
processed data files: lin_all.nucpos.bed, lin_all.nucsig.bw, lin_all.occ.bw, sacCer3.scores.bw, sacCer3.mappable.bed

extract_protocol:
5 million cells were washed twice in Sorbitol buffer (1.4 M Sorbitol, 40 mM HEPES-KOH pH 7.5, 0.5 mM MgCl2) before being incubated for 30 min at 30 degrees and shaking at 300 rpm with 0.5 mg/mL 100T zymolyase.  The cells were then washed twice in Sorbitol Buffer before being incubated with 2.5 ml of Nextera Transposase in 47.5 ml of 1x TD buffer at 37 degrees for 30 minutes.
Sequencing libraries were constructed using a modified version of the Illumina Nextera DNA Sample prep kit, as per Buenrostro et al. (2013).


title: GM12878_GSM1155959


title: S. cerevisiae lin6b


title: iEEC16_FAIRE_NA_rep2_1


title: GM12878_GSM1155958


title: GM12878_GSM1155957


title: S. cerevisiae lin3c


title: RNA-seq control siRNA

city: Tel- aviv

first: Gil

last: Ast

state: Israel

address: Ramat Aviv

department: Human Molecular Genetics & Biochemistry

institute: Tel-Aviv University

data_processing:
For MNase-seq: Fastq files of each condition were mapped to the human genome (hg19) using Bowtie2 (Langmead and Salzberg, 2012) with default settings. We constructed a perl script that calculates a single score for each over a section of 147 bp from the start of the read, thus creating .wg files containing the marking the position of a nucleosome on a fragmentof 147 bp.
For mRNA-seq: mapped the Fastq files to the human genome (hg19) using Bowtie2 (Langmead and Salzberg, 2012) with default settings.splicing analysis was performed using SpliceTrap (v0.90.5) software with default parameters (Cutoff-M, Junction reads-5) (Wu et al., 2011).
Genome_build: hg19
Supplementary_files_format_and_content: MNase-seq file: a gzip archive of .wg files (one file for each chromosome). Files are tab delimited text with the following format: chr_position, nucleosome score
Supplementary_files_format_and_content: RNA-seq file: contains the results.txt output of the SpliceTrap tool

source_name: HeLa cells

growth_protocol: HeLa cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal calf serum, 0.29 mg ml–1 L-glutamine, 100 U ml–1 penicillin, 0.1 mg ml–1 streptomycin, and 1 U ml–1 nystatin at 37 °C in a humidified atmosphere with 5% CO2.

extract_protocol:
Total RNA was extracted from the cells using TRI reagent (Sigma)
Deep sequencing libraries were prepared using standard Illumina TruSeq library preparation kits. 2 x 101-bp paired-end sequencing was performed using an Illumina HiSeq 2000.

treatment_protocol: Cells were transfected with 20nM control siRNA- Non Targeting (Thermo Scientific) using Lipofectamine RNAiMAX (Invitrogen). Following transfection, cells were incubated for 48 hours before RNA extraction.

tag: sirna knock down

value: HeLa


title: iEEC16_H3K4me1_NA_rep2_1


title: RNA-seq TSA treated

extract_protocol:
Total RNA was extracted from the cells using TRI reagent (Sigma)
Deep sequencing libraries were prepared using standard Illumina mRNA-seq library preparation kits. Sequencing of 50-bp single-end reads was performed using an Illumina Genome Analyzer II.

treatment_protocol: Cells were treated with 750 ng/ml TSA for 16 h


title: Osmotic Stress Time 0 A rep1


title: S. cerevisiae lin2c


title: RNA-seq Untreated


title: S. cerevisiae lin2b


title: iEEC16_INPUT_NA_rep1_1


title: S. cerevisiae lin6a


title: S. cerevisiae lin3b


title: S. cerevisiae lin2a


title: S. cerevisiae lin3a