title: ESCs_Pol II

description: ChIPed DNA

source_name: mouse embryonic pluripotent stem cells

value: Pol II (N20) Santa Cruze, SC-899

value: mouse embryonic pluripotent stem cells


title: Hela_RASL-seq_si-NC_rep3

status: Public on Oct 08 2014

type: SRA

city: Wuhan

first: Bo

last: Yang

state: Hubei

address: Luo-jia-shan, Wuchang

department: Biochemistry and Molecular Biology

country: China

institute: College of Life Sciences, Wuhan University

description:
RASL-seq library from NC cells
Library strategy: RASL-Seq

library_source: transcriptomic

data_processing:
The sequences were mapped to the oligo pool sequences by Bowtie. The processed data file contain both the genomic location (the first 6 columns) and counts for the short and long isoforms (last 2 columns) of each alternative splicing event.
Genome_build: hg18
Supplementary_files_format_and_content: bedGraph and bed files

molecule: total RNA

organism: Homo sapiens

source_name: Hela Cells

growth_protocol: Hela cells were cultured in high-glucose Dulbecco’s modified Eagle’s medium (DMEM, Gibico) supplemented with 10% fetal bovine serum (FBS) at 37 degress Centigrade and 5% CO2.

extract_protocol:
Total RNAs were extracted by Trizol from treated HeLa cells.
1 µg of Hela total RNA was mixed with 100 fmol of human RASL oligos, 2 pmol of biotinylated-oligo-dT and 5 µl of streptavidin magenetic beads. Followed by one hour of annealing at 45ºC, magnetic beads were washed 3 times by washing buffer. Then ligation was achieved by adding 5U of T4 DNA ligase for 1 hour at 37ºC. After 3 times of wash, ligated oligos were eluted to water by heating at 65ºC for 5 minutes, and then PCR amplified using primers containing sequencing adaptor and barcode sequences. PCR products were purified and sequenced.

treatment_protocol: Transfection of NC siRNA

tag: cell line

value: Hela

tag: transfection

value: NC siRNA


title: Hela_RASL-seq_WT_rep3

description:
RASL-seq library from WT cells
Library strategy: RASL-Seq


title: OSKM_D8_Pol II

status: Public on Oct 07 2014

city: ShenZhen

first: Minghui

last: He

state: GuangDong

address: Beishan Industrial Zone, Yantian District

department: Cancer Research Department

institute: BGI-Shenzhen

description: ChIPed DNA

library_source: genomic

data_processing:
Base calling was performed using CASAVA version 1.8.2 (illumina).
ChIP-seq reads were trimmed for adaptor sequence and mapped to mouse genome using BWA (v0.5.9) by default parameters.
Peaks were called using MACS (v1.4.2) with default parameters.
Genome_build: mm9
Supplementary_files_format_and_content: wig files were generated by MACS

molecule: genomic DNA

organism: Mus musculus

source_name: mouse reprogramming intermediates at Day8

growth_protocol:
OG2 MEFs (Silva et al., 2008) were obtained from E13.5 embryos carrying the Oct4-GFP transgene. MEFs were maintained in DMEM/High glucose (Hyclone) containing 10% fetal bovine serum (FBS, PAA), GlutaMax (Invitrogen), non-essential amino acids (Invitrogen) and penicillin/streptomycin (Hyclone).
Mouse ESCs were cultured in mouse ESC medium (DMEM with 15% FBS [Gibco], GlutaMax, nonessential amino acids, sodium pyruvate, 0.1 mM β-mercaptoethanol and 1,000 U ml-1 LIF [Millipore]).
For reprogramming, 15,000 OG2 MEFs in 12-well plates were transduced twice using retroviral supernatants of pMX-Oct4/Sox2/Klf4/c-Myc generated with Plat-E cells. The medium was changed to mouse ESC medium at day 2 post-infection and renewed daily.

extract_protocol:
Harvested cells were lysated and sonicated to 100-500bp. Protein-DNA complexes were isolated with antibody. ChIP complexes were eluted with 1% SDS in 100 mM NaHCO3 and crosslinks were reversed by incubating samples over night at 65C. DNA fragments were purified using phenol-chloroform extraction and ethanol precipitation
Libraries were constructed according to the Illumina ChIP-seq library preparation instructions. Briefly, pull-down DNA was end repaired using a combination of T4 DNA polymerase, T4 PNK and Klenow polymerase. The DNA was then purified and used for A-tailing by adding a dATP to the 3' end. An indexed adaptor with a "T" overhang at the 3' end was then ligated to each DNA sample. The ligated DNA was then subjected to PCR amplification followed by gel purification.

treatment_protocol: Singel cell suspension was crosslinked with 1% formaldehyde for 15 min at room temperature with rotation, then quenched with glycine at a final concentration of 0.125 M. Cells were rinsed twice with cold PBS.

tag: chip antibody

tag: cell stage

value: intermediate(Day 8 after OSKM infection)

tag: strain


title: OSKM_D5_Pol II

description: ChIPed DNA

source_name: mouse reprogramming intermediates at Day5

value: intermediate(Day 5 after OSKM infection)


title: OSKM_Cdk9-DN_D10_rep1

description: expression level (FPKM, Fragments Per Kilobase of exon per Million fragments mapped)

data_processing:
Base calling was performed using CASAVA version 1.8.2 (illumina)
RNA-seq reads were trimmed for adaptor sequence and mapped to mouse genome using Tophat (v2.0.10) and Ensembl genome annotation file(Mus_musculus.NCBIM37.67.gtf)
Cufflinks (v2.2.1) was used to estimated gene expression abundance(FPKM, numbers of fragment per kilobase of each gene per million mapped reads)
Genome_build: mm9
Supplementary_files_format_and_content: FPKM values for each gene were generated by Cufflinks

source_name: Reprogramming intermediates

growth_protocol: Cells were cultured in ESC medium (DMEM with 15% FBS [Gibco], GlutaMax, nonessential amino acids, sodium pyruvate, 0.1 mM β-mercaptoethanol and 1,000 U ml-1 LIF [Millipore])

extract_protocol:
Total mRNA was isolated using TRIzol following the manufacturer’s instruction
RNA libraries were prepared for sequencing using standard Illumina protocols

treatment_protocol: Mouse embryonic fibroblasts were transduced with exogenous factors Oct4, Sox2, Klf4, c-Myc together with a control vector or Cdk9-DN

value: Reprogramming intermediates (Day10 after infection)

tag: transduced


title: MEFs_Pol II

description: ChIPed DNA

source_name: mouse embryonic fibroblasts

value: mouse embryonic fibroblasts


title: OSKM_Ctrl_D10_rep2

value: control vector


title: Hela_RNA-Seq_si-NC

description: mRNA library from NC cell

data_processing:
Paired-end reads were separately mapped into a custom Bowtie index, which was built from UCSC knownGenes (hg18) not including introns. Under the constraint of the insert size, only the paired-end reads whose two ends are about 50 nt apart on the same gene were kept. For each pair, the coordinates of the two end reads were mapped back to the genome based on its gene's genomic location, and then joined together. Only the pair-end reads with one unique genomic location were kept.
Genome_build: hg18
Supplementary_files_format_and_content: bedGraph and bed files

molecule: polyA RNA

extract_protocol:
Total RNAs were extracted by Trizol from treated HeLa cells and polyA RNAs were enriched by oligod(T) selection.
After fragmentation, mRNAs were reversely transcribed to cDNA with Superscript II in the presence of random primer. After RNase H digestion, the second strand cDNA was synthesized by DNA pol I followed by adaptor ligation. Size-selected PCR products were sequenced by Illumila Genome Analyzer II.


title: OSKM_Ctrl_D10_rep1


title: RARa_WT_BiotinChIPseq_Dox_RA_E2_Exp2

status: Public on Oct 09 2014

city: La Jolla

first: Daria

last: Merkurjev

state: CA

address: 9500 Gilman Drive, Mail Code 0648

department: Medicine

country: USA

institute: UCSD

description: ChIP-seq Experiment 2, 24hrs treatment with 2μg/ml doxycycline, 1hr treatment with 1μM RA and 100nM E2

data_processing:
Basecalls performed using CASAVA version 1.4
ChIP-seq reads were aligned to the hg18 genome assembly using Bowtie version 2.0.1.
The begGraph files were generated by using Homer v3.9, which the total tags are normalized to 1.00e+07.
Genome_build: hg18

source_name: MCF7 Tet-On Inducible stable line

growth_protocol: MCF7 obtained from ATCC were cultured in DMEM media supplemented with 10% FBS in a 5% CO2 humidified incubator at 37°C.

extract_protocol:
Lysates were clarified from sonicated nuclei and protein-DNA complexes were isolated with antibody or by streptavidin magnetic beads pull-down.
The extracted ChIP DNA was ligated to specific adaptors of Truseq system from Illumina, according to the manufacturers instructions.

treatment_protocol: Before experiment, the MCF7 cells were hormone-stripped in phenol red free DMEM medium plus 5% charcoal-treated FBS for 3-4 days, followed by treatment of either 100nM 17β-estradiol (E2) or ethanol as vehicle control for 1hr. In some cases, we also treated cells with either 1uM all-trans Retinoic Acid (RA), or Dimethyl sulfoxide (DMSO) as vehicle control for 1 hour. To perform Biotin-ChIP experiments through Biotin-streptavidin pull-down, we used some BLRP-tagged MCF7 Tet-On inducible stable lines that can in vivo biotinylate BLRP-tagged proteins. To induce BLRP-tagged protein expression, 2μg/ml doxycycline was added into culture media for about 24 hours before hormone treatment and collection.

tag: cell description

value: beast cancer cell line

tag: chip antibody or biotin-streptavidin pulldown of blrp-tagged proteins

value: Biotin-ChIP using Streptavidin Magnetic beads (Solulink)

tag: treatment


title: OSKM_Cdk9-DN_D5_rep2

value: Reprogramming intermediates (Day5 after infection)


title: OSKM_Ctrl_D5_rep1


title: OSKM_Cdk9-DN_D4_rep2

value: Reprogramming intermediates (Day4 after infection)


title: OSKM_Cdk9-DN_D4_rep1


title: OSKM_Cdk9-DN_D10_rep2


title: OSKM_Ctrl_D5_rep2


title: Hela_RASL-seq_si-U2AF65+Flag-U2AF35_rep2

description:
RASL-seq library from cells of transfection of U2AF65 siRNA and Flag-U2AF35
Library strategy: RASL-Seq

treatment_protocol: Transfection of U2AF65 siRNA and Flag-U2AF35

value: U2AF65 siRNA and Flag-U2AF35


title: OSKM_Ctrl_D4_rep2


title: OSKM_Ctrl_D4_rep1


title: Ab(-)_HS_43

city: Bergen

first: Tomasz

last: Stokowy

address: Nygardsgaten 5

department: IT Division

country: Norway

institute: University of Bergen

description:
bibl15
negative control sample

data_processing:
Reads were mapped to the mouse genome (mm10) with Bowtie2.
Aligned files were converted to bam and sorted using Samtools.
Peaks detection was carried with case-control strategy (detected peaks are given in bed files)
HSF1 and HSF2 target sites were annotated to genomic regions using HOMER software (annotated binding sites are given in text file)
Genome_build: mm10
Supplementary_files_format_and_content: bed - detected peaks; txt - annotated binding sites.

source_name: mock ChIP from spermatocytes heat shocked at 43°C

growth_protocol: Testes (twenty males for one isolation) were decapsulated and cell suspension was obtained by enzymatic treatment (collagenase type IA, trypsin and DNase I). Population of cells enriched in spermatocytes (up to 80%) was obtained by unit gravity sedimentation in a Sta-Put chamber (SP-120, ProScience Inc, Ontario, Canada) containing a 1-3% (w/v) linear BSA gradient in PBS. Finally cells were suspended in CO2 saturated RPMI medium supplemented with 10% (v/v) fetal bovine serum, 0.004% (v/v) gentamycin, and 6mM sodium lactate, at the temperature 32°C (physiological temperature of testes).

extract_protocol:
Cells were fixed by adding of formaldehyde to a final concentration of 1% and incubated at room temperature for 10 minutes. Next 2.5 M glycine was added (1/20 volume) for 5 minutes. Cells were washed with ice-cold PBS and lysed following the manufacturer’s protocol. For analyses of HSF1 and HSF2 binding, the ChIP assay was carried out according to protocol for ChIP kit of Upstate Biotechnology (Lake Placid, NY) and protein A-sepharose (Amersham) was used. For 30µg of chromatin, sonicated to 100-500 bp fragments, 3 µg of rabbit anti-HSF1 (cat. no SPA-901, Enzo) or 5 µg of goat anti-HSF2 (cat. no AF5227, R&D Systems) polyclonal antibodies were used. Samples proceeded with normal goat serum served as negative controls.
Sequencing libraries were generated using ChIP-Seq Sample Prep Kit (Illumina). Template amplification and cluster generation were performed using the cBot and TruSeq SR Cluster Kit v2 cBot-GA for cBot, and 80 nucleotides were sequenced with Illumina Genome Analyzer IIx using TruSeq SBS Kit v5 sequencing kits.

treatment_protocol: An equal volume of CO2 saturated, pre-heated to 60°C medium was added to the cell suspension, which immediately raised the temperature from 32°C to 43°C. The tubes were sealed with parafilm and submerged in a water bath at 43°C for an additional 5, 10, or 20 min. Samples were combined in one heat shocked sample.

value: FVB/N

tag: gender

value: male

tag: tissue

value: testis

tag: cell type

value: spermatocytes

tag: developmental stage

value: adult

tag: age

value: 10-16 weeks

tag: temperature

value: normal goat serum (negative control)


title: Ab(-)_HS_38

description:
bibl14
negative control sample

source_name: mock ChIP from spermatocytes heat shocked at 38°C

treatment_protocol: An equal volume of CO2 saturated, pre-heated to 53°C medium was added to the cell suspension, which immediately raised the temperature from 32°C to 38°C. The tubes were sealed with parafilm and submerged in a water bath at 38°C for an additional 5, 10, or 20 min. Samples were combined in one heat shocked sample.


title: Ab(-)_C

description:
bibl13
negative control sample

source_name: mock ChIP from untreated spermatocytes

treatment_protocol: untreated

value: control


title: HSF2_HS_43

description:
bibl12
HSF2 binding sites in spermatocytes heat shocked at 43°C

source_name: HSF2 ChIP DNA from spermatocytes heat shocked at 43°C

value: anti-HSF2 (cat. no AF5227, R&D Systems)


title: Wrn-3-vitC

city: Quebec

first: Michel

last: Lebel

state: Qc

address: 2705 Laurier Blvd, Local T-2-04

department: Centre Hospitalier de l’Université Laval (CHUL)

country: Canada

institute: Centre de Recherche du CHU de Québec

data_processing:
Detailed statistics on the quality of the reads were calculated with FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). The 50 base pairs raw sequences were aligned on the C. elegans ce10/W220 genome with TopHat using the Ensembl annotations provided with the Illumina iGenomes. The htseq-count software (http://www-huber.embl.de/users/anders/HTSeq) was used to count the number of reads aligned to each gene. These counts were then normalized relative to the sequencing depth with DESeq. DESeq was also used to calculate the fold changes, log fold changes, and significance of the changes for each comparison.
Genome_build: C. elegans ce10/W220 genome
Supplementary_files_format_and_content: tab-delimited text files include DESeq values for each Sample

organism: Caenorhabditis elegans

source_name: whole worm

extract_protocol:
Worms were spun down in an eppendorf tube and lysed in TRIZOL (Invitrogen, Carlsbad, CA) to extract total RNA.
RNA libraries were prepared for sequencing using standard Illumina protocols

treatment_protocol: Eggs were isolated by standard bleach treatment and hatched overnight at room temperature. The resulting L1 larvae were then distributed over NGM plates supplemented or not with 10mM vitamin C (Sodium L-ascorbate) (Sigma-Aldrich, ST-Louis, MO) and grown at 25oC. The developmental stage of the animals was monitored visually until the worms reached the larvae L4 stage.

tag: develpmental stage

value: L4 stage

value: whole worm

tag: genotype

value: wrn-1 mutant wrn-1(gk99)

value: treated with 10 mM ascorbate


title: HSF2_HS_38

description:
bibl11
HSF2 binding sites in spermatocytes heat shocked at 38°C

source_name: HSF2 ChIP DNA from spermatocytes heat shocked at 38°C


title: p27 Ck- Empty vector R9

city: Portland

first: David

last: Ellison

state: OR

address: 3181 SW Sam Jackson Park Rd CR145

institute: Oregon Health and Science University

data_processing:
All reads with adapter contamination were removed. Three bp from the 5’ end and one bp from the 3’ end were trimmed prior to alignment. All samples were aligned to the unmasked mouse genome (mm10) using Subread (Version1.3.6-p1)
Counts for each sample were determined by using the UCSC gtf file and the featureCounts method from Subread.
The biomaRt package used to annotate UCSC Gene ID to Ensembl Gene ID
Differential Expression Analysis was conducted for BCR-ABL and Empty Vector separately: BCR-ABL CK- - versus BCR-ABL KO (BCR-ABL Analysis) and Empty CK- - versus Empty KO (Empty Vector Analysis). The EdgeR generalized linear model was utilized to identify putatively differentially expressed genes. P-values were adjusted for multiple comparison using Benjmani and Hochberg.
Genome_build: mm10
Supplementary_files_format_and_content: Tab delimited edgeR output for BCR-ABL and Empty Vector analysis. Includes UCSC Gene ID, Gene Name, logFC, logCPM,Pvalue, FDR

source_name: bone marrow

growth_protocol: Bone marrow cells were harvested from p27CK- and p27-/- mice bone marrow and transduced with retrovirus expressing BCR-ABL1 or empty vector control. Cells were cultured for five days and sorted for Lin-GFP+ cells

extract_protocol: RNA-seq libraries were constructed using the TruSeq protocol (Illumina). Poly(A)+ RNA was isolated using oligo-dT bound to magnetic beads. Poly(A)+ RNA was then chemically fragmented. RNA fragments were converted to double stranded cDNA using random hexamer priming. Fragment ends were treated to remove overhanging nucleotides and then a single “A” was added to the 3’ end of each strand to facilitate ligation. Illumina adaptors with barcode sequences was ligated to the fragments. The resulting library was then amplified using polymerase chain reaction with a limited number of round. Unincorporated nucleotides and adaptor dimers were removed using AMPure XP beads (Agencourt). Libraries were combined for multiplexing and sequenced on a HiSeq 2000 sequencer (Illumina). Base call data was assembled into fastq files using the CASAVA suite (Illumina).

tag: transduction

value: empty


title: OSKM_Cdk9-DN_D5_rep1

value: Reprogramming intermediates (Day5 after infection)


title: OSKM_Cdk9-DN_D3

value: Reprogramming intermediates (Day3 after infection)


title: HSF2_C

description:
bibl10
HSF2 binding sites in spermatocytes at physiological temperature

source_name: HSF2 ChIP DNA from untreated spermatocytes


title: Wrn-2-vitC


title: Wrn-1-vitC


title: Wrn-3

value: no treatment


title: HSF1_HS_38

description:
bibl8
HSF1 binding sites in spermatocytes heat shocked at 38°C

source_name: HSF1 ChIP DNA from spermatocytes heat shocked at 38°C

value: FVB/N

value: anti-HSF1 (cat. no SPA-901, Enzo)


title: RARg_WT_BiotinChIPseq_Dox_E2_Exp5

description: ChIP-seq Experiment 5, 24hrs treatment with 2μg/ml doxycycline, 1hr treatment with 100nM E2

tag: cell line

value: beast cancer cell line

value: Biotin-ChIP using Streptavidin Magnetic beads (Solulink)


title: HSF1_HS_43

description:
bibl9
HSF1 binding sites in spermatocytes heat shocked at 43°C

source_name: HSF1 ChIP DNA from spermatocytes heat shocked at 43°C

value: male

value: testis

value: spermatocytes

value: adult

tag: chip antibody


title: AP2g_ChIPseq_Veh_Exp8

description: ChIP-seq Experiment 8, 1hr treatment with vehicle control

value: Santa Cruz Biotech, SC-8977

value: 1hr treatment with vehicle control


title: HSF1_C

description:
bibl7
HSF1 binding sites in spermatocytes at physiological temperature

source_name: HSF1 ChIP DNA from untreated spermatocytes

value: 10-16 weeks

value: control


title: Wrn-1

value: L4 stage

value: whole worm

value: wrn-1 mutant wrn-1(gk99)


title: HEK293_repA_smallRNASeq

city: Berlin

first: Annalisa

last: Marsico

address: Ihnestraße 63-73

department: Computational Biology

country: Germany

institute: Max Planck Institute for Molecular Genetics

description: processed data file contains RPKM for mature miRNA abundance measurements

data_processing:
Sequenced reads were trimmed forthree-prime  adaptor sequence, masked for low-quality and short sequences, then mapped to hg19 whole genome using the short-read aligner Bowtie2. Reads that mapped perfectly to nucleotide position 1 to 18 of each annotated miRNA were used to quantify miRNA expression.
Reads per Kilobase per Milion reads were calculated for each mature miRNA according to the definition in Mortazavi et al., Nat Methods 2008, averaging the two rpkm values from the two replicates for each cell line.
Genome_build: hg19
Supplementary_files_format_and_content: tab-delimited text file includes RPKM values for each sample

source_name: Embryonic kidney derived cells (HEK293 cells)

growth_protocol: Hela, A549 and HEK293 cells were maintained under standard conditions at 37°C and 5% CO2 in DMEM containing 10% FCS and antibiotics.

extract_protocol:
The chromatin fraction was isolated as in Bhatt et al. (Cell 2012), and associated total RNA was isolated using Trizol extraction.
The libraries for sequencing small RNAs were constructed using the Illumina TrueSeq small RNA kit.

treatment_protocol: For Drosha RNAi, 5x10^5 cells were seeded per 10cm dish, transfected after 24h with 60nM siRNA, harvested by trypsinization 72h after transfection.

value: Embryonic kidney derived cells


title: BCR-ABL inhibitor-resistant KCL-22 single clone #2

city: Cambridge

first: Joshua

middle: M

last: Korn

state: MA

address: 250 Mass Ave

institute: Novartis

data_processing:
Illumina Casava1.8 software used for basecalling.
Sequenced reads were trimmed for adaptor sequence or low-quality sequence, then mapped to GRCh37 whole genome and transcriptome based on ucsc knownGene using a modified version of Tophat v1.3
Fragments Per Kilobase of exon per Megabase of library size (FPKM) were calculated using Cufflinks version 1.3.
Genome_build: GRCh37
Supplementary_files_format_and_content: tab-delimited text file includes FPKM values for each Sample.

source_name: Blood (chronic myeloid leukemia in blast crisis)

growth_protocol: Human CML cell line KCL-22 was transduced with ClonTracer library and the polyclonal pool was generated.

extract_protocol:
RNeasy Plus Mini Kit (QIAGEN) following the manufacturer's instructions
RNA libraries were prepared for sequencing using standard Illumina protocols

treatment_protocol: Replicate cell populations were treated with either 0.1% DMSO (vehicle), 2.5 µM imatinib, 0.3 µM nilotinib, or 2.5 µM GNF-2 as descirbed in "Characteristics" section, and single clones were isolated by FACS sorting.

value: CML cell line KCL-22

value: Single clone was isolated from 2.5 µM GNF-2-resistant ClonTracer-barcoded KCL-22 subpopulation and expanded. This clone is never exposed to catalytic BCR-ABL inhibitors.

tag: resistant to

value: 2.5 µM GNF-2


title: BCR-ABL inhibitor-resistant KCL-22 single clone #4

value: 2.5 µM imatinib/0.3 µM nilotinib/2.5 µM GNF-2


title: N2-2-vitC

value: wild type

value: treated with 10 mM ascorbate


title: OSKM_Ctrl_D3

tag: cell stage

value: Reprogramming intermediates (Day3 after infection)

tag: strain

value: control vector


title: RARa_WT_BiotinChIPseq_noDox_Exp2

description: ChIP-seq Experiment 2, no treatment with doxycycline, 1hr treatment with 1μM RA and 100nM E2

tag: cell description

value: beast cancer cell line

value: Biotin-ChIP using Streptavidin Magnetic beads (Solulink)


title: N2-3-vitC


title: BCR-ABL inhibitor-resistant KCL-22 single clone #1


title: HeLa_rep2_RNASeq

description: processed data file contains RPKM for primiRNA abundance measurements

data_processing:
Sequenced reads were trimmed for adaptor sequence, and masked for repeats or low-quality sequence, then mapped to hg19 whole genome using TopHat 2 and default parameters
Reads in the 100 bp regions upstream and downstream of the premiRNA Drosha cutting sites we counted by means of the HTSeq software v0.5.4p2
Reads per Kilobase per Milion reads were calculated for each miRNA primary transcript according to the definition in Mortazavi et al., Nat Methods 2008, averaging the two rpkm values from the regions upstream and downstream of each miRNA precursor.
Genome_build: hg19
Supplementary_files_format_and_content: tab-delimited text file includes RPKM values for each sample

source_name: HeLa cells

extract_protocol:
The chromatin fraction was isolated as in Bhatt et al. (Cell 2012), and associated total RNA was isolated using Trizol.
The library was constructed using the Illumina TruSeq Stranded Total RNA kit with Ribo-Zero Gold extraction.

value: HeLa

value: cervical epithelial


title: 0.3 µM nilotinib-resistant KCL-22

value: ClonTracer-barcoded KCL-22 cell population was treated with 0.3 µM nilotinib for 27 days to acquire nilotinib-resistant cell population.

value: 0.3 µM nilotinib


title: N2-3

value: L4 stage

value: whole worm

value: no treatment


title: 2.5 µM imatinib-resistant KCL-22

value: CML cell line KCL-22

value: ClonTracer-barcoded KCL-22 cell population was treated with 2.5 µM imatinib for 27 days to acquire imatinib-resistant cell population.

value: 2.5 µM imatinib


title: N2-1


title: N2-2


title: 2.5 µM GNF-2-resistant KCL-22

value: ClonTracer-barcoded KCL-22 cell population was treated with 2.5 µM GNF-2 for 21 days to acquire GNF-2-resistant cell population.

value: 2.5 µM GNF-2


title: Vehicle-treated KCL-22

value: ClonTracer-barcoded KCL-22 cell population was treated with 0.1% DMSO for 7 days.

value: Vehicle control - Treated with 0.1% DMSO


title: N2-1-vitC

value: wild type

value: treated with 10 mM ascorbate


title: Wrn-2

value: wrn-1 mutant wrn-1(gk99)


title: Control vector

city: New York

first: Yuheng

last: Lu

state: NY

address: 417 East 68th Street

institute: Memorial Sloan Kettering Cancer Center

description: Cross-linked RNA

data_processing:
Using the FASTX-Toolkit from the Hannon Laboratory (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY), 3' ends of reads were trimmed until the base quality score was not <30 and retained if the remaining read length was at least 20 nt. After trimming, we mapped CLIP reads to the hg19 human genome with Bowtie2 (Langmead and Salzberg, 2012).
We used a custom R package to call peaks in read coverage in the Msi2 HITS-CLIP experiments (Loeb et al., 2012), and we tested for differential binding between Msi2-overexpressing cells and control while correcting for differential expression using a joint generalized linear model of HITS-CLIP and RNA sequencing read counts in the candidate peak regions.  Peak windows that were differentially bound at an FDR threshold of 0.1 were reported unless otherwise noted.
Genome_build: hg19
Supplementary_files_format_and_content: CSV file containing peak site features and read counts from each sample

extract_protocol:
RNA was extracted using standard phenol/chloroform extraction methods
RNA libraries were prepared and barcoded at the Broad Institute Sequencing Core facility and then sent to the core facility at the Sloan- Kettering Institute for single-end Illumina sequencing.

value: Hematopoietic stem cells (HSCs)

tag: vector

value: Empty control

tag: antibody

value: anti–Flag M2 Ab


title: Erlotinib-resistant HCC827

source_name: Lung

growth_protocol: Human NSCLC cell line HCC827 was transduced with ClonTracer library and the polyclonal pool was generated.

treatment_protocol: Replicate cell populations were treated with either 0.1% DMSO (vehicle), 2 µM erlotinib or combination of 2 µM erlotinib/0.2 µM crizotinib as descirbed in "Characteristics" section.

value: NSCLC cell line HCC827

tag: treatment

value: ClonTracer-barcoded HCC827 cell population was treated with 2 µM erlotinib for 47 days to generate the erlotinib-resistant cell population.

value: 2 µM erlotinib


title: Vehicle-treated HCC827

value: ClonTracer-barcoded HCC827 cell population was treated with 0.1% DMSO for 4 days.


title: BCR-ABL inhibitor-resistant KCL-22 single clone #3

value: CML cell line KCL-22

value: Single clone was isolated from 2.5 µM imatinib-resistant ClonTracer-barcoded KCL-22 subpopulation and expanded. This clone is never exposed to allosteric BCR-ABL inhibitors.

value: 2.5 µM imatinib/0.3 µM nilotinib


title: FLAG-tagged MSI2 expression vector, replicate 2

description: Cross-linked RNA

value: FLAG-tagged MSI2 expression vector


title: FLAG-tagged MSI2 expression vector, replicate 1

description: Cross-linked RNA


title: GFP_2b

city: Boston

first: Michael

middle: E

last: Talkowski

address: 185 Cambridge Street

institute: Massachusetts General Hospital

data_processing:
Read quality was assessed using fastQC (Babraham Bioinformatics). One lane had a high proportion of N's at a particular position, those reads and their mate pairs were filtered out. Reads containg bases with map quality < 20 were also filtered out.
The multiple fastq files for each library were combined before alignment. Reads were aligned using Gsnap (Wu et al., 2010) version 2012-07-207. Uniquely mapping reads were kept. Insert size metrics below are for combined, uniquely aligned reads.
Counts mapping to genes were generated using the bedTools (Quinlan & Hall, 2010), version 2.17.0, function multibamcov.
Sample quality was assessed by sample clustering, correlation between technical replicates ("a" and "b" samples), and level of CHD8 knockdown achieved. As described in the accompanying publication, the samples CHD8sh3a, CHD8sh3b, and LacZb were excluded due to low concordance between technical replicates. In addition, the samples CHD8sh1a and CHD8sh1b were excluded from differential expression analysis due to low (~60%) CHD8 knockdown, resulting in these samples clustering together with controls. These samples are therefore absent from the processed data file.
The threshold for detection (>3 reads) was determined using ERCC spike-ins, as described in Blumenthal et al., 2014. Genes for which the threshold was met in all retained samples were kept. Genes that had no transcripts >=250 nt in length, as well as genes assigned a "tRNA" or "rRNA" biotype in the gtf file, were filtered out.
Differential expression analysis was carried out using DESeq (Anders & Huber, 2010) version 1.12.1. Due to observed batch effects, a full model of Count ~ Batch + Treatment was compared to a reduced model  Count ~ Batch to identify genes that were affected by treatment after accounting for batch. Genes meeting p < 0.05 were considered differentially expressed for all downstream analysis.
Gene expression values were reported in two ways: log2CPMs corrected for batch using the removeBatchEffect function in the edgeR package (Robinson et al., 2010; version 3.4.2), and FPKMs, without batch correction, generated using Cufflinks (Trapnell et al., 2010; version 2.0.2).
Genome_build: GrCH37, Ensembl transcriptome build 71.
Supplementary_files_format_and_content: Processed data files consist of Ensembl Genes with differential expression between knockdown (KD) and control samples (DESeq), log2CPMs in each sample, and FPKMs in each sample.

source_name: human NPCs

growth_protocol: GM8330-8 was cultured with neural expansion medium [70% DMEM (Invitrogen), 30% HAMS-F12 (Mediatech) supplemented with 2% B27 supplement (Invitrogen) and 1% Penicillin-Streptomycin-Glutamine] on poly-L-ornithine (20 g/ml, Sigma) and Laminin (5 g/ml, Sigma) coated culture plates. NPC media was supplemented with basic fibroblast growth factor (bFGF, 20 ng/ml, R&D Systems), epidermal growth factor (EGF, 20 ng/ml, Sigma) and heparin (5 g/ml, Sigma).

extract_protocol:
Total RNA was isolated using a TRIzol/chloroform extraction and quality monitored on an Agilent Tape Station.
RNA-seq libraries were prepared using a customized version of the originally published, strand specific dUTP method (Levin et al., 2010; Parkhomchuk et al., 2009). Standard Illumina paired-end library preparation is performed with barcoded adaptors. Each library also included 1 ul of a 1:10 dilution of ERCC RNA Control Spike-Ins (Ambion) that were added from one of two mixes, each containing the same 92 synthetic RNA standards of known concentration and sequence.

treatment_protocol: GM8330-8 was cultured in 6-well plates to about 80-90% confluency. 20ul of the designated virus stock (107 – 108) was added drop wise to each well and puromycin selection was started 48 hours post transduction.

value: iPSC (GM8330-8)-derived NPC

tag: passages

tag: shRNA target gene and trc accession

tag: batch


title: GFP_2a

tag: cell type


title: LacZb


title: LacZa


title: LacZ_2b


title: CHD8sh6a


title: GFPa


title: GFPb


title: CHD8sh6_2b


title: CHD8sh6_2a


title: CHD8sh3a


title: CHD8sh2b


title: CHD8sh1b


title: CHD8sh4b


title: CHD8sh6b


title: CHD8sh5a


title: CHD8sh1a


title: Erlotinib/crizotinib dual resistant HCC827

value: The erlotinib-resistant HCC827 cell population was treated with 2 µM erlotinib+0.2 µM crizotinib combination for 10 days to enrich for erlotinib/crizotinib dual-resistant subpopulations.


title: CHD8sh4a


title: CHD8sh3b


title: CHD8sh2a


title: LacZ_2a


title: Input

data_processing:
Alignment: Sequence reads were obtained and mapped to the human GrCH37 genome using bwa version 0.7.5a sampe with default parameters.
Aligned reads were filtered using the samtools command “samtools view -b -f 2 -F 4 -q 1” to filter out multiply mapped reads and reads not in proper pairs, then duplicates were removed using samtools rmdup.
Three of the libraries (CHD8_Novus60417, CHD8_Bethyl, and Input) were sequenced in two separate runs. Alignment and filtering of aligned reads were carried out separately for each run, and then combined before peak detection.
Peaks: Peak detection was performed with the Model-based Analysis of ChIP-Seq (MACS) algorithm (http://liulab.dfci.harvard.edu/MACS/) version 2.0.10.2013.9.13 using default parameters. Peaks that met q<0.05 were used. Peaks mapping to GL* contigs were removed before further analysis.
Genome_build: GrCH37
Supplementary_files_format_and_content: Processed data files consist of MACS2 output, after converting chromosome names to "chrN" for compatibility with UCSC-format tools, and removing peaks in GL* contigs. For CHD8, the final peak list was obtained by merging peak lists for the three individual antibodies, then selecting the merged peaks that overlapped a peak from each of the individual peak lists separately.

source_name: human control NPCs

extract_protocol:
Complexes were precipitated with Dynabeads Protein A beads (Invitrogen), and immunoprecipitated chromatin was eluted in elution buffer de-crosslinked at 65°C for 8 hr (or O/N), and treated with proteinase K (Roche). DNA was purified by extracting with phenol and chloroform and precipitating in ethanol.
Library preparation was carried out using the NEBNext Ultra DNA Library Prep Kit for Illumina (NEB Cat#E73705), according to the manufacturer's instructions.

tag: shRNA target gene

value: GFP


title: CHD8_Bethyl

value: CHD8 (Bethyl A301-224A)


title: CHD8_Novus60417

value: CHD8 (Novus Biological NB100-60417)


title: CHD8_Novus60418

value: CHD8 (Novus Biological NB100-60418)


title: ETS1_PANC1_ChIPseq

city: North Chicago

first: Josh

last: Plotnik

state: IL

address: 1 North Waukegan Rd, AP10/210A

institute: AbbVie Inc.

description: epithelial-like

data_processing:
Illumina Casava1.8.2 software used for basecalling
Reads were aligned to the Homo sapien genome (UCSC hg19) using Bowtie2 (v2.0.6)
Peak calling was accomplished using the Useq platform (v8.0.1).
Genome_build: UCSC hg19
Supplementary_files_format_and_content: tab-delimited text files include for each stated sample: chromosome, start, stop

source_name: PANC1 Pancreatic cancer cell line

growth_protocol: DU145 cells were maintained in DMEM media (Mediatech-Cellgro) with 10% FBS, 100 U/ml Penicillin/Streptomycin, and 5microgram/ml puromycin (if selected)

extract_protocol:
ChIP DNA was isolated as described in Hollenhorst et. al., Genes and Development, 2011
Sequencing libraries were generated using a modified Illumina Truseq sample preparation protocol. ChIP DNA's were sheared to ~150 nucleotides using a Diagenode BioRuptor and the size was confirmed by DNA gel electrophoresis. DNA end repair of the cDNA was performed using Klenow DNA polymerase (New England BioLabs), T4 DNA polymerase (New England BioLabs), and T4 DNA ligase (New England BioLabs) before the sample was subjected to QIAquick PCR purification (Qiagen). Adapters were ligated to DNA fragments using the T4 DNA ligase (New England BioLabs). The product was run on a 2% agarose gel and size selected between 200 and 300 nucleotides to then be purified by a Gel Extraction kit (Qiagen). Universal and indexing adapters were taken from the TruSeq sample preparation kit (Illumina).

value: ETS1 (Santa Cruz, sc-350, lot #F1312)


title: JUND_DU145_ChIPseq

description: epithelial

source_name: DU145 Prostate cancer cell line

value: JUND (Santa Cruz, sc-74, lot #K1111)


title: GABPA_DU145_ChIPseq

description: epithelial

value: GABPA (Santa Cruz, sc-13442, lot #B2009)


title: ELK4_DU145_ChIPseq

description: epithelial

value: ELK4 (Santa Cruz, sc-1426, lot #G2711)


title: ETS1_DU145_ChIPseq

description: epithelial

value: ETS1 (Santa Cruz, sc-350, lot #F2811)


title: DU145_input

description: epithelial


title: ELF1_DU145_ChIPseq

description: epithelial

value: ELF1 (Santa Cruz, sc-631, lot #L2702)


title: U0126 treated DU145 cells replicate 1

description: polyA selected mRNA

data_processing:
Illumina Casava1.8.2 software used for basecalling
Reads were aligned to the Homo sapien genome (UCSC hg19) using TopHat2 (v2.0.7) running BowTie2 (v2.0.6) and Samtools (v.0.1.18) using a modified protocol from Trapnell et al., Nature Protocols,  2012. The parameters used were -G -p 8
Differential expression of aligned reads was produced using Cufflinks (v2.1.1) using a modified protocol from Trapnell et al., Nature Protocols,  2012. The three replicates for each shRNA DU145 cell line were combined for the differential expression analysis. The parameters used were -b -p 8 -u -L
Genome_build: UCSC hg19
Supplementary_files_format_and_content: tab-delimited text files include for each combination of replicates: RPKM values, locus, status, log2(fold_change), test_stat, p-value, q-value, and significance

source_name: brain metastasis of grade IV prostate cancer

extract_protocol:
Total RNA was isolated from DU145 cells transduced with lentiviral shRNA knockdown vector or U0126 treatment using the Rneasy mini kit (Qiagen) according to manufacturer's instruction
Sequencing libraries were generated using a modified Illumina Truseq sample preparation protocol.Total RNA was DNAase treated with TURBO DNase (Invitrogen) The DNase treated RNA was polyA selected with oligo(dT) beads (Invitrogen). A Superscript III Reverse Transcriptase First-Strand Synthesis (Invitrogen) system was used to generate cDNA from the polyA selected RNA with random hexamer primers (Invitrogen). After first strand synthesis a second strand was generated using E. coli DNA ligase (New England BioLabs) and E. coli DNA polymerase I (New England BioLabs). The double stranded cDNAs were sheared to ~150 nucleotides using a Diagenode BioRuptor and the size was confirmed by DNA gel electrophoresis. DNA end repair of the cDNA was performed using Klenow DNA polymerase (New England BioLabs), T4 DNA polymerase (New England BioLabs), and T4 DNA ligase (New England BioLabs) before the sample was subjected to QIAquick PCR purification (Qiagen). Adapters were ligated to DNA fragments using the T4 DNA ligase (New England BioLabs). The product was run on a 2% agarose gel and size selected between 200 and 300 nucleotides to then be purified by a Gel Extraction kit (Qiagen). Universal and indexing adapters were taken from the TruSeq sample preparation kit (Illumina).

treatment_protocol: U0126 treated cells were treated with a concentration of 50mg/mL for 6hrs

tag: condition

value: U0126 treated


title: PANC1_input

description: epithelial-like


title: U0126 treated DU145 cells replicate 3

description: polyA selected mRNA


title: U0126 treated DU145 cells replicate 2

description: polyA selected mRNA


title: ETS1 knockdown in DU145 cells using shRNA replicate 3

description: polyA selected mRNA

value: ETS1 shRNA


title: Luciferase knockdown in DU145 cells using shRNA replicate 3

description: polyA selected mRNA

value: luciferase knockdown


title: Luciferase knockdown in DU145 cells using shRNA replicate 2

description: polyA selected mRNA


title: K562-DS10686

first: Matthew

middle: T

last: Maurano

address: 435 East 30th Street

department: Institute for Systems Genetics

institute: NYU School of Medicine

data_processing:
Peak: ChIP-seq affinity zones (HotSpots) were identified using the HotSpot algorithm described in Sabo et al. (2004). 1.0% false discovery rate thresholds (FDR 0.01) were computed for each cell type by applying the HotSpot algorithm to an equivalent number of random uniquely mapping 36mers. DNase data used unthresholded hotspots.
Genome_build: hg19
Supplementary_files_format_and_content: .bed: peak locations generated using the hotspot algorithm

source_name: K562 erythroleukemia cells

growth_protocol: Cells were grown according to the approved ENCODE cell culture protocols.

extract_protocol:
Cells were crosslinked with 1% formaldehyde, and the reaction was quenched by the addition of glycine. Fixed cells were rinsed with PBS, lysed in nuclei lysis buffer, and the chromatin was sheared to 200-500 bp fragments using Fisher Dismembrator (model 500). Sheared chromatin fragments were immunoprecipitated with specific polyclonal antibodies at 4 degrees C with gentle rotation. Antibody-chromatin complexes were washed and eluted. The cross linking in immunoprecipitated DNA was reversed and treated with RNase-A. Following proteinase K treatment, the DNA fragments were purified by phenol-chloroform-isoamyl alcohol extraction and ethanol precipitation. 20-50 ng of ChIP DNA was end-repaired, adenine ligated to Illumina adapters was added, and then a Solexa library was made for sequencing.
20-50 ng of ChIP DNA was end-repaired, adenine ligated to Illumina adapters was added, and then a Solexa library was made for sequencing.

tag: experiment

value: CTCF ChIP-seq

tag: replicate


title: Luciferase knockdown in DU145 cells using shRNA replicate 1

description: polyA selected mRNA

tag: cell line


title: K562-DS11508


title: K562-DS11506


title: K562-DS11247


title: CHD7

tag: chip antibody

value: CHD7 (Bethyl A301-223A)


title: CHD8sh5b

value: iPSC (GM8330-8)-derived NPC


title: UR

status: Public on Oct 06 2014

city: Kiel

first: Dan

last: Shen

address: Hermann-Rodewald-Straße 9

institute: Kiel University

description: mock treated B. napus root small RNA

data_processing:
The sequence reads were processed to get rid of the low quality tags and several kinds of contaminants, including 5' primer contaminants, sequences without 3' primer and insert tag, with ploy A.
The cleaning data from each libraries were mapped to B. rapa and B. oleracea genome by SOAP.
The data were aligned to GenBank, Rfam (10.1), repeat associated RNA, B. rapa and B. oleracea genomic mRNA to remove unexpected sRNA, such as rRNA, tRNA, snRNA, snoRNA and degraded mRNA.
Genome_build: Brassica rapa genome and Brassica oleracea genome (database: http://brassicadb.org/brad/)
Supplementary_files_format_and_content: small RNA reads in txt format sequence, and expressed reads without tRNAs, rRNAs, snRNAs, snoRNAs, rasiRNA and degrade mRNA

organism: Brassica napus

source_name: Root

growth_protocol: Seeds of oilseed rape (Express 617) were surface sterilized and plated on B5 medium for germination. Inoculated plants were replanted into 2×2 cm plastic tube containing sterilized soil and sand and placed into climatic cabinet with 16h photoperiod. 6 days post-infection (dpi)- and 12 dpi- plants roots were collected for RNA extraction.

extract_protocol:
Roots were cleaned and freezed by liquid N2 and total RNA were isolated by Trizol reagent (Invitrogen).Total RNA from 6dpi-old roots was mixed for sequencing. In total, two samples, untreated root (UR) and V. longisporum infected root (IR) were used to construct small RNA libraries.
small RNA libraries were prepared for sequencing using standard Illumina protocols

treatment_protocol: Ten-day-old plants were extracted from B5 medium and the root parts were placed into the conidia suspension (1×107 conidia ml-1) and water as mock-inoculation for 30min.

tag: tissue

value: Root

tag: infection

value: mock

tag: time

value: 6 days after mock-treatment

tag: species

value: B. napus Expressed 617


title: IR

description: V. longisporum infected B.napus root small RNA

value: Verticillium longisporum

value: 6 days after infection


title: ETS1 knockdown in DU145 cells using shRNA replicate 2

description: polyA selected mRNA


title: ETS1 knockdown in DU145 cells using shRNA replicate 1

description: polyA selected mRNA


title: ChIP-Seq H1.2 T30

city: Barcelona

first: Miguel

last: Beato

address: Aiguader 88

country: Spain

institute: CRG

description:
Sample 20
ChIP-seq
processed data file: Read_counts_per_100_kb.txt

data_processing:
Basecalls performed using Illumina pipeline version 1.4.0
ChIP-Seq Single-end reads were processed by aligning to the reference human genome (GRCh37/hg19) using Bowtie v1.0.0. ChIP-seq signals were summed in windows of 100 Kb. Summed reads were normalized for sequencing depth and divided by the corresponding signal obtained for an input DNA of T47D cells to determine the normalized signal over input enrichment/depletion. Homer (Hypergeometric Optimization of Motif EnRichment) v4.3 was used to find regions of significance enrichment using an input as a control.
HiC Paired-end reads were processed by first aligning to the reference human genome (GRCh37/hg19) using BWA. Reads filtered from analysis include those that were not uniquely mapped, mapped more than 500 bp from relevant restriction sites, had bad sequence quality (e.g. 40% or more of the bases with Sanger PHRED quality ≤ 2) or bad BWA mapping quality (≤ 20), or were located in regions classified to have exceptionally high sequence depth (top 0.1%) by the 1000 Genomes project’s data. To offset bias introduced by PCR amplification of the sequencing library, only one of the duplicated pairs were used for subsequent analyses. Datasets were used to generate contact matrices at 100 Kb.
RNA-Seq Paired-end reads were mapped using GEM mRNA Mapping Pipeline (v1.7) using last Gencode Annotation version 18, which followed specific steps: 1) Map reads to genome reference index (including chromosme 1 to X) with maximum mismatch ratio of 0.06 and base quality threshold of 26. 2) Map reads to a transcriptome index generated from previous reference index using same options as step 1. 3) Create de-novo trasncriptome index and map again the reads to this index using maximum mismatch ratio of 0.04, minimmum split size of 15 bp and maximmum of 500000 bp. 4) Map reads again to de-novo transcriptome index created using same options as step 1. 5) Merge mappings created and pair alignments using with base quality threshold of 26 and maximmum insertion size of 500000 bp. As a result BAM alignment files were obtained and used to generate strand-specific genome-wide normalized profiles using RSeQC3 software. Exon quantifications summarized by per-genes for expression level determination were obtained either as raw read counts and reads per kilobase per milion mapped reads (RPKM) using Flux Capacitor.
Genome_build: hg19
Supplementary_files_format_and_content: ChIP-seq data files are peak results “TSV” files with genomic coordinates of peaks. HiC data files are whole genome “TSV” matrices. RNA-Seq data file is a “TSV” table which summarizes read counts per genes.

source_name: Breast cancer cell line

growth_protocol: Cells were routinely grown in RPMI 1640 medium supplemented with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin

extract_protocol:
After hormone treatment, the medium was replaced by medium containing 1% formaldehyde and incubated 10 min at 37ºC for cross-linking. The reaction was stop by adding 125 mM glycine and incubated 5 min at room temperature. Cells were washed twice with cold PBS, and collected. Preparation of chromatin and ChIP experiments were performed as previously described (Strutt et al, Methods Mol Biol. 1999; 119:455-567). Immunoprecipitated DNA was purified by phenol:chloroform followed by ethanol precipitation
It was performed using the NEBNext DNA sample prep Reagents set 1

treatment_protocol: For the experiments, cells were plated in RPMI medium without phenol red supplemented with 10% dextran-coated charcoal-treated FBS and 48 hr later medium was replaced by fresh medium without serum. After 1 day in serum-free conditions, cells were incubated with R5020 (10 nM) for different times between 0 and 60 minutes

value: Breast cancer cell line T47D-MTVL

value: progestin R5020-stimulated

tag: time (minutes)

value: anti-Histone H1.2 (catalog# ab4086, Abcam)


title: ChIP-Seq CTCF T0

description:
Sample 21
ChIP-seq

value: unstimulated

value: anti-CTCF (catalog# 07-729, Millipore)


title: ChIP-Seq CTCF T60

description:
Sample 22
ChIP-seq


title: RNA-Seq R6

description:
Sample 11
mRNA-seq
processed data file: rna-seq.readCounts.txt

extract_protocol:
Total RNA was extracted using Trizol.
mRNA-Seq sample prep kit (ref. RS-930-1001, Illumina)

treatment_protocol: For the experiments, cells were plated in RPMI medium without phenol red supplemented with 10% dextran-coated charcoal-treated FBS and 48 hr later medium was replaced by fresh medium without serum.

value: Progesterone


title: ChIP-Seq H3K14ac T0

description:
Sample 15
ChIP-seq
processed data file: Read_counts_per_100_kb.txt

value: anti-H3K14ac T0 (catalog# 07-353, Millipore)


title: RNA-Seq R1

description:
Sample 10
mRNA-seq
processed data file: rna-seq.readCounts.txt


title: ChIP-Seq H3K36me2 T30

description:
Sample 14
ChIP-seq
processed data file: Read_counts_per_100_kb.txt

value: anti-H3K36me2 (catalog# 07-369, Millipore)


title: RNA-Seq E6

description:
Sample 12
mRNA-seq
processed data file: rna-seq.readCounts.txt

value: Estradiol


title: ChIP-Seq H3K36me2 T0

description:
Sample 13
ChIP-seq
processed data file: Read_counts_per_100_kb.txt


title: Hi-C NcoI T60

description:
Sample 7
HiC

data_processing:
Library strategy: Hi-C
Basecalls performed using Illumina pipeline version 1.4.0
ChIP-Seq Single-end reads were processed by aligning to the reference human genome (GRCh37/hg19) using Bowtie v1.0.0. ChIP-seq signals were summed in windows of 100 Kb. Summed reads were normalized for sequencing depth and divided by the corresponding signal obtained for an input DNA of T47D cells to determine the normalized signal over input enrichment/depletion. Homer (Hypergeometric Optimization of Motif EnRichment) v4.3 was used to find regions of significance enrichment using an input as a control.
HiC Paired-end reads were processed by first aligning to the reference human genome (GRCh37/hg19) using BWA. Reads filtered from analysis include those that were not uniquely mapped, mapped more than 500 bp from relevant restriction sites, had bad sequence quality (e.g. 40% or more of the bases with Sanger PHRED quality ≤ 2) or bad BWA mapping quality (≤ 20), or were located in regions classified to have exceptionally high sequence depth (top 0.1%) by the 1000 Genomes project’s data. To offset bias introduced by PCR amplification of the sequencing library, only one of the duplicated pairs were used for subsequent analyses. Datasets were used to generate contact matrices at 100 Kb.
RNA-Seq Paired-end reads were mapped using GEM mRNA Mapping Pipeline (v1.7) using last Gencode Annotation version 18, which followed specific steps: 1) Map reads to genome reference index (including chromosme 1 to X) with maximum mismatch ratio of 0.06 and base quality threshold of 26. 2) Map reads to a transcriptome index generated from previous reference index using same options as step 1. 3) Create de-novo trasncriptome index and map again the reads to this index using maximum mismatch ratio of 0.04, minimmum split size of 15 bp and maximmum of 500000 bp. 4) Map reads again to de-novo transcriptome index created using same options as step 1. 5) Merge mappings created and pair alignments using with base quality threshold of 26 and maximmum insertion size of 500000 bp. As a result BAM alignment files were obtained and used to generate strand-specific genome-wide normalized profiles using RSeQC3 software. Exon quantifications summarized by per-genes for expression level determination were obtained either as raw read counts and reads per kilobase per milion mapped reads (RPKM) using Flux Capacitor.
Genome_build: hg19
Supplementary_files_format_and_content: ChIP-seq data files are peak results “TSV” files with genomic coordinates of peaks. HiC data files are whole genome “TSV” matrices. RNA-Seq data file is a “TSV” table which summarizes read counts per genes.

extract_protocol:
After hormone treatment, the medium was replaced by medium containing 1% formaldehyde and incubated 10 min for cross-linking. The reaction was stop by adding 125 mM glycine. Hi-C librarires were prepared as described in Lieberman-Aiden et al. (Science, 2009).
Lieberman-Aiden et al., 2009

treatment_protocol: For the experiments, cells were plated in RPMI medium without phenol red supplemented with 10% dextran-coated charcoal-treated FBS and 48 hr later medium was replaced by fresh medium without serum. After 1 day in serum-free conditions, cells were incubated with R5020 (10 nM) 0 and 60 minutes


title: Hi-C HindIII T0

description:
Sample 1
HiC


title: HeLa GRO-Seq rep1

status: Public on Oct 04 2014

city: Copenhagen N

first: Robin

last: Andersson

address: Ole Maaloes Vej 5

country: Denmark

institute: University of Copenhagen

description: nascent RNA

data_processing:
Library strategy: GRO-seq
Pass Filter:  Reads that pass filter were selected from fastq files.
Read Trimming: reads were trimmed to 30 bases
rDNA filter: Reads were first mapped to a copy of rRNA gene (GenBank accession #: U13369.1) including external and interanl spacers, and only reads that were not mapped to the rDNA were kept.
mapping to the genome: Bowtie was used to map 30mers with up to two mismatches to the mm9 genome.
Genome_build: hg19
Supplementary_files_format_and_content: Processed files are Bedgraphs from combined replicates.  Each entry represents the number of reads at each base.

growth_protocol: HeLa cells were grown in DMEM media supplemented with 10% FBS and 1x Pen/strep.

extract_protocol:
To isolate nuclei, cells were re-suspended in 5 ml LYSO-A buffer (35 mM Tris-Cl, pH7.5, 150 mM sucrose, 80 mM KCl, 5 mM MgCl2, 0.5 mM DTT) and centrifuged for 5 min at 1000 g. They were then re-suspended in 5 ml LYSO-A buffer containing 0.05% Tween-20. Cells were incubated on ice for 1-5 min, with constant checking to ensure that 90% of cells were permeabilised, using a haemocytometer and tryphan blue (Life Technologies). Cells were washed in 5 ml LYSO-A buffer and transferred to siliconized tubes (Eppendorf) in 1 ml LYSO-A. Cells were centrifuged at 900 g for 5 min and re-suspended in 500 μl F80 buffer (50 mM Tris, pH8.3, 40% glycerol, 80 mM KCl, 5 mM MgCl2, 0.1 mM EDTA, 0.5 mM DTT). Cells were counted and then re-suspended at 5 x 106 cells/100 μl before being snap-frozen in liquid nitrogen in aliquots of 100 μl. All steps were carried out at 4°C and using wide-bore pipette tips to minimize the loss of sample.
GRO-seq libraries were prepared as in Core et al. (Core, Waterfall, and Lis, 2008), with the following modifications.  Trizol (invitrogen) was used to stop the reaction instead of DNase I and proteinase K treatment.  The RNA was further extracted once with acid phenol:chloroform, and once with chloroform before precipitating with 2.5 volumes of -20oC ethanol.  Bead binding buffers all contained 4units/ml of SUPERaseIN (ambion) and the following buffers were slightly modified.  Bead blocking buffer: 0.25X SSPE, 1mM EDTA, 0.05% tween, 0.1% PVP, and 1mg/ml ultrapure BSA (Ambion); Binding buffer: 0.25XSSPE, 37.5mM NaCl, 1mM EDTA, 0.05% tween; Low salt wash buffer: 0.2X SSPE, 1mM EDTA, 0.05% Tween.  High Salt wash buffer: 0.25% SSPE, 137.5mM NaCl, 1mM EDTA, 0.05% Tween.  The end repair steps were modified as follows.  Pelleted RNA from the first bead binding was resuspended in 20ul, and heated to 70oC for 5min, followed by incubation on ice for 2min.  1.5ul tobacco acid pyrophosphatase (TAP) buffer, 4.5ul water, 1 ul SUPERaseIn, and 1.5ul TAP (Epicentre) were then added and the reaction incubated at 37oC for 1.5 hours. 1ul 300mM MgCl2 and 1ul T4 polynucleotide Kinase (PNK) were added to the reaction for an additional protocols l 30 min. for phosphorylating the 5’-ends, 20ul T4 PNK buffer, 2ul 100mM ATP, 145ul water, 1ul SUPERaseIn, and an additional 2ul of PNK were added for 30 min at 37oC.  The reaction was then stopped by addition of 20mM EDTA followed by acid phenol extraction and precipitation.

treatment_protocol: no special treatments of cells were performed for this study

value: HeLa

tag: gro-seq antibody

value: anti-BrU (sc-32323-ac)


title: Hi-C HindIII T60

description:
Sample 3
HiC

tag: treatment


title: HeLa GRO-Seq rep2

description: nascent RNA


title: ChIP-Seq H1.2 T0

description:
Sample 19
ChIP-seq
processed data file: Read_counts_per_100_kb.txt


title: ChIP-Seq H3K27me3 T30

description:
Sample 18
ChIP-seq
processed data file: Read_counts_per_100_kb.txt

value: anti-H3K27me3 (catalog# 39156, Active Motif)


title: ChIP-Seq H3K27me3 T0

description:
Sample 17
ChIP-seq
processed data file: Read_counts_per_100_kb.txt

value: anti-H3K27me3 (catalog# 39155, Active Motif)


title: ChIP-Seq H3K14ac T30

description:
Sample 16
ChIP-seq
processed data file: Read_counts_per_100_kb.txt


title: RNA-Seq T0

description:
Sample 9
mRNA-seq
processed data file: rna-seq.readCounts.txt

value: No Treatment


title: Hi-C NcoI T0

description:
Sample 5
HiC


title: PSC from PDA patient [CA2]

status: Public on Oct 03 2014

first: Ruth

last: Yu

address: 10010 N Torrey Pines Rd

department: Gene Expression Lab

institute: Salk Institute

description:
processed data file available on Series record: CA.bw
PSC from PDA patient

data_processing:
Read alignment and junction mapping was accomplished using TopHat2 v2.0.4 using a 25 bp 5’ segment seed for initial mapping followed by differential gene expression analysis using Cuffdiff v2.0.2 to map reads to the reference genome annotation.
BEDTools was used to generate bedGraphs from the bam files generated from TopHat2.
Bigwig files were generated from bedGraphs using the bedGraph to bigwig command from UCSC.
Genome_build: NCBI mouse build 37.2 and human build 37.2.
Supplementary_files_format_and_content: Bigwig files for UCSC browser viewing

source_name: human PSCs

growth_protocol: Human PSCs, mouse PSCs, and MiaPaCa2 cells were cultured in DMEM containing 10% FBS.

extract_protocol: RNA extraction was with Trizol per manufacturer's instructions. Standard Illumina RNA library construction with multiplexing

treatment_protocol: Cells were treated with calcipotriol at a concentration of 100nM.

tag: strain

value: human pancreatic stellate cells

tag: condition/treatment

value: primary PSCs from cancer patient


title: PSC from non-cancer control [DI1]

description:
processed data file available on Series record: DI.bw
PSC from non-cancer control

value: primary PSCs from control patient


title: PSC from non-cancer control [DI2]


title: PSC from non-cancer control [DI3]


title: PSC from non-cancer control [DI4]


title: PSC from PDA patient [CA6]


title: MiaPaCa-2 cells treated with conditioned media [Mia-CMS]

value: human pancreatic adenocarcinoma cell line

value: conditioned media from calcipotriol-treated cells


title: MiaPaCa-2 cells treated with conditioned media  [Mia-CM]

value: conditioned media from untreated cells


title: MiaPaCa-2 cells treated with DMEM  [Mia-DMEM]

description: MiaPaCa-2 cells treated with DMEM

value: DMEM


title: PSC from PDA patient [CA3]


title: MiaPaCa-2 cells treated with calcipotriol  [Mia-CAL]

value: 100nM calcipotriol


title: PSC from non-cancer control [DI5]


title: PSC from PDA patient [CA7]


title: day 7 mouse PSCs treated with DMEM [mPSC-d7]

description: day 7 mouse PSCs treated with DMEM

source_name: mouse PSCs

value: mouse  primary pancreatic  stellate cells

value: day 7 untreated


title: control-rep2

city: Stanford

first: Mark

middle: M.

last: Davis

address: 279 Campus Drive

department: Department of Microbiology and Immunology

institute: Stanford University

data_processing:
RNA-seq reads were aligned to the known transcriptome sequence from UCSC mm9 RefSeq using Bowtie2 v2.1.0 with parameters  -q --no-mix --no-discordant --non-deterministic  --no-unal --omit-sec-seq
FPMK values were calculated using in-house Perl scripts
Genome_build: mm9
Supplementary_files_format_and_content: FPMK values table

source_name: liver

growth_protocol: Ten female BALB/c mice, 6-8 weeks of age (Jackson Laboratory), were randomly picked (for each biological repeat experiment).

extract_protocol:
Liver total RNA was isolated using Trizol (Invitrogen).
RNA-Seq library was created by using TruSeq kit (illumina). All samples were barcoded and then mixed into one library. Sequencing reads (101bp, pair-ended) were generated using the Illumina Hi-Seq (Stanford Cancer Institute).

treatment_protocol: Hydrodynamically infused with a mixture of 6mg of appropriate shRNA plasmids and 4mg pBluescript plasmid DNA (Stratagene) as carrier. The liver was harvested 7 days post-injection, frozen in liquid nitrogen and then grounded to powder.

value: BALB/c

tag: age

value: 6-8 weeks

value: Liver


title: sh-8165-rep2

description: shRNA Guide strand sequence (5’ to 3’) :UAUUUUCUCGCAGACCCGCUU


title: sh-4168-rep2

description: shRNA Guide strand sequence (5’ to 3’) :UUAAUGUUGGGAUUGAUGCUU


title: sh-1193-rep2

description: shRNA Guide strand sequence (5’ to 3’) :UAACAUUUGGGCUGCGAGCUU


title: sh-4161-rep2

description: shRNA Guide strand sequence (5’ to 3’) :UGGGAUUGAUGCCAUGUGCUU


title: day 3 mouse PSCs treated with DMEM [mPSC-d3]

description: day 3 mouse PSCs treated with DMEM

tag: cell type

value: day 3 untreated


title: sh-4224-rep2

description: shRNA Guide strand sequence (5’ to 3’) :UGCCAUAUGUGGAGUACGCUU


title: sh-7841-rep1

description: shRNA Guide strand sequence (5’ to 3’) :UUCAUAAUGGGCGUCGAGCUU


title: control-rep1


title: sh-4231-rep2

description: shRNA Guide strand sequence (5’ to 3’) :UGAAAUUUGCCAUAUGUGCUU


title: sh-4231-rep1


title: sh-8165-rep1


title: sh-5451-rep2

description: shRNA Guide strand sequence (5’ to 3’) :UAUCAAAAGCCUCAUACACUUU


title: sh-5445-rep2

description: shRNA Guide strand sequence (5’ to 3’) :UAGCCUCAUACAGGACCUCUU


title: sh-7841-rep2


title: sh-1193-rep1


title: sh-4168-rep1


title: sh-4224-rep1


title: sh-5445-rep1


title: sh-4161-rep1


title: day 7 mouse PSCs treated with calcipotriol [mPSC-d7-CAL]

value: day 7 calcipotriol treated


title: day 3 mouse PSCs treated with calcipotriol [mPSC-d3-CAL]

value: day 3 calcipotriol treated


title: sh-5451-rep1


title: 4Clong

city: Moscow

first: Nickolai

last: Tchurikov

address: 32, Vavilova str.

department: Department of Epigenetic Mechanisms of Gene Expression Regulation

country: Russia

institute: Engelhardt Institute of Molecular Biology

description: DNA fraction >400 bp

data_processing:
Base calling and quality control were performed in real time with standard Illumina analysis pipeline using a phiX control.
Sequenced reads were trimmed for adaptor sequences and low quality sequences by cutadapt v. 1.2.1 using the following options: --minimum-length=30 --trimmed-only --quality-base=33 --quality-cutoff=3 -n 2 -g CTGGTGAGGGCATG  -g GAATTCCCTGCTTCCCATT
Trimmed reads were mapped to rDNA and to hg19p10 by bwa 0.7.5a using the mem algorithm and by samtools 0.1.12a (r862)
Mapping and alignment results were converted to resulting table using ad hoc Perl scripts. Main results are in Text table files, bed files are supplied for more convenience.
Genome_build: Human ribosomal DNA complete repeating unit,  GenBank Accession number U13369
Genome_build: Homo Sapiens hg19/GRCh37.p10 genome with included to chr14 from address 1 Human ribosomal DNA U13369
Supplementary_files_format_and_content: Tab-delimited text file include the following features of each mapping: begin, end, length, coverage, number of reads, sequence. BED files are supplied too.

extract_protocol:
DNA samples were prepared as described before (Dekker et al., 2002; Osborne et al., 2004; http://www.protocol-online.org/cgi-bin/prot/view_cache.cgi?ID=3978).

Cross-linking
To suspension containing about 20x106 HEK293T cells in 40 ml of DMEM formaldehyde solution was added to final concentration 1.5%. After mixing incubation of cell suspension was performed for 10 min at room temperature with mixing. The quenching with 2.75 ml of 2M glycin (final concentration 0.125M) was performed. After incubation at room temperature for 5 min the suspension was cooled for 15 min in ice bath and then cells were collected by centrifugation for 15 min at 3500 rpm at +2°C.

Lysis
The pellet of cells was resuspended at °C in 1ml of buffer containing 10 mM tris-HCl buffer, pH 8, 10 mM NaCl, 0.2% NP-40 and freshly added protease inhibitors-- 0.1 mM PMSF and 1:500 protease inhibitor cocktail (Sigma). After incubation for 15 min cells were homogenized by passing through insulin syringe about 50 times into Eppendorf. Then nuclei were spin down by centrifugation for 5 min at 5000 rpm in Eppendorf centrifuge 5415 R at +2°C.

Digestion with EcoRI
The nuclei pellet was resuspended in 756 ul of solution containing 40 mM tris-HCl buffer, pH 7.4, 50 mM NaCl, 10 mM MgCl2 and 10 mM 2-mercaptoethanol. Then 20% SDS was added to final concentration 0.3% and incubation with shaking was performed for 1 h at +37°C. To sequester SDS 180 ul of 10% Triton X-100 was added and the incubation for 1 h at +37°C was performed. Before digestion 1 ul of BSA (5 mg/ml) was added with mixing. Then 50 ul of EcoRI (10 u/ul) was added and after mixing digestion was performed overnight at +37°C.

Ligation
To inactivate the restriction enzyme, 35'ul of 20% SDS was added (final concentration 0.7%) and the probe was heated to 65°C for 30 min. Then the mixture was transferred into 15 ml Nunc tube and consequently 375 ul of 20% Triton X-100 (to final concentration 1%), 750 ul of 10x ligase buffer, 7,5'ul of BSA (5 mg/ml), 80 ul of 100 mM ATP, and 5241 ul of milliQ water  were added and the final 7.5 ml solution was well mixed. Then 10 ul of T4 DNA ligase (200 u/ul) was added and after mixing incubation was performed for 5 h at +16°C and then for 30 min at room temperature. Usually during ligation DNA concentration was equal to 2 ng/ul.

DNA purification
For isolation of DNA 50 ul of proteinase K (10 mg/ml) was added (final concentration 50 ug/ml) and after mixing incubation was performed at 55°C overnight. For RNA digestion 40 ul of RNase A (10 mg/ml) was added (final concentration 0.5 ug/ml) and after mixing incubation was performed for 30 min at +37°C. After extensive extraction with phenol-chloroform extraction (three times with 7 ml each) DNA was precipitated by 2.5 vol. of ethanol after addition of 40 ul 10 mg/ml glycogen and 175 ul 4M NaCl. The final DNA pellet was washed twice with 70% ethanol and then dissolved in 0.1xTE.

Digestion with FaeI
About 15 ug of DNA was digested in 250 ul solution with 75 u of FaeI overnight at +37°C. Then the enzyme was inactivated by heating at 65°C for 30 min. DNA was isolated after phenol-chloroform extraction, precipitated by ethanol and dissolved in 100 ul of 0.1xTE.

Ligation
For circularization 15'ug of DNA was incubated in 8 ml of T4 DNA ligase buffer containing 400 u of T4 DNA ligase for 5 h at +16°C. DNA was isolated after phenol-chloroform extraction, precipitated by ethanol and dissolved in 50 ul of 0.1xTE.

PCR and library preparation
One or two rounds of PCR were used as described below. There are about 300-400 copies of rDNA. That is why a single round of PCR (with up to 35 cycles) was found to be sufficient. DNA concentration was titrated and finally about 30 ng of DNA was used for PCR with primers   5' TCTTTGAAAAAAATCCCAGAAGTGGT 3' and 5' AAGTCCAGAAATCAACTCGCCAGT 3' (for PCR-1), and 5' GCCTAAGCCTGCTGAGAACTTTC 3' and 5' CAGCATTCTGTAGGGAGATCAAATC 3' (for PCR-2). After separation in 2% agarose gels two DNA fractions (200-400 bp and higher 400 bp) were eluted using QIAqiuck gel extraction kit (Quigen). The libraries were prepared using TruSeq RNA Sample Preparation Kit v2 (Illumina) using adapter AR006 for 200-400 bp DNA fraction and adapter R007 for DNA fraction higher 400 bp. The samples were sequenced using MiSeq (Illumina) following the manufacturer's protocols.

value: kidney

value: embryo

tag: genotype

value: wild type


title: 4Cshort

description: DNA fraction 200-400 bp


title: cfa-diro

city: Sainte-Anne-de-Bellevue

first: Lucienne

last: Tritten

state: QC

address: 21111 Lakeshore Road

department: Institute of Parasitology

institute: McGill University

data_processing:
basecall using illumina CASAVA v1.8
The 3' adaptor was removed from raw reads and reads with the length less than 10 were excluded to constitute the mappable reads.
The mappable reads were aligned against dog genome and miRbase using Bowtie.
Genome_build: Dirofilaria immitis 2.2; miRbase v20; Canis familiaris 3.1;
Supplementary_files_format_and_content: unique_miRs.xls: summary of miRNA candidates identified in Dirofilaria immitis

organism: Canis lupus familiaris

source_name: plasma

extract_protocol:
Total RNA was extracted from plasma samples using a kit (Norgen Biotek Corp.).
Illumina Truseq Small RNA Preparation

tag: infection of d. immitis strain

value: MP3 and Missouri strains (lab strains)

tag: number of dogs used

value: pool of 4


title: cle40-2 replicate 3

status: Public on Oct 02 2014

city: Düsseldorf

first: Helge

last: Pallakies

address: Universitätsstrasse 1

department: Developmental Genetics

institute: CEPLAS / Heinrich-Heine-University Düsseldorf

data_processing:
Illumina CASAVA 1.8.2 software was used for base calling, demultiplexing and trimming
Mapping (TAIR10 annotation) was performed in the CLC genomics workbench
Differential expression analysis was performed in R (2.14.1) with the package DEseq (Anders and Huber, 2010)
Genome_build: TAIR 10
Supplementary_files_format_and_content: .csv-files show fold change expression between Col-0 wildtype and each mutant showing significance via the Benjamini-Hochberg adjusted (Reiner et al., 2003) p-value.

organism: Arabidopsis thaliana

source_name: root tip tissue

growth_protocol: Seeds were grown on plant agar containing germination medium consisting of Murashige and Skoog (2.2 g/l) salts with Gamborgs no. 5 vitamins , 2-(N-morpholino) ethanesulfonic acid (0.5 g/l) and sucrose (10 g/l) and grown in a vertical position at 21°C under constant light conditions.

extract_protocol:
RNA was isolated using the Qiagen RNeasy Plant Mini Kit with additional integrated DNAseI treatment and additionally treated with DNAseI from Roche Diagnostics.
RNA libraries were prepared for sequencing using standard Illumina protocols.

value: root tip tissue

tag: genetic background


title: ET1-CM, experiment3

city: Milwaukee

first: Ulrich

last: Broeckel

state: WI

address: 8701 Watertown Plank Road

institute: Medical College of Wisconsin

description: miRNA sequencing based expression data from ET-1 stimulated hiPSC-CMs from experiment 3

data_processing:
Ion Torrent's Torrent Suite version 3.6 was used for basecalling
Raw sequencing reads were aligned using the SHRiMP2 aligner and were aligned against the human reference genome (hg19) for novel miRNA prediction and then against a custom reference sequence file containing miRBase v.20 known human miRNA hairpins, tRNA, rRNA, adapter sequences and predicted novel miRNA sequences.
SHRiMP aligner's gmapper-ls was used with the following parameters: -s 00111111001111111100,00111111110011111100,00111111111100111100,00111111111111001100,00111111111111110000,11111111111 -o 1 -H -E -a -1 -q -30 -g -30 --qv-offset 33 --strata -N 6
miRNA quanitification was done using HTSeq v0.5.3p3 using the default union parameter.
Differential miRNA expression was analyzed using the DESeq (v.1.12.1) R/Bioconductor package
Genome_build: hg19, miRBase v.20 human miRNA hairpins
Supplementary_files_format_and_content: tab-delimited text files containing raw read counts for known mature human miRNAs.

source_name: hiPSC-CM from experiment3 after ET-1 stimulation

growth_protocol: iCell-CMs were obtained from one single batch of cells. iCell Cardiomyocytes were plated at 2.0 × 104 cells/well in a 96-well plate precoated with 0.1% gelatin solution.

extract_protocol:
18 h post ET-1 stimulation, cells were harvested with Total RNA Purification 96-Well Kit (Norgen Biotek Corp.).  Total RNA was extracted per manufacturer’s recommendations, resuspended in nuclease-free water, and quantified by UV spectrophotometry (NanoDrop™ 2000, Thermo Scientific).
MicroRNA libraries from rRNA depleted total RNA (Ribo-Zero rRNA removal Kit, Epicentre) were prepared using Ion Torrent Total RNA Seq small RNA kit as per manufacturer’s recommendations (Life Technologies).

treatment_protocol: After two days of recovery, the cells were cultured in William’s E medium supplemented with Cocktail B (1:25) from the Hepatocyte Maintenance Supplement Pack (Life Technologies). After an additional 11 days of recovery, cells were stimulated for 18 h with ET-1 (Sigma Aldrich) at 10-8M as recommended by the manufacturer. Experiments were performed in triplicate for both unstimulated controls (control-CM) and ET-1 stimulated cells (ET1-CM).

value: hiPSC-derived cardiomyocytes


title: control-CM, experiment1

description: miRNA sequencing based expression data from unstimulated hiPSC-CMs from experiment 1

source_name: hiPSC-CM from experiment1 before ET-1 stimulation


title: cle40-2 replicate 1


title: coryne-3 replicate 2


title: cfa-dim

value: natural infection  (co-infection with hookworm)


title: clavata2-gabi replicate 2


title: ET1-CM, experiment1

description: miRNA sequencing based expression data from ET-1 stimulated hiPSC-CMs from experiment 1

source_name: hiPSC-CM from experiment1 after ET-1 stimulation


title: control-CM, experiment3

description: miRNA sequencing based expression data from unstimulated hiPSC-CMs from experiment 3

source_name: hiPSC-CM from experiment3 before ET-1 stimulation


title: WT_IC_RC12h.2011_11_30

city: Flagstaff

first: Bridget

middle: Marie

last: Barker

state: AZ

address: 3051 Shamrell Blvd

department: CFP

institute: TGEN-North

description: replicate 3

data_processing:
Illumina HiSeq fastq files were quality filtered using Trimmomatic-0.30 using parameters as in the paired end example in the trimmomatic manual, keeping only reads >= 36 bp and requiring each of the paired reads pass the filtering.
Trimmomatic quality filtered fastq reads were aligned to the Aspergillus_fumigatusa1163.CADRE.18.dna.toplevel.fa genome (http://fungi.ensembl.org/info/website/ftp/index.html) using bowtie2-2.1.0, keeping only concordantly mapped read pairs.
The concordantly aligned paired end bam files were subsampled using samtools-0.1.19 view -s option such that number of reads in input control approximately matched number of reads in ChIP sample within replicate.
Subsampled bam files were filtered to genearate deduplicated bam files using samtools-0.1.19 rmdup followed by picard-tools-1.93 MarkDuplicates with REMOVE_DUPLICATES=TRUE.
Deduplicated bam files for each replicate were combined to obtain a single input control bam file and single ChIP bam file.
The combined single ChIP bam file and combined single IC bam file were used as input for peak calling using macs2 version 2.0.10.20131216 (parameters: mfold 5,50; format bampe; keep-dup all; call-summits).
Genome_build: Aspergillus_fumigatusa1163.CADRE.18.dna.toplevel.fa (http://fungi.ensembl.org/info/website/ftp/index.html)
Supplementary_files_format_and_content: tab separated text file. The peaks file from macs2 with run details and called peak locations and scores.

organism: Aspergillus fumigatus

source_name: mycelia

growth_protocol: All strains are routinely grown in liquid glucose minimal media (LGMM) that contains 1% glucose, salt solution and trace minerals, at 37°C, under agitation (200RPM) in baffle flasks.

extract_protocol:
After reverse crosslinking, samples were treated with 2.5 mg of RNase A and incubated for 30 minutes at room temperature.  DNA was extracted either by using PCR purification kit (Qiagen) following the high pH protocol, or EtOH precipitation, with 50 mL as final volume.  5mL of sample was assayed on Qubit using high sensitivity dsDNA kit (Invitrogen).  To check sonication, 10μL of IC was run on E-gel EX 2% agarose gel (Invitrogen) and fragment size ranged from 1kb to 100bp
fragmented ends were repaired, an adenine molecule was added to the repaired end, PAGE-purified adapters were added to the overhang, and the mixture amplified with primer 1.0 and primer 2.0 with an individual index tag to allow for multiplex of samples in a single lane. Samples were gel purified to obtain a size range between 200-400bp.  Libraries were validated by real time PCR, concentration was determined with Qubit (Invitrogen) and integrity was checked with an Agilent Bioanalyzer.
76 bp paired end sequencing and four ChIP samples were indexed per lane on the Illumina HiSeq 2000.

treatment_protocol: hypoxia, 1% oxygen, 5%CO2


title: ET1-CM, experiment2

description: miRNA sequencing based expression data from ET-1 stimulated hiPSC-CMs from experiment 2

source_name: hiPSC-CM from experiment2 after ET-1 stimulation


title: WT_ChIP_RC4H.2011_05_31

description: replicate 1


title: control-CM, experiment2

description: miRNA sequencing based expression data from unstimulated hiPSC-CMs from experiment 2

source_name: hiPSC-CM from experiment2 before ET-1 stimulation


title: Col-0 replicate1

description: crn_diffexp.csv; clv2_diffexp.csv; cle40_diffexp.csv

value: wildtype Col-0


title: WT_IC_RC4H.2011_05_31

description: replicate 1


title: WT_ChIP_RC4H.2011_11_11

description: replicate 2


title: WT_ChIP_RC12h.2011_11_30

description: replicate 3


title: clavata2-gabi replicate 3


title: clavata2-gabi replicate 1


title: WT_IC_RC4H.2011_11_11

description: replicate 2


title: cle40-2 replicate 2


title: IlluminaDSB01

data_processing:
Illumina Casava 1.8 software used for basecalling.
Sequenced reads were trimmed for adaptor sequences and low quality sequences by cutadapt v. 1.2.1 using the following options: --minimum-length=30 --trimmed-only --quality-base=33 --quality-cutoff=3 -n 2 -g CCCAAGCTTAAGCGGCCGCAAAC -g CCGAATTCTCCTTATACTGCAGGGG
Trimmed reads were mapped to rDNA and to hg19/GRCh37p10 by bwa 0.7.5a using the mem algorithm and by samtools 0.1.12a (r862)
Mapping and alignment results were converted to resulting table using ad hoc Perl scripts with.
Obtained mapping table to hg19 have been checked for presense of Sau3AI recognition sequence (GATC), it has to be presented in final mapping near the only one end, so DSB have to be at the opposite end of alignment. All alignments without Sau3AI sequence have been removed from the final table.
Main results are in table_rDNA.txt and table_hg19.txt text tables, wig and bed files are supplied for more convenience.
Genome_build: Human ribosomal DNA complete repeating unit,  GenBank Accession number U13369
Genome_build: Homo Sapiens hg19/GRCH37p10 genome with included to chr14 from address 1 Human ribosomal DNA U13369
Supplementary_files_format_and_content: Tab-delimited text file include the following features of each mapping: begin, end, length, coverage, number of reads, sequence. BED and WIG files are supplied too.

source_name: HEK293T cells

growth_protocol: The cells were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemente+B29d with 10% FBS penicillin (100 U/ml), streptomycin (100 ug/ml) and L-glutamine (4 mM).

extract_protocol:
DNA-agarose plugs were prepared as described before [Tchurikov NA and Ponomarenko NA (1992) Detection of DNA domains in Drosophila, human and plant chromosomes possessing mainly 50- to 150-kilobase stretches of DNA. Proc Natl Acad Sci USA 89: 6751-6755]. About 6 millions of HEK293T cells in 2 ml of culture medium were pelleted by centrifugation at 2000 rpm, resuspended in 0.3 ml of the same medium, gently mixed at 42°C with an equal volume of a 1% agarose L (LKB) in PBS solution, and distributed on a mold containing 100-microliters wells. The mold was placed on ice for 2-5 min, covered with parafilm. The agarose plugs were then placed in Petri dishes with 5 ml of solution containing 0.5 M EDTA (pH 9.5), 1% sodium laurylsarcosine, and 1-2 mg of proteinase K solution per ml for 40-48 hr at 50°C, and stored at 4°C in the same solution. Each DNA-agarose plus usually contained about 15 microgramms of DNA corresponding to about 1 millions of cells.
To test the quality of isolated DNA the fractionation in the pulsed-field gels was performed as described before (Tchurikov and Ponomarenko, 1992). Portions of the original agarose-DNA plugs (5-50 microliters) containing 1-10 microgramms of DNA were used for electrophoresis without any restriction enzyme digestion. The DNA samples were run in 0.8% agarose gels on an LKB Pulsaphor system using hexagonal electrode and switching times of 25 or 450 sec.
For elution of DNA preparations the fractionation in 1% agarose conventional mini-gel was performed. One-half of DNA-agarose plug was washed in 1xTE 3 times (for 15 min each) following by 3 times washing in the same solution containing 17.4 microgramms/ml PMSF in ethanol. After fractionation in the mini-gel, the ethidium-bromide stained DNA band was excised and electoeluted inside the dialysis cellulose membrane bag. After overnight dialysis without stirring against 1 liter of 0.01 x TE at 4°C, the DNA was concentrated with PEG (4°C) and redialyzed.

Rapid amplification of forum domains termini (RAFT) procedure:
About 1.5 microgramms of isolated DNA (see above) was treated with Klenow fragment and then ligated with 70 ng of double-stranded oligonucleotide (25 bp long 5'-phosphorylated 5' pCCCCTGCAGTATAAGGAGAATTCGGG 3' oligonucleotide annealed with 26 bp long 5' biotinylated 5' bio-CCGAATTCTCCTTATACTGCAGGGG 3' oligonucleotide) in 150 microliters of solution containing 0.1 M NaCl, 50 mM Tris HCl (pH 7.4), 8 mM MgCl2, 9 mM 2-mercaptoethanol, 7 microM ATP, 7.5 % PEG, and 40 units of T4 DNA ligase at 20°C for 16 hr. After heating at 65°C for 10 min the DNA preparation was digested with Sau3A enzyme to shorten the forum domain to the termini attached to the ligated oligonicleotide. The selection of such termini was performed in 0.5 ml Eppendorf tubes using 300 microliters of suspension containing streptavidin magnespere paramagnetic particles, SA-PMP (Promega) according to the manufacturer's recommendations. After extensive washing with 0.5xSSC removing DNA fragments corresponding to internal parts of forum domains, the forum termini (FT) DNA preparation was eluted from the SA-PMP using digestion with EcoRI enzyme in final volume of 50 mictolitres (double-stranded FT). The FT were then ligated with 100x molar excess of double-stranded Sau3A adaptor (5'-phosphorylated 5' pGATCGTTTGCGGCCGCTTAAGCTTGGG 3' oligonucleotide annealed with 5' CCCAAGCTTAAGCGGCCGCAAAC 3' oligonucleotide). In some experiments (FT) DNA preparation was eluted from the SA-PMP using heating by incubation at 100°C for 3 min in 50 microliters of 0.01xTE (single-stranded FT). Before heating the FT preparation was ligated with100x molar excess of double-stranded Sau3A adaptor in suspension with SA-PMP (see above). Both final DNA samples (double-stranded FT or single-stranded FT) were used for PCR amplifications. 40 cycle PCR amplification in 30 microlitersl of a solution containing 67 mM Tris-HCl (pH 8.4); 6 mM MgCl2; 10 mM 2-mercaptoethanol; 16.6 mM ammonium sulfate; 6.7 microM EDTA; 5 microg/ml BSA; 1 mM dNTPs; 1 microg of primer corresponding to Sau3A adaptor (5' CCCAAGCTTAAGCGGCCGCAAAC 3'); 1 microg of primer corresponding to biotinilated oligonucleotide (5' CCGAATTCTCCTTATACTGCAGGGG 3') and 1 u of Taq polymerase was performed using Eppendorf Mastercycler Personal. Amplification conditions were 90°C for melting, 65°C for annealing and 72°C for extension, for 1 min each.

Libraries were prepared according to Illumina's instructions accompanying the DNA Sample Kit (Part# 0801-0303). Briefly, DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (32 to 52 exo minus) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3â€™ end. After adapter ligation DNA was PCR amplified with Illumina primers for 15 cycles and library fragments of ~200 -400 bp (insert plus adaptor and PCR primer sequences) were band isolated from an agarose gel. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Genome Analyzer IIx following the manufacturer's protocols.

treatment_protocol: HEK 293 cells were seeded in 10 cm culture plates 1-2 days before experiment in DMEM containing 10% FBS, and used at approximately 60-80% confluency.

tag: cell line


title: coryne-3 replicate 1


title: Col-0 replicate3


title: Col-0 replicate2


title: Polistes metricus ad libitum nutrition manipulation 5th instar larvae - EE1 sample

city: Ames

first: Ali

last: Berens

state: IA

address: 251 Bessey Hall

department: Ecology, Evolution, and Organismal Biology

institute: Iowa State University

data_processing:
We removed the adapter sequences before transcriptome assembly using the fastx_clipper tool from the Fastx Toolkit (Version 0.0.13). We filtered reads for quality (threshold greater than or equal to 20) using the Trim Perl script (Nik Joshi, unpublished; code available from http://wiki.bioinformatics.ucdavis.edu/index.php/Trim.pl).
We assembled the groomed transcriptomic short reads de novo using Trinity (default parameters, Version r2012-06-08).
The final Trinity-produced Transcriptome Shotgun Assembly (TSA) was annotated with the mRNAmarkup protocol (Volker Brendel, unpublished; code available from http://brendelgroup.org/bioinformatics2go/mRNAmarkup.php). mRNAmarkup: parameter option –F 5 5 10 10 90 60; MuSeqBox option –A 45 10 75
We aligned the raw paired-end reads to the TSA using Bowtie2 (default options, Version 2.1.0) and quantified the abundances of the transcripts for each library with eXpress (default options, Version 1.3.1)
Genome_build: GI:659332307
Supplementary_files_format_and_content: tab-delimited text files include raw count values for each Sample

organism: Polistes metricus

source_name: head

extract_protocol:
Larvae were flash frozen in liquid nitrogen and stored at -80C until RNA extraction. We extracted total RNA from individual larval heads using an RNeasy Kit (Qiagen). Illumina TruSeq RNA Sample Prep Kit (Cat#FC-122-1001) was used with 1 ug of total RNA for the construction of sequencing libraries.
RNA libraries were prepared for sequencing using standard Illumina protocols

treatment_protocol: The nutrition samples come from nests that were collected from North Carolina in late Spring 2009 and reared in the laboratory of James Hunt to control the nutrition level provided to the nest. The low nutrition nests were restricted to protein (worm every three days), and the high nutrition nests were provided protein ad libitum (worm every day). The caste samples were collected from P.metricus nests at the Iowa 4-H Center (Madrid, IA). Based on the colony life cycle of paper wasps [15], foundress-reared (worker-destined) and worker-reared (queen-destined) larvae were collected late Spring and late Summer 2010, respectively.

value: head

value: 5th Instar Larvae

tag: Sex

value: female

tag: biome

value: laboratory reared

value: USA:North Carolina:Raleigh


title: Polistes metricus ad libitum nutrition manipulation 5th instar larvae - CC1 sample

tag: tissue


title: DGCR8-CLIP-seq replicate11 (No RNase 1 treated)

city: Seoul

first: Young-Soo

last: Kwon

address: Nungdongro 209, Gwangjingu

department: Department of Bioscience and Biotechnology

country: South Korea

institute: Sejong University

description:
CLIP enriched RNA
DGCR8_CLIP_long_cov_neg_wo_RNase_hg19.bed, DGCR8_CLIP_long_cov_pos_wo_RNase_hg19.bed

data_processing:
Basecalls performed using CASAVA version 1.8.2
Reads were demuliplexed by using codes in the id of each reads and first 2 reads by using the ShortRead package in the BioConductor. Identical sequences were collapsed using the occurrenceFilter function in the ShortRead package.
3' adapter sequences were trimmed using the trimLRPatterns function in the ShortRead package. The 3' adapter sequences trimmed were sequences containing CCTGTGGTCGTAGCATCAGCTA.
The trimmed sequences were converted to reverse complementary sequences.
CLIP-seq and small RNA reads were aligned to the 199 genome assembly using Bowtie verions 1.12.7  with the following configurations -q –m 50 -n 2 -l 44 -5 1 -3 5 -a --best --strata -p 7 --sam for noncoding RNAs and -q –m 1 -n 2 -l 44 -5 1 -3 5 -a --best --strata -p 7 --sam for miRNA precursors and unique genomic sequences.
Genome_build: hg19
Supplementary_files_format_and_content: bedGraph files were generated by converting mapped data of DGCR8-CLIP-seq , Drosha-CLIP-seq, and DGCR8-RIP seq to coverage data The scores represent coverage on each position of the human genome (hg19).
Supplementary_files_format_and_content: csv files were generated by counting reads mapping to mature miRNAs (miRBase release 20)

source_name: H1 human embryonic stem cells

growth_protocol: H1 human embryonic stem cells were maintained on Matrigel (BD Biosciences, Cat# 354277)-coated plates in MEF-conditioned medium. Medium was exchanged every day and colonies were passaged by Collagenase Type IV (Cat# 17104-019) every 3-4 days. For generation of MEF-conditioned medium, gamma-irradiated MEFs of CF1 strain were used. 2.5 x 106 MEFs were plated on a gelatin-coated 100mm plate containing 10ml of D-MEM (Cat# 10569-010) supplemented with 10% FBS. The following day, the medium was replaced with DMEM:F12 (Cat# 10565-018) containing 20% Knockout Serum Replacement (KSR)(Cat# 10828-028), 1mM glutamine (Cat# 25030-081), 0.1mM β-mercaptoethanol (Cat# 21985-023), 1x non-essential amino acids (Cat# 11140-050) and 4ng/ml bFGF (Cat# PHG0264). The medium was collected after 24 hours of conditioning for four days, filter-sterilized and stored at -20C until use. 20ng/ml of bFGF was freshly supplemented before use. All reagents were purchased from Life Technologies unless stated otherwise.

extract_protocol:
Two million cells were resuspended in 250 μl lysis buffer (50 mM Tris-HCl pH 7.4, 100 mM NaCl, 1 mM MgCl2, 0.1 mM CaCl2, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) containing EDTA-free protease inhibitor cocktail (Roche) and RNase inhibitor (Ambion) and briefly sonicated by using the Bioruptor. The lysates were treated with 5 μl of Turbo DNase (Ambion) and 10 μl of 10 U/μl or 1 U/μl RNase I (Ambion) at 37°C for 3 min. Cellular debris was removed by centrifugation at 13,000 rpm for 15 min. Protein-RNA complexes were immunopurified with an anti-Flag or anti-DGCR8 antibody (Bethyl laboratories) bound to Protein A/G Dynabeads. Beads were washed twice with high-salt wash buffer (50 mM Tris-HCl pH 7.4, 1 M NaCl, 1 mM EDTA, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS) followed by two washes with PNK buffer (20 mM Tris-HCl pH 7.4, 10 mM MgCl2, and 0.2% Tween-20). For dephosphorylation of 3’ ends, 1U of FastAP alkaline phosphatase (Fermentas) was added to the beads and incubated at 37°C for 20 min. After washing the beads twice with PNK buffer, protein-bound RNAs were ligated to a pre-adenylylated 3’ adaptor overnight at 16°C. Beads were washed twice with PNK buffer again and labeled with [γ- 32P] ATP for visualization. The labeled RNAs were separated by 4-12% NuPAGE Bis-Tris gel (Invitrogen), transferred to a nitrocellulose membrane, and excised from the membrane. The protein was degraded by proteinase K treatment, and RNA was recovered by phenol/chloroform/isoamyl alcohol extraction and ethanol precipitation. H1 cells were washed twice with PBS and cross-linked with 1% formaldehyde at room temperature for 10 min. After washing twice with PBS, cells were collected and sonicated in 0.2 ml lysis buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA, 1% SDS, 0.5% Empigen BB). After centrifugation at 13,000 rpm for 15 min, the supernatant was diluted 2.5-fold in IP buffer (20 mM Tris-HCl pH 8.0, 100 mM NaCl, 2 mM EDTA, 0.5% Triton X-100) and incubated with an anti-DGCR8 antibody or IgG bound to Protein A/G Dynabeads at 4°C for 2 hours. Beads were serially washed with 1 ml washing buffer I (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM EDTA, 0.1% SDS, 1% Triton X-100), washing buffer II (20 mM Tris-HCl pH 8.0, 2 mM EDTA), washing buffer III (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 1% NP-40, 1% deoxycholate, 0.25 M LiCl), and TE buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA). Beads were then treated with 5 μl of Turbo DNase at 37°C for 3 min and washed twice with PNK buffer. FastAP alkaline phosphatase was added and incubated at 37°C for 20 min. Beads were washed twice with PNK buffer again. DGCR8-bound RNAs were ligated to a pre-adenylylated 3’ adaptor overnight at 16°C and washed twice with PNK buffer. Protein-RNA complexes were removed from the beads through 30 min incubation with 1% SDS. Crosslinked proteins were eliminated by proteinase K treatment at 65°C for 2 hrs, and RNA was recovered by phenol/chloroform/isoamyl alcohol extraction and ethanol precipitation. For AGO2 immunoprecipitation flag-AGO2 H1 cells were lysed in IP buffer containing 1% Empigen BB, 0.1 mM DTT, and protease inhibitor cocktail (Roche) in PBS. After centrifugation at 13,000 rpm for 15 min, the supernatant was incubated with an anti-Flag antibody (Wako) or IgG bound to Protein A/G Dynabeads (Invitrogen). Beads were washed five times with IP buffer. AGO2-bound RNAs were extracted with phenol/chloroform/isoamyl alcohol (Sigma) and precipitated with ethanol.
Protein-associated RNAs purified by IP, CLIP, or RIP were ligated to a pre-adenylylated 3’ adaptor that contains a 2-nt adapter code for de-multiplexing and a 3-nt random sequence to minimize the loss of information after collapsing identical sequences.  The RNA-adapter hybrids were converted into cDNAs with RT primer and SuperScript III reverse transcriptase (Invitrogen). The cDNAs were size-fractionated by electrophoresis on a 12% denaturing polyacrylamide gel and circularized by single-stranded DNA ligase (Epicentre). Circularized cDNAs were amplified with PCR primers, in which one of them contained a 6-nt primer code for de-multiplexing. PCR products were purified by agarose gel elution and subjected to next-generation sequencing.

treatment_protocol: For CLIP experiments H1 cells were irradiated with 300 mJ/cm2 of UVC light (254 nm) by using the CL-1000 ultraviolet crosslinker (UVP). For RIP experiments cells were cross linked using 1% formaldehyde for 10 min at room temperature.

tag: antibody


title: Polistes metricus ad libitum nutrition manipulation 5th instar larvae - LL1 sample


title: DGCR8-CLIP-seq replicate 12 (Low RNase 1 treated)

description:
CLIP enriched RNA
DGCR8_CLIP_long_cov_neg_w_RNase_hg19.bed, DGCR8_CLIP_long_cov_pos_w_RNase_hg19.bed

value: anti-DGCR8 antibody (Bethyl laboratories)


title: pif1-RNA

city: Albany

first: james

middle: m

last: tepperman

address: 800 Buchanan St.

department: Plant and Microbial Biology

institute: Plant Gene Expression Center/UC Berkeley

description: 3'-end polyA RNA

data_processing:
single-end 50-nt reads
Reads passed quality filtering were aligned to the TAIR10 transcriptome assembly using Bowtie-0.12.7 with the following settings: -v 0 --norc -a --best.
Only reads mapped uniquely to the 3’-end 500-bp region of the single locus were counted for gene expression.
differential expression between two genotypes was determined using EdgeR (Bioconductor)
Genome_build: TAIR10
Supplementary_files_format_and_content: Tab-delimited text files containing raw reads counts of every locus in each biological triplicate sample - reads from technical duplicates are combined.

source_name: whole seedlings

growth_protocol: Stratified seeds were irradiated with WL at 21ºC for 3 h to induce germination, followed by a FR pulse for 15 min to suppress pseudo dark effects, and grown in darkness at 21 ºC for 2 d before harvest.

extract_protocol:
Rneasy Plus mini kit was used to prepare total RNA from ~100 mg ttissue (fw).  20 ug total RNA was used for construction of each library. There are technical duplicate runs for each biological triplicate sample.
The sequencing library construction was adapted from Illumina’s directional mRNA-seq sample preparation protocol. The mRNA was fractionated from 20 µg of total RNA using Dynabeads Oligo (dT)25 (Invitrogen), and fragmented using Fragmentation Reagents (Ambion) at 70 ºC for 2.5 min in 20 µl of reaction. The polyA-tailed 3’-end fragments were captured by another run of mRNA purification as described above, and then treated by Antarctic Phosphatase and T4 Polynucleotide Kinase (NEB) at 37 ºC for 1 h and 2 h, respectively. The sample was purified using RNeasy MinElute Cleanup Kit (Qiagen) according to Illumina’s protocol. The eluted mRNA fragments were ligated with 2.5 µM of Illumina’s SRA 5’ adaptor by T4 RNA Ligase 1 (NEB) at 20 ºC for 4 h. The 3’ cDNA adapter derived from Illumina’s v1.5 sRNA 3’ adapter was conjugated with the anchored oligo (dT)20 primer, and introduced through reverse transcription using the SuperScript III First-Strand Synthesis System (Invitrogen). The first-strand cDNA was purified using the Agencourt AMPure XP system (Beckman Coulter Genomics), and then amplified by PCR reaction using Phusion High-Fidelity DNA Polymerase (NEB) with Illumina’s sRNA PCR primer set. The size of purified library DNA was validated by Bioanalyzer 2000 (Agilent).
Since only short fragments from the 3' end of all mRNA's are sequenced in this protocol there is no need to adjust (RPKM)  for length of message.  Construction strategy is  directional - mRNA's are sequenced only 5' to 3' to avoid ambiguities in overlapping ORFS.

treatment_protocol: 2d continuous darknes (48 hours post-stratification) at 21ºC

tag: developmental stage

value: 2-day old

tag: growth conditions

value: true dark

value: seedling

value: Columbia


title: pif134-RNA

description: 3'-end polyA RNA


title: pif3-RNA

description: 3'-end polyA RNA


title: pif5-RNA

description: 3'-end polyA RNA


title: pifpif3pif4pif5(pifq)-RNA

description: 3'-end polyA RNA


title: pif145-RNA

description: 3'-end polyA RNA


title: pif4-RNA

description: 3'-end polyA RNA


title: pif345-RNA

description: 3'-end polyA RNA


title: WT-RNA

description: 3'-end polyA RNA

value: WT