title: pif135-RNA

description: 3'-end polyA RNA


title: Polistes metricus ad libitum nutrition manipulation 5th instar larvae - 01 sample

status: Public on Oct 02 2014

type: SRA

city: Ames

first: Ali

last: Berens

state: IA

address: 251 Bessey Hall

department: Ecology, Evolution, and Organismal Biology

country: USA

institute: Iowa State University

library_source: transcriptomic

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 We filtered reads for quality (threshold greater than or equal to 20) using the Trim Perl script (Nik Joshi, unpublished; code available from http://wiki.bioinformatics.ucdavis.edu/index.php/Trim.pl).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final Trinity-produced Transcriptome Shotgun Assembly (TSA) was annotated with the mRNAmarkup protocol (Volker Brendel, unpublished; code available from http://brendelgroup.org/bioinformatics2go/mRNAmarkup.php).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

molecule: total RNA

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.

tag: tissue

value: head

tag: age

value: 5th Instar Larvae

tag: Sex

value: female

tag: biome

value: laboratory reared

value: USA:North Carolina:Raleigh


title: Polistes metricus restricted nutrition manipulation 5th instar larvae - FF1 sample


title: Polistes metricus foundress reared 5th instar larvae - 4H7L4 sample

value: late spring 2010

value: field at Iowa 4H Center


title: Polistes metricus restricted nutrition manipulation 5th instar larvae - M1 sample


title: Polistes metricus foundress reared 5th instar larvae - 4H18L1 sample


title: Polistes metricus foundress reared 5th instar larvae - 4H8L2 sample


title: Polistes metricus foundress reared 5th instar larvae - 4H11L2 sample


title: Polistes metricus restricted nutrition manipulation 5th instar larvae - L1 sample


title: DGCR8-CLIP-seq negative control (Low 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_IgG_hg19.bed, DGCR8_CLIP_long_cov_pos_IgG_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)

organism: Homo sapiens

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

value: rabbit IgG (Bethyl laboratories)


title: Polistes metricus worker reared 5th instar larvae - 4HW11L2 sample

value: late summer 2010


title: Polistes metricus worker reared 5th instar larvae - 4HW3L1 sample


title: Polistes metricus worker reared 5th instar larvae - 4HW12L4 sample


title: Polistes metricus restricted nutrition manipulation 5th instar larvae - W1 sample


title: DNA_Ae356_small_workers_bodies

city: Shenzhen

first: Hong

middle: zhi

last: Cao

state: Guangdong

address: F5, Main Building, Beishan Industrial Zone, Beishan Road, Yantian District

department: Bioinformatics

country: China

institute: BGI-SHENZHEN

description: small worker_DNA replicate B

library_source: genomic

data_processing:
library strategy: DNA-Seq for DNA samples and Strand-specific RNA-Seq for RNA samples
RNA and DNA reads were aligned to the genome of Acromyrmex Echinatior (Aech_2.0_scaffolds.fa.gz from http://hymenopteragenome.org/acromyrmex/) using BWA (Version: 0.5.9-r16), allowing 5 mismatches and 0 gap for the 90-bp reads. Only uniquely mapped reads with mapping quality >= 20 and with no suboptimal hits found by BWA were kept for further analysis. Reads resulted from PCR duplications (i.e. read pairs that map to identical genomic locations) were removed except for the one with the highest sequencing quality. Then, we cut the first and last 6-bp of the aligned reads, as it is known that (a) the error rate of Illumina sequencing is relatively higher on both end of a read, especially on the 3’end, (b) some 5’ end mismatches may be introduced by the random hexamer during the first- and second-strand syntheses during RNA library construction and (c) mapping errors around insertions/deletions relative to a reference genome can lead to mismatches occurring towards the beginning and ends of a read. In addition, to improve the accuracy of reads alignment, we used BLAT to re-align all the uniquely mapped reads to the reference genome. As the fundamental algorithmic between BLAT and short read aligners (e.g. BWA) are different, their read mapping results are often different and complementary, especially in handling reads derived from spliced junctions. The reads that could be aligned to positions that are different from BWA alignments with mismatch rates no more than that reported by BWA were removed.
Genome_build: Aech_2.0_scaffolds.fa.gz from http://hymenopteragenome.org/acromyrmex/
Supplementary_files_format_and_content: tab-delimited text files include DNA read coverage and RNA read coverage for each RNA editing site for each sample.

molecule: genomic DNA

organism: Acromyrmex echinatior

source_name: small worker whole body

growth_protocol: Colonies of Acromyrmex echinatior (numbers Ae322, Ae356 and Ae363) were collected in Gamboa, Panama in 2006-2008 and maintained in the laboratory under standard conditions of ca. 25°C and ca. 70% relative humidity where they were supplied with a diet of bramble leaves, rice and pieces of apple.

extract_protocol:
Heads from 50 gynes, 50 large workers or 200 small workers originating from the same colony were pooled and used for RNA extraction as described previously (Yek et al. 2013). The remaining body parts were likewise pooled per caste and colony and subsequently used for DNA extraction. Genomic DNA was extracted using a Blood & Cell Culture Midi kit (Qiagen) following the instructions enclosed in the kit, with the modification that sample lysis was performed overnight.
For DNA samples, 5 μg genomic DNA per sample was fragmented by sonication with a Covaris S2 system (Covaris, MA) to a mean size of approximately 500 bp, followed by end-repair, 3’-end addition of dA, and adapter ligation. The ligated fragments were size selected at 500 bp on an agarose gel and amplified by 10 cycles of PCR to yield the DNA libraries. All libraries were subjected to 90 bp pair-end sequencing on an Illumina HiSeq 2000 platform.
For RNA samples, strand-specific RNA-Seq libraries were constructed according to Parkhomchuk D, et al (2009). Briefly, 2 μg total RNA per sample was first treated with DNase I (New England BioLabs) to remove the possible contamination of genomic DNA. Then poly (A+) mRNA was purified from the total RNA using the Dynabeads mRNA Purification Kit (Invitrogen), followed by fragmentation using the RNA fragmentation kit (Ambion). Next, the first cDNA strand was synthesized using random hexamer primers and reverse transcriptase (Invitrogen), and second-strand cDNA was synthesized using DNA polymerase I (New England BioLabs) where dUTP was used instead of dTTP. After that, the synthetic double-strand cDNA was end repaired, followed by 3’-end addition of dA, adapter ligation and size selection according to Illumina’s protocols. Finally, the selected products were treated by UNG (Applied Biosystems) and amplified by 15 cycles of PCR to yield the strand-specific RNA-Seq libraries. All the libraries were subjected to 90 bp pair-end sequencing on the Illumina HiSeq 2000 platform.

treatment_protocol: RNA and DNA samples were collected from three castes: (i) gynes (AeG), (ii) large workers (AeL) and (iii) small workers (AeS). Animals were flash frozen in liquid nitrogen, and the heads separated from the bodies with a pair of forceps.

tag: colony id

tag: caste

value: small worker

value: whole body


title: DNA_Ae363_small_workers_bodies

description: small worker_DNA replicate C


title: RNA_Ae356_small_worker_heads

description: small worker_RNA replicate B

source_name: small worker heads

value: heads


title: RNA_Ae363_small_worker_heads

description: small worker_RNA replicate C


title: DNA_Ae363_gynes_bodies

description: gyne_DNA replicate C

source_name: gyne whole body

value: gyne


title: RNA_Ae322_small_worker_heads

description: small worker_RNA replicate A


title: Polistes metricus worker reared 5th instar larvae - 4HW10L2 sample


title: RNA_Ae356_large_worker_heads

description: large worker_RNA replicate B

source_name: large worker heads

value: large worker


title: DNA_Ae356_large_workers_bodies

description: large worker_DNA replicate B

source_name: large worker whole body


title: DNA_Ae322_large_workers_bodies

description: large worker_DNA replicate A


title: RNA_Ae363_gyne_heads

description: gyne_RNA replicate C

source_name: gyne heads


title: RNA_Ae322_large_worker_heads

description: large worker_RNA replicate A


title: RAD21_96h_rep3_input

city: Dresden

first: Maciej

last: Paszkowski-Rogacz

address: Tatzberg 47/49

department: UCC / Medical Faculty and University Hospital Carl Gustav Carus

country: Germany

institute: Technische Universität Dresden

data_processing:
Reads were aligned to the mm9 genome version using Bowtie 2 suite with "--very-sensitive" parameter
From each generated alignment file a set of 7 million unique, randomly picked positions was selected for further processing. Additionally, pooled data sets were generated by merging alignment files from replicates of the same samples.
Peak calling was performed with MACS 2 (using default parameters) on each of the individual replicates and pooled samples.
Genome_build: mm9
Supplementary_files_format_and_content: bigWig files contain MACS-generated pileup and control lambda tracks
Supplementary_files_format_and_content: bed files contain MACS-generated peak locations

organism: Mus musculus

source_name: Splenic

extract_protocol:
B cells were cross-linked with 1 % formaldehyde at room temperature for 10 minutes and cross-linking was quenched by 125 mM glycine. Cells were lysed in SDS lysis buffer (1 % SDS, 10 mM EDTA, 50 mM Tris-HCl, pH 8.1) and the chromatin was sonicated to an average fragment length of 500 base pairs (bp) using a Branson Sonifier 450. Sonicated chromatin was pre-cleared and incubated with 2 µg of anti-RAD21 antibody (ab992, Abcam), 4 µl anti-CTCF (07729, Millipore), 2 µg anti-PAX5 (sc-1974, Santa Cruz) and 1 µg anti-IgG (sc-2027, Santa Cruz) overnight. The complexes were washed with increasing salt stringency and cross-links were reversed in the presence of 0.2 M NaCl at 65 °C for 6 hours. Proteinase K treatment was carried out in the presence of 10 µM EDTA, 40 µM Tris-HCl, pH 6.5 and 20 µg Proteinase K. DNA fragments were recovered with phenol-chloroform extraction following one chloroform extraction. DNA was precipitated with 2 volumes of ethanol, 1/3 volumes of 7.5 M ammonium acetate and 20 µg glycogen with an overnight incubation at -20 °C. Precipitated DNA was washed with 70 % Ethanol to remove excess salt and DNA was resuspended in 30 µl TE buffer supplemented with 0.3 µg RNAseA.
Immunoprecipitated fragments were end-repaired with NEBnext End Repair Module (NEB) and purified by using Agencourt Ampure XP - beads (Beckman Coulter) and A-tailed using the NEBnext dA-Tailing Module according to manufacturers’ instructions. After purification, adaptors were ligated (Adaptor-Oligo 1: 5'ACACTCTTTCCCTACACGACGCTCTTCCGATCT-3', Adaptor-Oligo 2: 5'-P-GATCGGAAGAGCACACGTCTGAACTCCAGTCAC-3') by using 1x NEBnext Quick Ligation Buffer (NEB), 10x excess of DNA Adaptors, 5 µl Quick T4 DNA Ligase (NEB) at 50 µl total volume. Large-scale amplification of library constructs was performed by using the PCR Enrich Adaptor Ligated cDNA Library module (NEB).

tag: strain

value: Spleen

value: None (Input DNA)

tag: time


title: DNA_Ae363_large_workers_bodies

description: large worker_DNA replicate C


title: DNA_Ae322_small_workers_bodies

description: small worker_DNA replicate A


title: RAD21_48h_rep3_input


title: DNA_Ae322_gynes_bodies

description: gyne_DNA replicate A


title: RNA_Ae322_gyne_heads

description: gyne_RNA replicate A


title: DNA_Ae356_gynes_bodies

description: gyne_DNA replicate B


title: RNA_Ae363_large_worker_heads

description: large worker_RNA replicate C


title: RNA_Ae356_gyne_heads

description: gyne_RNA replicate B


title: RAD21_48h_rep1_input


title: RAD21_96h_rep2_input


city: San Francisco

first: UCSF-UBC

last: CENTER

state: CA

address: UCSF-UBC

institute: UCSF-UBC

description:
design description: MeDIP-Seq analysis of breast myoepithelial cell from RM071 (HS2369) using Illumina Genome Analyzer IIx
library name: HS2369
EXPERIMENT_TYPE: DNA Methylation
EXTRACTION_PROTOCOL: Refer to document 'MeDIP-seq library construction protocol v2' by Costello Lab at the Roadmap Epigenomics Project site, Experimental Protocols page (URL: http://www.roadmapepigenomics.org/protocols/type/experimental/)
EXTRACTION_PROTOCOL_TYPE_OF_SONICATOR: Diagenode Bioruptor
EXTRACTION_PROTOCOL_SONICATION_CYCLES: 25 min
MeDIP_PROTOCOL: Refer to document 'MeDIP-seq library construction protocol v2' by Costello Lab at the Roadmap Epigenomics Project site, Experimental Protocols page (URL: http://www.roadmapepigenomics.org/protocols/type/experimental/)
MeDIP_PROTOCOL_DNA_AMOUNT: 2 ug
MeDIP_PROTOCOL_BEAD_TYPE: Protein A/G Beads
MeDIP_PROTOCOL_BEAD_AMOUNT: 100 ul
MeDIP_PROTOCOL_ANTIBODY_AMOUNT: 1 ug
MeDIP_ANTIBODY: Methylcytidine
MeDIP_ANTIBODY_PROVIDER: Eurogentec
MeDIP_ANTIBODY_CATALOG: BI-MECY-0100
MeDIP_ANTIBODY_LOT: 80604
****************
For data usage terms and conditions, please refer to:
http://www.drugabuse.gov/funding/funding-opportunities/nih-common-fund/epigenomics-data-access-policies
****************

data_processing:
ANALYSIS FILE NAME: GSM1517153_UCSF-UBC.Breast_Myoepithelial_Cells.MeDIP-Seq.RM071.bed
ANALYSIS ALIAS: GSM1517153_hg19.level.1
ANALYSIS TITLE: Mapping of Breast Myoepithelial Cells MeDIP-Seq Data
ANALYSIS DESCRIPTION: Illumina reads produced by MeDIP-Seq on Breast Myoepithelial Cells, Donor RM071 were mapped to the human genome using Pash.
ANALYSIS TYPE: REFERENCE_ALIGNMENT
DATA_ANALYSIS_LEVEL: 1
EXPERIMENT_TYPE: MeDIP-Seq
GENOME_ASSEMBLY: NCBI Build GRCh37/UCSC Build hg19
SOFTWARE: Pash
SOFTWARE_VERSION: 3.0
MAXIMUM_ALIGNMENT_LENGTH: Read length
MISMATCHES_ALLOWED: 10% of read length
ALIGNMENTS_ALLOWED: 10
TREATMENT_OF_MULTIPLE_ALIGNMENTS: If a read maps to more than 10 positions it is removed from consideration.
TREATMENT_OF_IDENTICAL_ALIGNMENTS_OF_MULTIPLE_READS: None
ALIGNMENT_POSTPROCESSING: None
RELEASE_NUMBER: Human Epigenome Atlas 2
NUMBER_OF_MEDIP-SEQ_EXPERIMENTS_SCORED: 24
FINDPEAKS_SCORE: 0.3948
FINDPEAKS_PERCENTILE: 75
HOTSPOT_SCORE: 0.4128
HOTSPOT_PERCENTILE: 79
IROC_SCORE: 0.963
IROC_PERCENTILE: 66
POISSON_SCORE: 0.7116
POISSON_PERCENTILE: 83

ANALYSIS FILE NAME: GSM1517153_UCSF-UBC.Breast_Myoepithelial_Cells.MeDIP-Seq.RM071.wig
ANALYSIS ALIAS: GSM1517153_.hg19.level.2
ANALYSIS TITLE: Raw Signal Density Graphs of Breast Myoepithelial Cells MeDIP-Seq Data
ANALYSIS DESCRIPTION: Illumina MeDIP-Seq read mappings from Breast Myoepithelial Cells, Donor RM071 were processed into density graphs of raw signal representing the aligned read density.
ANALYSIS TYPE: ABUNDANCE_MEASUREMENT
DATA_ANALYSIS_LEVEL: 2
EXPERIMENT_TYPE: MeDIP-Seq
GENOME_ASSEMBLY: NCBI Build GRCh37/UCSC Build hg19
SOFTWARE: In house programs and scripts
SOFTWARE_VERSION: NA
READ_EXTENSION: 200bp
GENOMIC_WINDOW: 20bp
TREATMENT_OF_REGIONS_PRONE_TO_MULTIPLE_ALIGNMENTS: None
RELEASE_NUMBER: Human Epigenome Atlas 2
BROWSER_TRACK_NAME: BMC MeDIP 71
BROWSER_TRACK_DESCRIPTION: UCSF-UBC-UCD Breast Myoepithelial Cells MeDIP-Seq Donor RM071 EA Release 2
NUMBER_OF_MEDIP-SEQ_EXPERIMENTS_SCORED: 24
FINDPEAKS_SCORE: 0.3948
FINDPEAKS_PERCENTILE: 75
HOTSPOT_SCORE: 0.4128
HOTSPOT_PERCENTILE: 79
IROC_SCORE: 0.963
IROC_PERCENTILE: 66
POISSON_SCORE: 0.7116
POISSON_PERCENTILE: 83

extract_protocol: library construction protocol: Refer to document 'MeDIP-seq library construction protocol v2' from the Costello Lab at the Roadmap Epigenomics Project site, Experimental Protocols page (URL: http://www.roadmapepigenomics.org/protocols/type/experimental/)

tag: submitted sample id

tag: body site

value: Breast

tag: histological type

value: Myoepithelial cell

tag: is tumor

value: No

value: primary cell

value: breast myoepithelial cell


title: RAD21_96h_rep1_input


description:
design description: MeDIP-Seq analysis of breast luminal epithelial cell from RM071 (HS2367) using Illumina Genome Analyzer IIx
library name: HS2367
EXPERIMENT_TYPE: DNA Methylation
EXTRACTION_PROTOCOL: Refer to document 'MeDIP-seq library construction protocol v2' by Costello Lab at the Roadmap Epigenomics Project site, Experimental Protocols page (URL: http://www.roadmapepigenomics.org/protocols/type/experimental/)
EXTRACTION_PROTOCOL_TYPE_OF_SONICATOR: Diagenode Bioruptor
EXTRACTION_PROTOCOL_SONICATION_CYCLES: 25 min
MeDIP_PROTOCOL: Refer to document 'MeDIP-seq library construction protocol v2' by Costello Lab at the Roadmap Epigenomics Project site, Experimental Protocols page (URL: http://www.roadmapepigenomics.org/protocols/type/experimental/)
MeDIP_PROTOCOL_DNA_AMOUNT: 1.5 ug
MeDIP_PROTOCOL_BEAD_TYPE: Protein A/G Beads
MeDIP_PROTOCOL_BEAD_AMOUNT: 100 ul
MeDIP_PROTOCOL_ANTIBODY_AMOUNT: 1 ug
MeDIP_ANTIBODY: Methylcytidine
MeDIP_ANTIBODY_PROVIDER: Eurogentec
MeDIP_ANTIBODY_CATALOG: BI-MECY-0100
MeDIP_ANTIBODY_LOT: 80604
****************
For data usage terms and conditions, please refer to:
http://www.drugabuse.gov/funding/funding-opportunities/nih-common-fund/epigenomics-data-access-policies
****************

data_processing:
ANALYSIS FILE NAME: GSM1517154_UCSF-UBC.Breast_Luminal_Epithelial_Cells.MeDIP-Seq.RM071.bed
ANALYSIS ALIAS: GSM1517154.hg19.level.1
ANALYSIS TITLE: Mapping of Breast Luminal Epithelial Cells MeDIP-Seq Data
ANALYSIS DESCRIPTION: Illumina reads produced by MeDIP-Seq on Breast Luminal Epithelial Cells, Donor RM071 were mapped to the human genome using Pash.
ANALYSIS TYPE: REFERENCE_ALIGNMENT
DATA_ANALYSIS_LEVEL: 1
EXPERIMENT_TYPE: MeDIP-Seq
GENOME_ASSEMBLY: NCBI Build GRCh37/UCSC Build hg19
SOFTWARE: Pash
SOFTWARE_VERSION: 3.0
MAXIMUM_ALIGNMENT_LENGTH: Read length
MISMATCHES_ALLOWED: 10% of read length
ALIGNMENTS_ALLOWED: 10
TREATMENT_OF_MULTIPLE_ALIGNMENTS: If a read maps to more than 10 positions it is removed from consideration.
TREATMENT_OF_IDENTICAL_ALIGNMENTS_OF_MULTIPLE_READS: None
ALIGNMENT_POSTPROCESSING: None
RELEASE_NUMBER: Human Epigenome Atlas 2
NUMBER_OF_MEDIP-SEQ_EXPERIMENTS_SCORED: 24
FINDPEAKS_SCORE: 0.2993
FINDPEAKS_PERCENTILE: 50
HOTSPOT_SCORE: 0.3537
HOTSPOT_PERCENTILE: 54
IROC_SCORE: 0.941
IROC_PERCENTILE: 45
POISSON_SCORE: 0.7111
POISSON_PERCENTILE: 79

ANALYSIS FILE NAME: GSM1517154_UCSF-UBC.Breast_Luminal_Epithelial_Cells.MeDIP-Seq.RM071.wig
ANALYSIS ALIAS: GSM1517154.hg19.level.2
ANALYSIS TITLE: Raw Signal Density Graphs of Breast Luminal Epithelial Cells MeDIP-Seq Data
ANALYSIS DESCRIPTION: Illumina MeDIP-Seq read mappings from Breast Luminal Epithelial Cells, Donor RM071 were processed into density graphs of raw signal representing the aligned read density.
ANALYSIS TYPE: ABUNDANCE_MEASUREMENT
DATA_ANALYSIS_LEVEL: 2
EXPERIMENT_TYPE: MeDIP-Seq
GENOME_ASSEMBLY: NCBI Build GRCh37/UCSC Build hg19
SOFTWARE: In house programs and scripts
SOFTWARE_VERSION: NA
READ_EXTENSION: 200bp
GENOMIC_WINDOW: 20bp
TREATMENT_OF_REGIONS_PRONE_TO_MULTIPLE_ALIGNMENTS: None
RELEASE_NUMBER: Human Epigenome Atlas 2
BROWSER_TRACK_NAME: BLEC MeDIP 71
BROWSER_TRACK_DESCRIPTION: UCSF-UBC-UCD Breast Luminal Epithelial Cells MeDIP-Seq Donor RM071 EA Release 2
NUMBER_OF_MEDIP-SEQ_EXPERIMENTS_SCORED: 24
FINDPEAKS_SCORE: 0.2993
FINDPEAKS_PERCENTILE: 50
HOTSPOT_SCORE: 0.3537
HOTSPOT_PERCENTILE: 54
IROC_SCORE: 0.941
IROC_PERCENTILE: 45
POISSON_SCORE: 0.7111
POISSON_PERCENTILE: 79

value: female

value: Luminal epithelial cell

value: breast luminal epithelial cell


title: RAD21_48h_rep2_input


title: RAD21_0h_rep3_input


title: RAD21_0h_rep2_input


title: RAD21_96h_rep3_ChIP

value: RAD21 (Abcam, ab992)


title: RAD21_0h_rep1_input


title: RAD21_96h_rep2_ChIP


title: RAD21_48h_rep2_ChIP


title: RAD21_96h_rep1_ChIP


title: RAD21_0h_rep3_ChIP


title: RAD21_48h_rep3_ChIP


title: RAD21_48h_rep1_ChIP


title: RAD21_0h_rep2_ChIP


title: RAD21_0h_rep1_ChIP


title: polysome gld2(RNAi)_rep3

first: Marco

last: Nousch

address: Pfotenhauerstrasse 108

institute: MPI-CBG

description:
fractionated RNA
DEgenes_Filtered_theta0.65_P_C_P_G2.txt

data_processing:
Illumina Casava1.8.2 software used for basecalling.
fastq files were mapped to the Caenorhabditis elegans genome downloaded from Ensembl (WS220) using the TopHat2 algorithm (version v2.0.10) allowing only unique mapping
Using the featureCount algorithm from the SubRead software package (version 1.4.0) the reads were counted from the bam files (tophat2 output) on exon-level based on the gene annotation from Ensembl, resulting in a read count for each gene
Using the DESeq package (R 3.0.2, DESeq version 1_14_0) the count data was normalized by the size factor to estimate the effective library size
Genome_build: Worm base WS220
Supplementary_files_format_and_content: tab-delimited text files include raw and norm read count for each gene

organism: Caenorhabditis elegans

source_name: polysome gld2(RNAi)_rep3

extract_protocol:
RNA was extracted from whole animal lysate using Trizol and poly(A) enriched using oligo dT beads
RNA libraries were prepared for sequencing using standard Illumina protocols

tag: Stage

value: young adult

value: whole worm


title: non-polysome gld4(RNAi)_rep3

description:
fractionated RNA
DEgenes_Filtered_theta0.7_NP_C_NP_G4.txt

source_name: non-polysome gld4(RNAi)_rep3


title: polysome gld4(RNAi)_rep3

description:
fractionated RNA
DEgenes_Filtered_theta0.7_P_C_P_G4.txt

source_name: polysome gld4(RNAi)_rep3


title: polysome gld4(RNAi)_rep2

source_name: polysome gld4(RNAi)_rep2


title: hMeDIP-seq_adipocytes_eWAT

city: Lille

first: Jérôme

last: Eeckhoute

address: Bâtiment J&K, Faculté de Médecine de Lille-Pôle Recherche, Boulevard du Professeur Leclerc

country: France

institute: CNRS

data_processing:
hMeDIP-seq reads were aligned to the mm9 genome assembly using Bowtie.
After duplicate removing using Picard tools (http://picard.sourceforge.net), wig file was generated at 25 bp resolution and normalized to the total number of uniquely mapped sequenced reads (extended to 200 bp).
Genome_build: mm9

source_name: Adipocytes from eWAT

growth_protocol: eWAT depots were collected from C57BL/6J mice and stored on ice-cold 1x phosphate buffer saline (PBS). eWAT was rapidly cut into l mm 3 pieces in 15 mL Hank’s balanced salt solution (HBSS) medium containing 1% bovine serum albumin (BSA), l mg/mL collagenase type I (Sigma, Saint-Louis, MO, USA), 100 U/mL penicillin, 0.l mg/mL streptomycin, 0.25µg/mL fungizone (Gibco, Life Technologies, Carlsbad, CA, USA) then incubated with gentle shaking at 37°C for l h. Digested samples were then filtered (250 µm) and adipocytes were separated from the stromal vascular fraction (SVF) by centrifugation at 300 g for 5 minutes at room temperature.

extract_protocol:
Genomic DNA was prepared using a DNA extraction kit (Dneasy Blood and Tissue kit, Qiagen) with a supplementary step of RNAse digestion. Genomic DNA was then sonicated to produce fragments ranging from 200 bp to 500 bp. Methylated DNA were then immunoprecipitated using antibody directed against 5-hydroxymethylcytosine (Diagenode, CS-HMC-100).
Libraries were prepared according to Illumina's instructions

tag: developmental stage

value: adult

tag: cell-type

value: Adipocytes

value: eWAT


title: polysome control_rep2

source_name: polysome control_rep2


title: polysome gld2(RNAi)_rep1

source_name: polysome gld2(RNAi)_rep1


title: PNST Twist1

city: Cincinnati

first: Katherine

last: Yutzey

state: OH

address: 240 Albert Sabin Way

department: Molecular Cardiovascular Biology

institute: Cincinnati Children's Medical Center

data_processing:
50bp sequence reads were mapped to the mouse genome (NCBI Build 37, mm9) with Illumina gapped aligner Eland v2.
Peaks were called using Model-based Analysis of ChIP-seq (MACS, v1.4.2) with the following parameters, band width=300, and p-value cut off of 1.00e-05.
Bowtie analysis was utilized to identify the nearest transcriptional start site upstream and downstream of the peak.
Genome_build: mus musculus (NCBI Build 37, mm9)
Supplementary_files_format_and_content: The bigWig (.bw) and peak txt files were generated using MACS analyisis.

source_name: Peripheral nerve sheath tumor cell line

growth_protocol: PNST cells were grown in DMEM with 10%FBS and harvested for ChIP at 80% confluency.

extract_protocol:
For ChIP experiments, ECCs, limb buds, and PNST cells were cross-linked and isolated according to manufacturer’s protocols (Magnify ChIP kit, Invitrogen).  Crosslinked DNA complexes were sheared by sonication (Virsonic 60, Virtis, output of 5) 6 times for 5 seconds with a 2-minute refractory period on ice.  Samples were sheared to a size of 150-250 basepairs (bp). ChIP was then performed according to the manufacturer’s protocol (Magnify ChIP Kit, Invitrogen).
A single read genomic DNA library was generated using an IntegenX robot (Apollo 324) and PrepX DNA library kit (IntegenX 400040) was utilized according to manufacturer's protocols on mouse genomic DNA.

tag: chip antibody

value: Twist1 (Sigma, T6451, 5 μg)

tag: cell type

value: peripheral nerve sheath tumor cells

value: FVB/N


title: polysome control_rep1

source_name: polysome control_rep1

tag: tissue


title: polysome control_rep3

source_name: polysome control_rep3


title: E12.5 ECC Twist1

source_name: E12.5 atrioventricular canal endocardial cushion

value: E12.5 endocardial cushion cells


title: non-polysome gld4(RNAi)_rep1

source_name: non-polysome gld4(RNAi)_rep1


title: non-polysome gld4(RNAi)_rep2

source_name: non-polysome gld4(RNAi)_rep2


title: E10.5 Limb bud Twist1

source_name: E10.5 forelimb buds

value: E10.5 limb bud


title: polysome gld2(RNAi)_rep2

source_name: polysome gld2(RNAi)_rep2


title: non-polysome control_rep2

description:
fractionated RNA
DEgenes_Filtered_theta0.65_NP_C_NP_G2.txt

source_name: non-polysome control_rep2


title: polysome gld4(RNAi)_rep1

source_name: polysome gld4(RNAi)_rep1


title: non-polysome gld2(RNAi)_rep2

source_name: non-polysome gld2(RNAi)_rep2


title: non-polysome gld2(RNAi)_rep1

source_name: non-polysome gld2(RNAi)_rep1


title: Input_gld4(RNAi)_rep3


title: non-polysome gld2(RNAi)_rep3

source_name: non-polysome gld2(RNAi)_rep3


title: Input_gld2(RNAi)_rep2


title: Input_gld2(RNAi)_rep1


title: Input_gld4(RNAi)_rep2


title: Input_control_rep3


title: Input_gld2(RNAi)_rep3


title: Input_gld4(RNAi)_rep1


title: Input_control_rep2


title: Liver in C57BL/6J mice under standard chow

city: Okayama

first: Jun

last: Wada

address: 2-5-1 Shikata-cho, Kita-ku

department: Department of Nephrology, Rheumatology, Endocrinology and Metabolism

country: Japan

institute: Okayama University

description: Sample 6

data_processing: The obtained data were mapped to mouse genome sequence and annotated (bowtie-0.12.7).

source_name: Liver

growth_protocol: Male C57BL/6J (Charles River Laboratories Japan, Yokohama, Japan) mice were housed in cages and maintained on a 12-hour light-dark cycle. STD (NMF; Oriental Yeast) and HFHS chow (D12331; Research Diet, New Brunswick, NJ) were used, the mice were sacrificed at 20 weeks of age.

extract_protocol:
Total RNAs were isolated from serum and various tissues by using QIAamp Circulating Nucleic Acid Kit and miRNeasy Mini kit (Qiagen, Hilden, Germany), respectively.
Total RNAs were subjected to Illumina TruSeq Small RNA Sample Preparation protocol (Illumina, San Diego, CA), including 3’- and 5’- adapter ligation, reverse transcription, PCR amplification, and pooled gel purification to generate a library product.

value: Liver

tag: age

value: 20 weeks

tag: gender

value: male


title: Liver in C57BL/6J mice under high fat high sucrose chow

description: Sample 2


title: Serum in C57BL/6J mice under standard chow

description: Sample 5

source_name: Serum

value: Serum


title: AGO2-associated small RNA seq IgG control replicate 2

description: IP enriched RNA

source_name: H1 human embryonic stem cells (knock-in cells containing a Flag tag at the N terminus of AGO2)

tag: antibody

value: rabbit IgG (Bethyl laboratories)


title: Serum in C57BL/6J mice under high fat high sucrose chow

description: Sample 1


title: AGO2-associated small RNA seq IgG control replicate 1

description: IP enriched RNA


title: non-polysome control_rep3

source_name: non-polysome control_rep3


title: AGO2-associated small RNA seq replicate 1

description: IP enriched RNA

value: M1 anti-Flag (Waco)


title: AGO2-associated small RNA seq replicate 2

description: IP enriched RNA


title: non-polysome control_rep1

source_name: non-polysome control_rep1


title: Drosha-CLIP-seq replicate 2

description:
CLIP enriched RNA
Drosha_CLIP_cov_pos_hg19.bed, Drosha_CLIP_cov_neg_hg19.bed

source_name: H1 human embryonic stem cells (knock-in cells containing a Flag tag at the C terminus of Drosha)


title: Drosha-CLIP-seq replicate 4


title: DGCR8-RIP-seq

description:
RIP enriched RNA
DGCR8_RIP_cov_pos_hg19.bed, DGCR8_RIP_cov_neg_hg19.bed

value: anti-DGCR8 antibody (Bethyl laboratories)


title: Drosha-CLIP-seq replicate 3


title: DGCR8-CLIP-seq replicate 10 (Low RNase 1 treated)

description:
CLIP enriched RNA
DGCR8_cov_pos_hg19.bed, DGCR8_cov_neg_hg19.bed

source_name: H1 human embryonic stem cells


title: DGCR8-CLIP-seq replicate 9 (Low RNase 1 treated)


title: Input_control_rep1

value: young adult


title: DGCR8-CLIP-seq replicate 2 (Low RNase 1 treated)


title: Epididymal fat in C57BL/6J mice under high fat high sucrose chow

description: Sample 3

source_name: Epididymal fat

value: Epididymal fat


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


title: DGCR8-CLIP-seq replicate 3 (High RNase 1 treated)


title: DGCR8-CLIP-seq replicate7 (High RNase 1 treated)


title: DGCR8-CLIP-seq replicate 5 (High RNase 1 treated)


title: DGCR8-CLIP-seq replicate 4 (Low RNase 1 treated)


title: DGCR8-CLIP-seq replicate 6 (Low RNase 1 treated)


title: WT2_BRD4_ChIPSeq

city: Bethesda

first: Patricia

last: Fernandez Ferri

state: MD

address: 41 Library Drive, B513

institute: NCI/NIH

data_processing:
base-calling: RTA 1.8.70.0; Alignment: Illumina Casava 1.8.2
read alignment: Novoalign/Novocraft/3.00.02
sorting and deduplication: SAMtools/Picard MarkDuplicates
peak-calling: SICER v1.1
peak intersections: BEDtools/intersectBED
peak annotation/tag density profiles: HOMER v4.0
Genome_build: hg19
Supplementary_files_format_and_content: BED format; called peaks

source_name: WT fibroblasts_BRD4_ChIPSeq

growth_protocol: Cells were grown in MEM containing 15% fetal bovine serum (FBS), 2mM L-glutamine, 100 Uml-1 penicillin and 100µgml-1 streptomycin, at 37ºC in 5% CO2.

extract_protocol:
Lysates were clarified from sonicated cells and histone-DNA complexes were isolated with antibody.
Library construction was performed following Illumina TruSeq 2.0 protocol

tag: cell type source

value: age-matched control wild-type individual 2 (TRS-WT)

value: WT dermal fibroblasts transformed by retroviral introduction of TERT, HRAS and SV40 T antigens (TRS-WT1)

tag: chip antibody vendor

value: Bethyl labs

tag: chip antibody cat. #


title: HG1_BRD4_ChIPSeq

source_name: HG fibroblasts_BRD4_ChIPSeq

value: HGPS patient 1 (TRS-HGPS)

value: HGPS dermal fibroblasts transformed by retroviral introduction of TERT, HRAS and SV40 T antigens (TRS-HGPS1)


title: HG2_BRD4_ChIPSeq

value: HGPS patient 2 (TRS-HGPS)


title: DGCR8-CLIP-seq replicate 8 (Low RNase 1 treated)


title: WT1_BRD4_ChIPSeq

value: age-matched control wild-type individual 1 (TRS-WT)


title: HG1 INPUT

source_name: HG fibroblasts_INPUT


title: WT2 INPUT

source_name: WT fibroblasts_INPUT


title: Adar2 -/-_whole_brain_miRNA_3

city: Vienna

first: Fritz

last: Sedlazeck

state: Vienna

address: Dr. Bohr-Gasse 9

department: CIBIV

country: Austria

institute: MFPL

data_processing:
Basecalling: Illumina Genome Analyzer 2 Platform
Small RNA sequences were mapped to mature miRNA sequences annotated in miRBase Release 17 (miRBase, http://www.mirbase.org/). The mapping was performed using NextGenMap (Sedlazeck FJ, Talloji P, von Haeseler A, Bachmair A: Benefit-of-doubt (BOD) scoring: a sequencing-based method for SNP candidate assessment from high to medium read number data sets. Genomics 2013, 101:204-209.) and allowed for up to two mismatches.
Count tables for mature miRNAs were created using R (www.r-project.org/)
Genome_build: mm9 / miRBase Release 17
Supplementary_files_format_and_content: The files are in .txt format. Files "replicate 1_raw reads_geo", "replicate 2_raw reads_geo" and "replicate 3_raw reads_geo" contain the following information for biological replicates 1-3: The first column contains the miRNA name, the second and third column gives the total read numbers for each miRNA in wildtype and ADAR2-/- mice respectively.

source_name: whole brain

extract_protocol:
Female mice were dissected and whole brain was homogenized. Total RNA was extracted using peqGOLD TriFast reagent according to manufacturer´s instructions (PEQLAB Biotechnologie GmbH, Erlangen, Germany). Total RNA was enriched for small RNAs (<200 nucleotides) using the mirVana™ miRNA Isolation Kit (Ambion®, Applied Biosystems).
Small RNAs were 5’-end labeled with 20μCi [γ-32P] ATP using T4 polynucleotide kinase (Fermentas™). Radiolabeled RNA was separated on 15% 19:1 AA:bisAA gels (8M UREA in 1xTBE) and exposed to X-ray films. RNA bands between 19-25 nucleotides were cut out and eluted in 400μl elution buffer (500mM NH4OAc, 0.2%SDS, 100mM EDTA) at room temperature over night. Eluted samples were purified over Sephadex G-25 spin-columns and precipitated. The size selected RNA was ligated to a 5’ pre-adenylated and 3’ blocked 3´-end adapter (5´-Appp- UCGUAUGCCGUCUUCUGCUUGUidT-3´) using truncated T4 RNA Ligase 2, (New England Biolabs) with the supplied buffer and a final concentration of 20% PEG6000. Ligations were incubated for 6 hours at room temperature followed by 4°C over night. Ligation products were purified on a 10% denaturing PAGE next to radiolabeled markers and ligated to the 5´adapter (5´-GUUCAGAGUUCUACAGUCCGACGAUC- 3´) using T4 RNA Ligase 1 (New England Biolabs) with the supplied buffer and 20% PEG. Again, the ligation product was purified on a denaturing PAGE. The final product was reverse transcribed using RevertAid™ H Minus Reverse Transcriptase (Fermentas™) using RT primer 5´-CAAGCAGAAGACGGCATACGA-3´ following manufacturer´s instructions. The cDNA was used as template for 15 cycles of PCR amplification (forward primer: 5´- AATGATACGGCGACCACCGACAGGTTCAGAGTTCTACAGTCCGA-3´, reverse primer: 5´-CAAGCAGAAGACGGCATACGA3´) using Phusion® High-Fidelity DNA Polymerase (Finnzymes) according to manufacturer´s protocol. PCR products were separated on a non-denaturing 6% AA TBE gel and stained with ethidiumbromide in 1x TBE. Bands of approximately 100bp corresponding to the final mature miRNA library were cut out and eluted in 400 μl elution buffer (0.5M NH4OAc, 1mM EDTA, pH 8.1) over night at room temperature. DNA was precipitated and subsequently resuspended in 10μl 1x TE.

tag: strain

value: brain

value: adult

value: female


title: Adar2 -/-_whole_brain_miRNA_2


title: Adar2 -/-_whole_brain_miRNA_1


title: wildtype_whole_brain_miRNA_2


title: wildtype_whole_brain_miRNA_1


title: Drosha-CLIP-seq replicate 1


title: wildtype_whole_brain_miRNA_3


title: Ycjw+ (YR2,YR3)

status: Public on Oct 01 2014

city: Palo Alto

first: Zheng

last: Zuo

address: Stanford

department: Genetics

institute: Stanford university

data_processing:
Library strategy: Spec-seq
Extracting binding sites sequence from raw reads. Each read follows the format of barcode-GATAGTCTCATTTTCACC-binding site-PE2. The barcode is typically NN dinucleotide or NNN trinucleotide.
Counting reads number for each variant, classified by bound/unbound
Calculating Bound/Unbound ratio for each variant
Ranking variants according to their relative affinities
Supplementary_files_format_and_content: For filetype 'processed.txt', each line has typically 5 items separated by tab, namely sequence, bound, unbound, bound/unbound ratio, relative binding energy;
Supplementary_files_format_and_content: For filetype 'paper.txt', each variant is further classified based on its library property, namely R2, R3,R4;

organism: Escherichia coli K-12

source_name: The recombinant protein is in vitro expressed by NEB Purexpress kit with estimated yield 100ng/ul

extract_protocol:
Methods: In our current work, all electromobility shift assay (EMSA) experiments were done in 1X NEB4 buffer [specify what this is] supplemented with 5% glycerol, and the purified protein of interest (LacI, PurR, and YcjW) was sequentially titrated into 15ul binding reaction system by 2 fold increase per lane. For the PurR protein guanine was included at 20uM. At least 30min incubation on ice can help establish the equilibrium state of protein-DNA interaction. All EMSA experiments were run in 6% 0.5X TBE PAGE gels at 120V for 30mins in the cold room.
5’ FAM-labeled DNA fragments can be visualized by Biorad Imager with 520nm bandpass filter (Supplemental Figure S1). Shifted and unshifted bands were cut, extracted and purified separately using acrylamide gel extraction buffer and Qiagen PCR columns. Each fraction of DNA was barcoded and amplified respectively by indexed-Illumina primers (PE1-Genetics1/2, PE2.0). To minimize possible PCR bias, we recommend no more than 10 cycles amplification (although in independent tests we found very minimal PCR bias).
For typical randomized DNA libraries with diversity up to 2000, a standard 1X75 Miseq run is sufficient to produce ~15M reads covering all possible variants with sufficient counts to get reliable ratios. Each read is sorted according to its barcode and binding site content. All sequences with variations in the conserved regions are filtered out. For each unique variant in the library, the ratio between bound reads and unbound reads is calculated and designated as relative binding affinity, compared to the preferred binding sites. The negative logarithm of each ratio is used to represent relative binding energy for each variant.
R2 library: AATTGTNAN CG NTNACAATT
R3.1 library: AATTGTGAG CNG NNNNCAATT
R3.2 library: AATTGTTAT CNG NNNNCAATT
R4 library: AATTGTNAN CCGG NTNACAATT
PR2 library: ACGCAAA CG NNNNCGT
PR3 library: ACGCAAA CGG NNNNCGT
YR2 library: TTCATGGGAC CG NNNNCACGGA
YR3 library: TTCATGGGAC CGC NNNNCACGGA


title: PurR+ (PR2,PR3)


title: Lac repressor+LacO (R2,R4)

description: 2 independent runs with 2X protein difference


title: WT1 INPUT


title: S2 input sequencing

city: Giessen

first: Marek

last: Bartkuhn

state: Hessen

address: Aulweg 132

department: Biomedical Informatics and Systems Medicine

institute: Justus-Liebig-University Giessen

description: ChIP-seq

data_processing:
Quality control of FASTQ files was done using FastQC.
Removal of low quality reads was done using fastx_toolkit-0.06
Reads were aligned to a pre-compiled index of the mouse hg19 genome using Bowtie (options: -k 1 -m 1) 0.12.5.
Duplicate reads were removed with Samtools' rmdup function.
Samtools was used to convert aligned reads to BAM format.
PeakRanger 1.15 was used at standard settings for peak calling
Genome_build: dm3
Supplementary_files_format_and_content: WIG files were produced using Macs 1.4 at 10bp resolution
Supplementary_files_format_and_content: BED files describing peaks were produced using PeakRanger 1.15.

organism: Drosophila melanogaster

source_name: S2 cells

growth_protocol: S2 were cells grown in SFX medium

extract_protocol:
Samples of 107 S2 cells in 10 mL of SFX medium were treated with 37% formaldehyde added to a final concentration of 1% and incubated on a rotator at room temperature for 10 min. Cross-linking was stopped by 0.125 M glycine, and the samples were washed with three portions of PBS (pH 8), with 0.5 mM PMSF and pelleted at 1000 rpm, 4°C, for 5 min. The pellet was resuspended in 10 mL of buffer I (25 mM HEPES, pH 7.8; 1.5 mM MgCl2, 10 mM KCl, 0.1% NP-40, 1 mM DTT, 0.5 mM PMSF, Calbiochem Cocktail V) and placed on ice for 10 min. The suspension was then homogenized by 20 strokes in Dounce homogenizer with pestle B and centrifuged at 2000 rpm, 4°C, for 5 min. The pellet was resuspended in 5 mL of buffer II (50 mM HEPES, pH 7.8; 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 0.5 mM PMSF, Calbiochem Cocktail V), sonicated in a Bioruptor sonifier (Diagenode, Belgium) for 15 min, with alternating 30-s ON/OFF intervals, and centrifuged at 14 000 rpm, 4°C, for 5 min to remove cell debris. A 50-µL aliquot of the supernatant was used to test the results of sonication and measure DNA concentration: the sample was diluted with 350 µL of elution buffer  (50 mM Tris, pH 8.0; 1 mM EDTA, 1% SDS, 50 mM NaHCO3) and treated with RNase A (1 µL from 10 mg/mL stock) at 37°C for 1 h and then with proteinase K (1 µL from 20 mg/mL stock) at 42°C for 2 h. After subsequent incubation at 65°C for 6 h, DNA was isolated by phenol–chloroform–isoamyl alcohol extraction, concentrated by ethanol precipitation with glycogen (each tube was supplemented with 5 µL of glycogen (from 20 mg/mL stock), 40 µL of sodium acetate (from 3 M stock), and 1 mL of ethanol, vortexed, and placed at –20°C for 4–5 h). The precipitated DNA was pelleted at 14 000 rpm for 30 min, washed with 80% ethanol, and resuspended in 50 µL of MilliQ water. Protein A Sepharose (Pierce) was washed with three portions of buffer II and incubated with 1 mg/mL BSA in the same buffer on a rotator at 4°C for 4 h. Chromatin samples containing 10–20 µg of DNA equivalent were each diluted with buffer II to a final volume of 1 mL, their 50-µL aliquots were stored as input, and then the samples were incubated overnight, at 4°C, with appropriate antibodies (rabbit antibodies against Pita (1:1000), ZIPIC (1:200), or CP190 (1:500), or with nonspecific IgG purified from rabbit preimmune sera (control)) in the presence of blocked Protein A Sepharose beads (40 µL). After incubation, the beads were washed on a rotator, at 4°C, with three 1-mL portions of buffer II and two 1-mL portions of buffer III (50 mM HEPES, pH 7.8; 500 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1 % SDS, 0.5 mM PMSF, Calbiochem Cocktail V), each wash for 10 min. Then 400 µL of elution buffer was added, and the beads were treated with RNase A (1 µL from 10 mg/mL stock) at 37°C for 1 h and, after adding 20 µL of NaCl (from 4 M stock), with proteinase K (1 µL from 20 mg/mL stock) at 42°C for 2 h. The samples were incubated on a thermoshaker at 65°C overnight, and DNA was isolated by phenol–chloroform–isoamyl alcohol and chloroform–isoamyl alcohol extraction in phase-lock tubes. The samples were concentrated by ethanol precipitation with glycogen (as described above), and resuspended in MilliQ water.
Sequencing libraries were prepared from 10 ng of immunoprecipitated DNA with the Illumina ChIPSeq DNA Sample Prep Kit according to Illuminas’ instructions

tag: cell line


title: HG2 INPUT


title: Lac repressor+LacO (R2,R3.1,R3.2,R4)


title: ChIP-Seq analysis of JMJD3 in CUTLL1 cells replicate1

city: NEW YORK

first: Panagiotis

last: Ntziachristos

address: 522 FIRST AVENUE

department: PATHOLOGY

institute: NYU MEDICAL SCHOOL

description: processed data file: CUTLL1-JMJD3-control-combined.wig.gz

data_processing:
Basecalls performed using CASAVA
ChIP-seq reads were aligned to the hg19 genome assembly using Bowtie version 0.12.7
MACS version 1.4.2 was used for ChIP-sequencing peak discovery. GenomicTools version 2.7.2 was used for performing genomic interval mathematical operations, genomic interval annotation, ChIP-seq heatmap generation and ChIP-seq profile construction.
Genome_build: hg19
Supplementary_files_format_and_content: wig format containg relative enrichments

source_name: ChIP-SEQ_JMJD3 CUTLL1

growth_protocol: cells in suspension, growing in RPMI

extract_protocol:
JMJD3 ChIP: 10 million cells were used for the ChIP and precipitated using 10micrograms of antibody against human JMJD3.
Libraries were generated as described before, including end-repair, A-tailing, adapter (Illumina Truseq system) ligation and PCR amplification.

treatment_protocol: CUTLL1 T cell leukemia cells treated with control GSKJ5 inhibitor

value: human T cell leukemia cells

value: abgent


title: ChIP-seq of Pita in S2 cells

description: ChIP-seq

value: anti-Pita


title: ChIP-Seq analysis of JMJD3 in CUTLL1 cells replicate 2


title: ChIP-seq of ZIPIC in S2 cells

description: ChIP-seq

value: anti-ZIPIC


title: ChIP-Seq analysis of H3K27me3 in CUTLL1 cells upon treatment with GSKJ4 replicate 2

description: processed data file: CUTLL1-H3K27me3-GSKJ4-combined.wig.gz

extract_protocol:
Micrococcal nuclease treated chromatin was used for the ChIP studies in an adaptation of the Upstate ChIP protocol.
Libraries were generated as described before, including end-repair, A-tailing, adapter (Illumina Truseq system) ligation and PCR amplification.

treatment_protocol: cells were untreated or treated with the GSKJ4 chemical inhibitor against H3K27me3 demethylases

value: abcam


title: ChIP-Seq analysis of H3K27me3 in CUTLL1 cells upon treatment with short hairpin against RENILLA replicate2

extract_protocol:
Micrococcal nuclease (MNASE) treated chromatin was used for the ChIP studies in an adaptation of the Upstate ChIP protocol
Libraries were generated as described before, including end-repair, A-tailing, adapter (Illumina Truseq system) ligation and PCR amplification of the libraries.

treatment_protocol: cells infected with retroviruses expressing short haiprins against human JMJD3 and Renilla

tag: infected with

value: retroviruses expressing short haiprins against Renilla


title: RNASEQ_CUTLL1-untreated-replicate3

description:
processed data file: degexp.CUTLL1-GSKJ4_48h-RNA.vs.CUTLL1-untreated-RNA.output_score.txt
RNASEQ_CUTLL1-untreated-rep3-RNA_R1.fastq.gz

data_processing:
Basecalls performed using CASAVA
RNA-seq reads were aligned to the mm9 genome assembly using Tophat
Differential gene expression analysis was performed for knockout vs wildtype. Analysis was performed using DEGseq.
Genome_build: hg19
Supplementary_files_format_and_content: TAB-delimited text, Degexp output containing normalized expression values

extract_protocol:
RNeasy kit (Qiagen)
Poly-A+ (magnetic oligodT-containing beads (Invitrogen)) RNA was used for library preparation. cDNA preparation and strand-specific library construction was performed using the dUTP method. Libraries were sequenced on the Illumina HiSeq 2000 using 50bp single-read method.

tag: treated with

value: none (untreated control)


title: ChIP-Seq analysis of H3K27me3 in CUTLL1 cells upon treatment with GSKJ4 replicate 1


title: ChIP-Seq analysis of H3K27me3 in CUTLL1 cells upon treatment with short hairpin against RENILLA replicate1


title: Sequencing analysis of Input in CUTLL1 cells replicate1

source_name: ChIP-SEQ_Input CUTLL1


title: RNASEQ_CUTLL1-GSKJ4_72h-replicate3

description:
processed data file: degexp.CUTLL1-GSKJ4_48h-RNA.vs.CUTLL1-untreated-RNA.output_score.txt
RNASEQ_CUTLL1-GSKJ4_72h-rep3-RNA_R1.fastq.gz

value: 2µM GSKJ4 for 72hr


title: ChIP-Seq analysis of H3K27me3 in CUTLL1 cells upon treatment with short hairpin against JMJD3 replicate1

value: retroviruses expressing short haiprins against human JMJD3


title: RNASEQ_CUTLL1-GSKJ4_72h-replicate2

description:
processed data file: degexp.CUTLL1-GSKJ4_48h-RNA.vs.CUTLL1-untreated-RNA.output_score.txt
RNASEQ_CUTLL1-GSKJ4_72h-rep2-RNA_R1.fastq.gz


title: ChIP-Seq analysis of H3K27me3 in CUTLL1 cells upon treatment with short hairpin against JMJD3 replicate2


title: RNASEQ_CUTLL1-untreated-replicate2

description:
processed data file: degexp.CUTLL1-GSKJ4_48h-RNA.vs.CUTLL1-untreated-RNA.output_score.txt
RNASEQ_CUTLL1-untreated-rep2-RNA_R1.fastq.gz


title: RNASEQ_CUTLL1-GSKJ4_72h-replicate1

description:
processed data file: degexp.CUTLL1-GSKJ4_48h-RNA.vs.CUTLL1-untreated-RNA.output_score.txt
RNASEQ_CUTLL1-GSKJ4_72h-rep1-RNA_R1.fastq.gz


title: RNASEQ_CUTLL1-GSKJ4_48h-replicate3

description:
processed data file: degexp.CUTLL1-GSKJ4_48h-RNA.vs.CUTLL1-untreated-RNA.output_score.txt
RNASEQ_CUTLL1-GSKJ4_48h-rep3-RNA_R1.fastq.gz

value: 2µM GSKJ4 for 48hr


title: RNASEQ_CUTLL1-GSKJ4_48h-replicate2

description:
processed data file: degexp.CUTLL1-GSKJ4_48h-RNA.vs.CUTLL1-untreated-RNA.output_score.txt
RNASEQ_CUTLL1-GSKJ4_48h-rep2-RNA_R1.fastq.gz


title: RNASEQ_CUTLL1-untreated-replicate1

description:
processed data file: degexp.CUTLL1-GSKJ4_24h-RNA.vs.CUTLL1-untreated-RNA.output_score.txt
RNASEQ_CUTLL1-untreated-rep1-RNA_R1.fastq.gz


title: RNASEQ_CUTLL1-GSKJ4_24h-replicate1

description:
processed data file: degexp.CUTLL1-GSKJ4_24h-RNA.vs.CUTLL1-untreated-RNA.output_score.txt
RNASEQ_CUTLL1-GSKJ4_24h-rep1-RNA_R1.fastq.gz

value: 2µM GSKJ4 for 24hr


title: RNASEQ_CUTLL1-GSKJ4_48h-replicate1

description:
processed data file: degexp.CUTLL1-GSKJ4_48h-RNA.vs.CUTLL1-untreated-RNA.output_score.txt
RNASEQ_CUTLL1-GSKJ4_48h-rep1-RNA_R1.fastq.gz


title: RNA-Seq analysis in CEM cells upon treatment with short hairpin against RENILLA replicate 1

description:
processed data file: degexp.CEM-shJMJD3-RNA.vs.CEM-renilla-RNA.output_score.txt
processed data file: degexp.CEM-shUTX-RNA.vs.CEM-renilla-RNA.output_score.txt

data_processing:
Basecalls performed using CASAVA
RNA-seq reads were aligned to the hg19 genome assembly using Tophat
Differential gene expression analysis was performed for each matched knockdown vs control pairs, separately in each biological or technical replicate in each of two cell lines (CUTLL1, CEM). Three types of comparisons were tested: (a) Jmjd3 knockdown vs renilla, (b) Jmjd3 knockdown vs Utx knockdown, and (c) Utx knockdown vs renilla. Analysis was performed using both DEGseq25 package leading to very similar conclusions.
Three types of comparisons were tested: (a) JMJD3 knockdown vs Renilla, (b) JMJD3 knockdown vs UTX knockdown, and (c) UTX knockdown vs Renilla. Analysis was performed using both DEGseq package leading to very similar conclusions.
Genome_build: hg19
Supplementary_files_format_and_content: TAB-delimited text, Degexp output containing normalized expression values

source_name: CEM cells_short hairpin against RENILLA

extract_protocol:
RNeasy kit (Qiagen)
Poly-A+ (magnetic oligodT-containing beads (Invitrogen)) or Ribominus RNA was used for library preparation. cDNA preparation and strand-specific library construction was performed using the dUTP method. Libraries were sequenced on the Illumina HiSeq 2000 using 50bp single-read method.

treatment_protocol: cells infected with retroviruses expressing short haiprins against human JMJD3 and UTX as well as Renilla

value: CEM

value: retroviruses expressing short haiprin against RENILLA


title: RNA-Seq analysis in CEM cells upon treatment with short hairpin against UTX replicate 1

description:
processed data file: degexp.CEM-shUTX-RNA.vs.CEM-renilla-RNA.output_score.txt
processed data file: degexp.CEM-shJMJD3-RNA.vs.CEM-shUTX-RNA.output_score.txt

source_name: CEM cells_short hairpin against UTX

value: retroviruses expressing short haiprin against UTX


title: RNA-Seq analysis in CEM cells upon treatment with short hairpin against UTX replicate 2


title: RNA-Seq analysis in CEM cells upon treatment with short hairpin against RENILLA replicate 2


title: RNA-Seq analysis in CEM cells upon treatment with short hairpin against JMJD3 replicate 2

description:
processed data file: degexp.CEM-shJMJD3-RNA.vs.CEM-renilla-RNA.output_score.txt
processed data file: degexp.CEM-shJMJD3-RNA.vs.CEM-shUTX-RNA.output_score.txt

source_name: CEM cells_short hairpin against JMJD3

value: retroviruses expressing short haiprin against JMJD3


title: RNA-Seq analysis in CUTLL1 cells upon treatment with short hairpin against RENILLA replicate 3

description:
processed data file: degexp.CUTLL1-shJMJD3-RNA.vs.CUTLL1-renilla-RNA.output_score.txt
processed data file: degexp.CUTLL1-shUTX-RNA.vs.CUTLL1-renilla-RNA.output_score.txt

source_name: CUTLL1 cells_short hairpin against RENILLA


title: RNA-Seq analysis in CUTLL1 cells upon treatment with short hairpin against RENILLA replicate 2


title: RNA-Seq analysis in CEM cells upon treatment with short hairpin against JMJD3 replicate 1


title: Sequencing analysis of Input in CUTLL1 cells replicate2


title: RNA-Seq analysis in CUTLL1 cells upon treatment with short hairpin against RENILLA replicate 1


title: RNA-Seq analysis in CUTLL1 cells upon treatment with short hairpin against UTX replicate 2

description:
processed data file: degexp.CUTLL1-shUTX-RNA.vs.CUTLL1-renilla-RNA.output_score.txt
processed data file: degexp.CUTLL1-shJMJD3-RNA.vs.CUTLL1-shUTX-RNA.output_score.txt

source_name: CUTLL1 cells_short hairpin against UTX


title: RNA-Seq analysis in CUTLL1 cells upon treatment with short hairpin against UTX replicate 1


title: RNA-Seq analysis in CUTLL1 cells upon treatment with short hairpin against JMJD3 replicate 3

description:
processed data file: degexp.CUTLL1-shJMJD3-RNA.vs.CUTLL1-renilla-RNA.output_score.txt
processed data file: degexp.CUTLL1-shJMJD3-RNA.vs.CUTLL1-shUTX-RNA.output_score.txt

source_name: CUTLL1 cells_short hairpin against JMJD3


title: RNASEQ_CUTLL1-GSKJ4_24h-replicate2

description:
processed data file: degexp.CUTLL1-GSKJ4_24h-RNA.vs.CUTLL1-untreated-RNA.output_score.txt
RNASEQ_CUTLL1-GSKJ4_24h-rep2-RNA_R1.fastq.gz


title: RNA-Seq analysis in CUTLL1 cells upon treatment with short hairpin against JMJD3 replicate 1


title: RNAseR.K562.Rep2

city: Sydney

first: Tim

middle: R.

last: Mercer

state: New South Wales

address: 384 Victoria Street Darlinghurst NSW 2010

country: Australia

institute: Garvan

data_processing:
Illumina Casava1.7 software used for basecalling.
Sequenced reads were aligned to the human genome using Tophat2 with default parameters and using reference aided assembly (gecode.v12.comprehensive assembly). Alignment files (.bam) were converted to processed bigWig files using the BEDtools genomeCoverageBed utility and the UCSC Genome Bioinformatics utility wigToBigWig (for visualization in UCSC Genome Browser).
Genome_build: hg19
Supplementary_files_format_and_content: .bigwig files

growth_protocol: Human K562 and Hela cells were cultured in RPMI and DMEM respectively plus 10% fetal bovine serum and penicillin/streptomycin at 37°C, 5% C02. K562 cells were collected by centrifugation, washed with PBS, centrifuged again and lysed in TRIzol (Life Technologies). HeLa cells were lysed in TRIzol by scraping of the cell culture flask. A standard TRIzol extraction was conducted and RNA resuspended in 200ul of RNase free water. Purity was confirmed by NanoDrop (Thermo scientific). RNA was DNase treated with Turbo™ DNase (Life Technologies), repurified with phenol/chloroform, nanodroppped again and the integrity of the RNA confirmed by Agilent 2100 Bioanalyzer (Agilent Technologies). The DNA free nature of the RNA was confirmed by the absence of product from a PCR for a section of NEAT1 gDNA with 200ng of purified RNA for 35 cycles. Five microgram lots of high quality RNA were treated with Ribo-Zero™ (Epicentre) to remove rRNA, purified by RNeasy Minelute (Qiagen) with successful ribodepletion confirmed by Bioanalyzer Pico chip.

extract_protocol:
A standard TRIzol extraction was conducted and RNA resuspended in 200ul of RNase free water. Purity was confirmed by NanoDrop (Thermo scientific). RNA was DNase treated with Turbo™ DNase (Life Technologies), repurified with phenol/chloroform, nanodroppped again and the integrity of the RNA confirmed by Agilent 2100 Bioanalyzer (Agilent Technologies).
RNA sequencing libraries were made with the TruSeq® RNA Sample Preparation v2 Kit (Illumina). Given the RNA was previously ribodepleted, library preparation was begun at the fragmentation step and the standard procedure followed. Successful generation of sequencing libraries from digested and RNaseR negative mock-digestion samples were confirmed by Agilent 2100 Bioanalyzer. Samples were sequenced on an Illumnia® HiSeq. Given the very small amount of RNA remaining after RNAseR digestion, multiple digested samples were combined to reach the minimum yield requirement for sequencing library preparation. Where necessary samples digested with different conditions that all provided satisfactory digestion were also pooled.

treatment_protocol: RNaseR (Epicentre) digestion was conducted on batches of 100 ng ribodepleted RNA. A number of digestion conditions were tested with the standard digestion procedure being 30U enzyme: 1 µg RNA for 30 min at 37°C. Mock digestion controls lacking RNaseR were also performed. RNA was purified by RNeasy Minelute and digestion of RNA by RNaseR confirmed by Bioanalyzer picochip.

tag: protocol

value: RNaseR treated

value: K562 (Human erythromyeloblastoid leukemia cell line)


title: RNA-Seq analysis in CUTLL1 cells upon treatment with short hairpin against JMJD3 replicate 2

value: human T cell leukemia cells


title: RNAseR.K562.Rep1


title: MockDigest.K562.Rep1

value: Untreated


title: MockDigest.K562.Rep2


title: RNAseR.Hela.Rep1

source_name: Hela

value: HeLa (Human cervical carcinoma cell line)


title: MockDigest.Hela.Rep2


title: DA respiratory tree

city: QINGDAO

first: Muyan

last: Chen

address: 5 YUSHAN ROAD

institute: Ocean University of China

data_processing:
The original image data was transferred into sequence data by base calling(using CASAVA version 1.5+), (defined as ‘raw reads’) and saved as fastq files that include the detailed reads sequences and the reads quality information.
The raw reads obtained from Solexa sequencing were processed by trimming poor quality reads, 5’ adapter pollution reads, reads without 3’ adapters, reads without insert fragments, reads containing poly(A) stretches, and reads less than 18nt. rRNA, tRNA, snRNA and snoRNA were removed by blasting against the GenBank database (http://blast.ncbi.nlm.nih.gov) and the Rfram database (http://sanger.ac.uk/software/Rfam).
Considering that no miRNA information of the sea cucumber was in miRBase19.0, the remaining clean reads were aligned to search all known precursor/mature miRNAs of all animal species in miRBase 19.0 with perfect matches.
Chose the highest expression miRNA for each mature miRNA family which was regarded as a temporary miRNA database. Clean data was aligned to the above temporary miRNA database and the expression of miRNA is generated by summing the count of reads which can align to the temporary miRNA database without mismatches.
Potentially novel miRNAs were identified using MIREAP (http://sourceforge.net/projects/mireap/) with stem-loop structure prediction
Genome_build: n/a
Supplementary_files_format_and_content: abundance

organism: Apostichopus japonicus

source_name: Apostihopus japonicus respiratory tree

extract_protocol:
Total RNA from each tissue sample was extracted using miRNeasy Mini kit (Qiagen) according to the manufacturer’s instructions.
Recovered Small RNAs were ligated sequentially to 5’ and 3’ adapters. Reverse transcription was performed followed by PCR amplification. The purified PCR products were used directly for cluster generation and sequencing analysis using the Illumina’s Solexa Sequencer according to the manufacturer’s instrunctions

value: degenerated respiratory tree

tag: status

value: deep-aestivation


title: NA respiratory tree

value: normal respiratory tree

value: non-aestivation


title: FH

city: Beijing

first: Jie

last: Lu

address: Shuangqiaozhonglu

institute: CAIQ

data_processing:
The program HCS1.1.37 and RTA1.7.45 was used to perform base-calling.
The adaptors, empty tags (no tag sequence between the adaptors), low quality tags (tags with unknown nucleotide ‘‘N’’) and tags with a copy number of 1 were removed from raw data to obtain 21bp clean tags. The copy numbers of clean tags were counted.
Supplementary_files_format_and_content: clean tag copy number

organism: Paulownia

source_name: healthy midribs and petioles

growth_protocol: The tissue cultured paulownias TH (healthy) and TD (diseased) were maintained in vitro on MS medium and grown in a greenhouse at 25℃ with a cycle of 16 h light and 8 h dark. In august 2011, the field-grown paulownias FH (healthy) and FD (diseased) were collected from field in Beijing.

extract_protocol: Four tag libraries of samples (TH, TD, FH and FD) were constructed in parallel. Total RNA was extracted from 3g of central leaf midribs and petioles from each sample using a TRIzol® Reagent (Invotrigen) following the manufacturer’s instructions, and then treated with DNase I (Invitrogen). RNA concentration and integrity were analyzed by Agilent 2100 Bioanalyzer (Agilent Technologies). Magnetic beads with oligo(dT) were used to purify poly(A)-containing mRNA and oligo-dT primer was used to sythesize double-stranded cDNA. The bead-bound cDNAs were then digested by endonuclease NlaIII, which recognizes and cuts CATG sites, to generate sticky 5' ends. The cut-off fragments were washed away and the bead-bound fragments were ligated to the Illumina adaptor 1 through the sticky 5' end. The bead-bound fragments with adaptor 1 were then digested by MmeI, which cuts at 17bp downstream of CATG site. After that, the tags with adaptor 1 were purified and ligated to Illumina adaptor 2 through the 3' end of the tag. After 15 cycles of linear PCR amplification, 95bp fragments were purified by 6% TBE polyacrylamide gel electrophoresis to obtain the final tag libraries. After denaturation, the single-stranded DNAs were fixed onto the Illumina Sequencing Chip (flowcell).

value: midribs and petioles

tag: disease state

value: healthy


title: TD

source_name: diseased midribs and petioles

value: infected with Paulownia Witches'-Broom Phytoplasma (PaWB)


title: MockDigest.Hela.Rep1


title: BP_CaptureSeq.Rep3

data_processing:
Library strategy: BP_CaptureSeq
Illumina Casava1.7 software used for basecalling.
Alignment to identify branchpoint nucleotide - The alignment approach to identify branchpoints is based on the Bowtie2 read aligner 39 and TopHat2 splice junction mapper 40. This pipeline proceeds as follows  Sequenced reads (.fastq file) was firstly aligned to the human genome using Tophat2: {$ tophat2 –x hg19.index –g gencode v12.comprehensive.gtf -1 sequences.1.fastq -2 sequences.2.fastq} Reads aligning to the reference genome are omitted from further analysis.  An index corresponding to unaligned reads is then assembled (unaligned_reads.index). The 5’ (23nt) sequence of each unique intron (23nt_5’introns.fa, using Gencode v12 comprehensive assembly) is then aligned to unaligned read index: {$ bowtie2 -x unaligned_reads.index -U 23nt_5’intron.fa } Unaligned reads with no match to an intron 5’ sequence are omitted. For reads to which a 5’ intron sequence aligns, the downstream sequences to the region aligning to the 5’ intron sequence is trimmed (trimmed_reads.fa). This sequence that remains following trimming is required to be longer than 20nt and is then aligned to the reference human genome: {$ bowtie2 -x introns.index -U trimmed_reads.fq } The.sam output is then analyzed for intronic alignments. Read alignments are required to occur <250 nt of the 3’ splice site of an intron whose 5’ termini is required to match the original 5’ sequence that was trimmed from the read (ie. both the splice 5’ intron sequence and trimmed read alignment are required derive from single intron). The 3’ nucleotide of the final alignment indicates the predicted branchpoint nucleotide.  As a secondary filter for spurious alignments, we then generated a lariat junction index centered on predicted branchpoint (lariat.index). This lariat junction index comprises the 100nt upstream to the branchpoint nucleotide followed by the 100nt sequence from the matched intron 5’ termini, together constituting the expected sequence to traverses the intron lariat junction for each branchpoint. Lariat sequences were required to have less than 80% homology to human genome. We then re-aligned all reads that do not align to genome: {$ bowtie2 -x lariat.index -U unaligned_reads.fq } The .sam output was filtered for reads requiring a full-length and unique match, with a requisite 20nt minimum overlap across branch junction. This provides the final annotation of branchpoints across which lariat reads align.
Alignment files (.bam) were converted to processed bigWig files using the BEDtools genomeCoverageBed utility and the UCSC Genome Bioinformatics utility wigToBigWig.
Genome_build: hg19
Supplementary_files_format_and_content: bigWig files

growth_protocol: Human K562 cells were cultured in RPMI and DMEM respectively plus 10% fetal bovine serum and penicillin/streptomycin at 37°C, 5% C02. K562 cells were collected by centrifugation, washed with PBS, centrifuged again and lysed in TRIzol (Life Technologies).

extract_protocol:
A  TRIzol extraction was conductedaccording to manufacturer's protocol and RNA resuspended in 200ul of RNase free water. Purity was confirmed by NanoDrop (Thermo scientific). RNA was DNase treated with Turbo™ DNase (Life Technologies), repurified with phenol/chloroform, nanodroppped again and the integrity of the RNA confirmed by Agilent 2100 Bioanalyzer (Agilent Technologies). The DNA free nature of the RNA was confirmed by the absence of product from a PCR for a section of NEAT1 gDNA with 200ng of purified RNA for 35 cycles. Five microgram lots of high quality RNA were treated with Ribo-Zero™ (Epicentre) to remove rRNA, purified by RNeasy Minelute (Qiagen) with successful ribodepletion confirmed by Bioanalyzer Pico chip
Capture sequencing was performed by combining and modifying the NimbleGen SeqCap EZ Library SR User’s Guide V3.0 and the NimbleGen Arrays User’s Guide: Sequence Capture Array Delivery v3.2. RNA sequencing libraries of ribodepleted total RNA from three K562 biological replicates were created using the TruSeq® Stranded mRNA Sample Preparation Kit (Illumina). Different index adaptors (4,6,12) were added to each K562 biological replicate. Library input consisted of ribodepleted RNA from 5 μg original total RNA after quality checking. ERCC RNA Spike-In Control mix 1 (Invitrogen) was added to ribodepleted RNA to give a final ERCC concentration of 1% (replicate 1 and 2) and 1.08% (replicate 3). Library preparation was begun at the fragmentation step by the addition of 9 μl EPH buffer and the standard protocol followed until “Enrich DNA Fragments”. The 20 μl obtained at the end of the “Ligate Adaptors” step was increased to 21 μl with resuspension buffer and mixed well. One microliter of this solution was added to 19 μl resuspension buffer in a new 0.3ml PCR plate and mixed. This new plate was utilised for the “Enrich DNA Fragments” step to create amplified test libraries to guide PreCapture LMPCR with the remaining 20 μl. Precapture LMPCR and QIAquick PCR Purification Kit (Qiagen) were performed as described by the NimbleGen SeqCap EZ Library SR User’s Guide V3.0. To test LMPCR yields K562 biological replicate 3 libraries were amplified for 8, 9 or 10 cycles, quantified on the bioanalyzer and then pooled. K562 biological replicate 1 and 2 libraries were thus amplified for 9 cycles and the yield quantified by bioanalyzer. Capture hybridization was modified from the NimbleGen Arrays User’s Guide: Sequence Capture Array Delivery V3.2. The NimbleGen Hybridization System was set to 42°C and allowed 3 h to equilibrate. Equal nanograms of library from each K562 biological replicate were pooled, some of pooled library was allocated for pre-capture sequencing and 1ug of the pooled library utilized for capture. The library for capture was mixed with 300 µg human Cot-1 DNA (Invitrogen) and 3.34 µl 100 μm TS-INV-HE (hybridisation enhancing) Index Oligos (IDT) to each index adaptor plus 1 µl 1000 μm TS-HE Universal Oligo 1 (IDT) in a 1.5 ml tube. Lid of tube was pierced with an 18 gauge needle and the sample dried at 60 °C in a vacuum concentrator. Once dry the tube was given a new lid to prevent contamination through the needle hole. Library/Cot/Oligo mix was resuspended in 11.2 µl of nuclease and nucleic acid free water, vortexed for 10 s and centrifuged at max speed for 10 s. Sample was solubilised at 70 °C for 10 m before repeating vortexing and centrifugation. Nimblegen 2 x SC Hybridization Buffer (18.5 µl) and SC Hybridization Component A (7.3 µl) were added and vortexing and centrifugation repeated. Sample was denatured at 95°C for 10 m. During this time the “Prepare Mixers” procedure from the NimbleGen Arrays User’s Guide was performed with the omission of the compressed gas step. After denaturation samples was vortexed for 10 s, centrifuged at max speed for 10 s and incubated at 42°C until ready to load onto the NimbleGen Hybridization system. The ‘Load and Hybridize Samples’ procedure from the NimbleGen Arrays User’s Guide was performed to begin array hybridization. Hybridization was conducted for ~3 days. The NimbleGen Arrays User’s Guide protocol was followed to prepare the elution chamber, disassemble, wash and elute the captured DNA from the microarray, with the following modifications. Gasket and elution chamber was setup in a DNA-free laminar flowhood. All washes were conducted with 50 ml buffer in 50 ml falcon tubes. Minelute purification (Qiagen) was conducted using a microfuge. Captured DNA was recovered with two 30 µl elutions from a Minelute column and the final volume adjusted to 60 µl. Post-capture LMPCR and cleanup was performed similar to the NimbleGen SeqCap EZ Library SR User’s Guide V3.0, with the following modifications. LMPCR was run using 5x Phusion buffer for 17 cycles. Each PCR contained 70 µl of master mix and 30 µl of captured DNA. Quantity and quality of amplified captured DNA was determined by bioanalyzer.  Enrichment of captured transcripts was measured by qPCR using SYBR Green PCR Master Mix and real time cyclers (Applied Biosystems). Enrichment was determined by the ratio of transcript abundance between the pre and post capture samples using equal nanograms of each. Enrichment was determined for three Nimblegen capture controls and 3 sequences captured specifically by the branchpoint array. Two transcripts not targeted by the capture array were also tested to examine the specificity of capture. Concentrations and volumes were as per the NimbleGen SeqCap EZ Library SR User’s Guide V3.0. Precapture and post capture samples (3 K562 biological replicates) were each sequenced on a single lane of an Illumnia® HiSeq.

treatment_protocol: Oligonucleotide probes were designed in conjunction with Dr. Ryan Bannen at Roche/Nimblegen using proprietary bioinformatics to optimise array probe sequence and omit repetitive regions. All human genome regions from the 100nt 5’ and 3’ termini of publicly annotated introns (Gencode v12 comprehensive assembly) were targeted. Any regions overlapping an annotated exon or a region of high transcription (as determined from publicly available Human K562 RNAseq alignments11) was excluded (See above). This final design covered 36.8Mb and targets both 3’ and 5’ 100nt termini for 76.4% (206,747) publicly annotated introns, or a single terminus for 90.2% (244,125) introns. Additional control probes were included within the design to assess CaptureSeq performance. Probes targeting all ERCC controls 38 as well 30 complex spliced genes for internal control of splice junction and transcript assembly were included. Final design, including controls, were manufactured on a Custom Sequence 2.M Array (Roche/Nimblegen. Cat #05329841001).

value: Capture Sequencing


title: FD


title: RNASEQ_CUTLL1-GSKJ4_24h-replicate3

description:
processed data file: degexp.CUTLL1-GSKJ4_24h-RNA.vs.CUTLL1-untreated-RNA.output_score.txt
RNASEQ_CUTLL1-GSKJ4_24h-rep3-RNA_R1.fastq.gz

value: 2µM GSKJ4 for 24hr


title: TH


title: BP_CaptureSeq.Rep2


title: Aged_29_3

city: Cork

first: Roman

middle: M

last: Stilling

address: Western Road

department: Anatomy and Neuroscience

country: Ireland

institute: University College Cork

data_processing:
Base calling was achieved through Illumina CASAVA pipeline
Alignment was achieved by using STAR aligner version 2.3.0 with following non-default parameters: --outFilterMismatchNmax 2,  --outSAMstrandField intronMotif
Read counting in genes was achieved using HTSeq with the following non-default parameters:  -m intersection-nonempty, –stranded=no
Differential expression calling for pairwise comparisons was done using DESeq2 with the following non-default parameters: cooksCutoff=FALSE
Genome_build: GRCm38.p2
Supplementary_files_format_and_content: .txt - tab delimited text file with read counts
Supplementary_files_format_and_content: .txt - tab delimited text file with expression values, fold change, p-values and gene annotation

molecule: polyA RNA

source_name: hand-dissected hippocampus

extract_protocol:
Total RNA was extracted using TRI reagent (Sigma-Aldrich) accoding to the suppliers manual. Folling DNase1 (life Technologies) treatment for 20min at 37deg C, RNA was again purified using phenol-chloroform extraction.
TruSeq

value: Hippocampus

tag: genotype

value: Wild type

tag: treatment

value: 29 months


title: Aged_29_1


title: HCT116 RNA-seq repPF

city: Los Angeles

first: Peggy

middle: J

last: Farnham

address: 1450 Biggy Street NRT 6503

department: GMCB

institute: University of Southern California Graduate Student

data_processing:
Illumina Casava1.8 software used for basecalling.
Alignment with Tophat 2.0.8b: ex: tophat --no-coverage-search -p 11 -r 150 hg19 HCT116_CAGATC_L001_R1_001.1.fastq HCT116_CAGATC_L001_R2_001.1.fastq
we used Cufflinks, a program of “alignment to annotation” having discontinuous mapping to the reference genome. mRNA abundance was measured by calculating FPKM (expected fragments per kilobase of transcripts per million fragments sequenced), to allow inter-sample comparisons. We specified the –G option with the GENCODE V15 comprehensive annotation so that the program will only do alignments that are structurally compatible with the reference transcript provided.
Genome_build: hg19
Supplementary_files_format_and_content: tab-delimited text files include RPKM values for each gene

source_name: HCT116 cell line

growth_protocol: HCT116 cells were grown in DMEM supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin until 80% confluent.

extract_protocol:
For HCT116 colon cancer cells, RNA was prepared using Trizol (Life Technologies, Carlsbad, CA),  paired-end libraries were prepared using the Illumina TruSeqV2 Sample Prep Kit (Catalog #15596-026), starting with 1 ug total RNA. Libraries were barcoded, pooled, and sequenced using an Ilumina Hiseq.
RNA libraries were prepared for sequencing using standard Illumina protocols

tag: cell type

value: colon cancer


title: RNAseR.Hela.Rep2

value: RNaseR treated


title: Aged_29_2

tag: tissue


title: Young_control_pe_2

tag: age

value: 3 months


title: Young_control_pe_1


title: Aged_24_5

value: 24 months


title: Aged_24_3


title: HCT116 RNA-seq repPJ


title: Aged_24_6


title: Young_control_pe_3


title: Aged_24_2


title: Aged_24_1


title: Young_control_2


title: Young_control_4


title: Young_control_1


title: BP_CaptureSeq.Rep1

value: K562 (Human erythromyeloblastoid leukemia cell line)


title: P17 EED

city: Chapel Hill

first: Joshua

last: Starmer

state: NORTH CAROLINA

address: 120 Mason Farm Road

institute: University of North Carolina at Chapel Hill

data_processing:
Initial processing and basecalling was done using the Illumina Pipeline (v.1.8.2)
Alignment to the mm9 genome was performed using Bowtie
BAM files were created from bowtiefiles using samtools
Peaks were called using MACS using the input and the combined replicates for each developmental stage/histone modification combination with default parameters
Reads were counted at features using HTseq-count and multiBamCov
Genome_build: mm9

source_name: Spermatocytes

extract_protocol:
Spermatocytes were isolated from p12 and p17 old testes which were digested with trypsin and collagenase, and fractionated through 5% percoll. The cells were  fixed in 0.6% Formaldehyde for 10 min at RT, then sonicated, and immunoprecipitated with the antibodies indicated.
DNA was end repaired, A-tailed, then Illumina barcode adaptors were ligated and amplified.

tag: developmental stage

value: WT

tag: chip antibody

value: EED (Millipore, 17-663)


title: P17 H3K4me3 rep1+rep2

value: H3K27me3 (Abcam, ab6002)


title: P17 Input rep1+rep2

value: Input


title: P12 Input


title: P12 H3K4me3

value: H3K4me3 (Abcam, ab8580)


title: P12 H3K27me3


title: P17 H3K27me3 rep1+rep2


title: ER4_Mitotic_Dnase rep3

city: University Park

first: Ross

last: Hardison

state: PA

address: 303 Wartik Lab

institute: Pennsylvania State University

description: processed data file: G1E-ER4_Mitotic_pooled_peaks.bed.gz; G1E-ER4_Mitotic_pooled.bigWig

data_processing:
Raw reads from DNase-seq libraries were first groomed using FASTQ Groomer on Galaxy Giardine (2005); Blankenberg et al. (2001);Goecks et al. (2010). This program verifies that each base call has a corresponding quality value, and that the quality value is in the Sanger, Phred+33 format. Groomed reads were then trimmed as only the first twenty bases of each read are genomic DNA. The trimmed reads were then mapped to mouse mm9 genome using Bowtie Langmead et al. (2009) using the parameters -m = 4 , -k = 1, and -best, thus allowing the 20 basepair reads to map to at most 4 locations, but reporting only the single, best alignment. This option was chosen to allow reads to map in duplicated regions, while still excluding reads to highly repetitive DNA. The mapped reads from Bowtie were then filtered to retain only those reads successfully mapped to the mm9 genome.
Using DNase2Hotspots as described in Baek et al., 2011, we initially called hotspots using mapped reads pooled from biological triplicates for each experimental condition. After testing a range of z-score thresholds, we decided on a final stringent threshold of 0% FDR that still captured many mildly DNase-sensitive areas. From within hotspots that meet the 0% FDR threshold, we called DNase peaks further enriched from the surrounding hotspot, using a thresholded at a z-score >20.
Reads pooled from biological triplicates for each experimental condition were converted to bed files that were used as input to F-seq (Boyle et al., 2008), which generates smoothed signals based on library-size normalized density of reads. From these pooled reads we used F-seq to identify a set of top 100,000 read-enriched regions. For visualization in browser tracks, genome-wide signal track produced by F-seq for individual replicates as well as the reads pooled from replicates are available as bigwig files at https://mery.genome-browser.bx.psu.edu/cgi-bin/ hgTrackUi?hgsid=187170_w5iejxXAHeIanNii3mA1Otjcfthv&c=chr17&g=chopDnase2. Note that F-seq signal is linearly proportional to RPKM.
Genome_build: mm9
Supplementary_files_format_and_content: bigwig files contain genome wide DNase signals calculated by F-seq as described above.  Bed files contain the chromosone, start, and end positions of the peak signal regions called by F-seq.

growth_protocol: G1E and G1E-ER4 cells were grown as described in Weiss et al., 1997.

extract_protocol:
At harvest, nocodazole-treated and asynchronous cells were cross-linked with 0.1% formaldehyde at room temperature for 10 minutes, then quenched with 1 M glycine. Fixed cells were washed with PBS and resuspended in 1X Cell Lysis Buffer (60 mM KCl, 15 mM NaCl, 5 mM MgCl2, 10 mM Tris pH 7.4, 300 mM sucrose, 0.1 mM EGTA, 0.5 mM PMSF, 0.1% NP40, and 2 μL/mL protease inhibitor cocktail (Sigma)). For mitotic samples, cells were then stained with anti-H3S10Phos antibody (Millipore 04-817) and Dy488 F(ab’)2 anti-rabbit IgG secondary antibody (Jackson 711-485-152), and sorted by FACS for H3S10Phos-positive cells as shown in Fig. 1. DNase I digestion was performed based on protocol from (Cockerill, 1999), with details outlined as follows. 2-10 million asynchronous cells (in Cell Lysis Buffer) or 2 million mitotic cells (collected from the FACS machine in PBS) were resuspended in 50 μL Nuclei Lysis Buffer (300 mM sodium acetate, 5mM EDTA pH 7.4, 0.5% SDS), added to 5 μL of 100 mM CaCl2, equilibrated at room temperature for 10minutes. A range of units of DNase I were added (see DNase-seq library preparation below for the range of units selected for sequencing) and the digestion reaction proceeded for 10 minutes at room temperature, then terminated by adding 350 μL of 0.1 mg/mL proteinase K in Nuclei Lysis Buffer. Samples were gently mixed by inversion, then incubated at 55◦Cfor 5 minutes, then overnight at 65◦Cfor reversal of formaldehyde crosslinks. Additional proteinase K was added to a final concentration of 0.1 mg/mL and incubated at 55◦Cfor 1h. DNA fragments were isolated by phenol-chloroform extraction and ethanol precipitation.
DNase-seq library construction was performed as described in (Song and Crawford, 2010), with the following modifications. Standard 0.8% agarose gels were run for 2h at 80V with 5 μL of each sample and stained with ethidium bromide to check extent of chromatin digestion. A range of 3 different DNase I concentrations were chosen for each condition that best matched digestion patterns between conditions. For mitotic samples, this was 2U, 4U, and 8U of DNase. For asynchronous samples, this was between 4U to 40U, adjusted proportionally to the number of cells in the sample. 70 μL of each sample (for each DNase I concentration) were subjected to blunt-end reaction containing 20 μL NEB Buffer 2, 7 μL 10 mM dNTP’s, 6 μL T4 DNA polymerase (NEB M0203), 2 μL BSA (100x), 4 μL 50mMMgCl2, and 95 μL dH2O, incubated at room temperature for 3.25h. 200 μL TE buffer was added and samples placed at 65◦Cfor 15 minutes to deactivate enzyme. Reactions were cleaned up by phenol-chloroform extraction and DNA resuspended in 30 μL 10 mM Tris-Cl. The samples corresponding to the three chosen DNase I concentrations chosen for each condition were measured by nano drop and pooled at equimolar concentrations into a single tube for overnight ligations to the first DNase-seq linker, as per (Song and Crawford, 2010). The one modification from (Song and Crawford, 2010) is that we added a 5’ phosphate to linker 1 to increase ligation efficiency.

treatment_protocol: G1E-ER4 cells were treated with 100 nM estradiol for 22h prior to harvest. Then, for mitotic arrest, both G1E and G1E-ER4 cells were treated with nocodazole (200 ng/ml) for 7h prior to harvest.

tag: strain background

value: inducible GATA-1 rescue subline of G1E cells

tag: cell markers

value: 100 nM estradiol for 22h and nocodazole (200 ng/ml) for 7h prior to harvest


title: Aged_24_4


title: ER4_Mitotic_Dnase rep1


title: ER4_Async_Dnase rep2

description: processed data file: G1E-ER4_Async_pooled_peaks.bed.gz; G1E-ER4_Async_pooled.bigWig

value: 100 nM estradiol for 22h prior to harvest


title: ER4_Async_Dnase rep1


title: G1E_Mitotic_Dnase rep2

description: processed data file: G1E_Mitotic_pooled_peaks.bed.gz; G1E_Mitotic_pooled.bigWig

tag: cell line source

value: targeted disruption of GATA-1 in strain 129 embryonic stem cells

value: GATA-1 (null) cell line

value: nocodazole (200 ng/ml) for 7h prior to harvest


title: rat pancreatic primary beta cell batch 3 with Nova1 knock-down

city: Bruxelles

first: Olatz

last: Villate

address: Route de Lennik 808 CP618

department: Faculty of Medecine

country: Belgium

institute: Universite Libre de Bruxelles

description: Nova1 knockdown

data_processing:
Sequencing reads were mapped to the Rattus norvegicus genome (version Rnor_5.0) using the program Tophat (http://ccb.jhu.edu/software/tophat/index.shtml) using default options and Ensembl 71 rat annotation. The Tophat mapper reports all mappings and split-mappings.
GTF files: Mapped reads were used to quantify transcripts from the RefSeq reference database, using the Flux Capacitor approach that deconvolves reads mapping to exonic regions shared by multiple transcripts by optimizing a system of linear equations and thus obtains a number of reads specifically assigned to each alternative spliceform (http://flux.sammeth.net). All genes and transcripts have been assigned a relative coverage rate as measured in RPKM units ("reads per kilobase per million mapped reads").
Genome_build: Rnor_5.0
Supplementary_files_format_and_content: The processed data files are in standard transcript GTF format (for the full format description see http://www.ensembl.org/info/website/upload/gff.html). The attributes column contains informations on the transcript length, transcript abundance quantification RPKM (reads per kilobase per million mapped reads) and the number of read that mapped to it.

organism: Rattus norvegicus

growth_protocol: rat pancreatic primary beta cells FACS-purified (FACSAria; BD Bioscience, San Jose, CA, USA) and cultured as described Cunha, D.A et al. 2008 J. Cell Sci., 121, 2308-2318.

extract_protocol:
Total RNA was isolated using the RNeasy Micro Kit (Qiagen) which favors purification of RNA molecules longer than 200 nucleotides, and sample preparation was done as described by the manufacturer (Illumina, Eindhoven, The Netherlands).
mRNA was purified from 1 µg total RNA using oligo (dT) beads, before it was fragmented and randomly primed for reverse transcription followed by second-strand synthesis to create ds cDNA fragments. The generated cDNA had undergone paired-end repair to convert overhangs into blunt ends. After 39- monoadenylation and adaptor ligation, cDNAs were purified on a 2% agarose gel and 200 basepair (bp) products were excised from the gel. Following gel digestion, purified cDNA was amplified by PCR using primers specific for the ligated adaptors. The generated libraries were submitted to quality control with the Agilent bio- analyzer 2100 (Agilent Technologies, Wokingham, UK) before sequencing. The RNA integrity number (RIN) values for all samples were 7.5 and above. 1 µl cDNA was loaded on an Agilent DNA chip (DNA-1000) to verify cDNA quality and quantity. Only libraries reaching satisfactory conditions were used for sequencing, on an Illumina HiSeq 2000 system

treatment_protocol: Cells were transfected with siRNA (30nM) using the Lipofectamine RNAiMAX lipid reagent (Invitrogen). Allstars Negative Control siRNA (Qiagen, Venlo, The Netherlands) was used as negative control (siCTL). This siCTL does not affect beta cell gene expression or insulin release, as compared with non-transfected cells. After 16h of transfection, cells were cultured for 48h recovery period before RNA-seqencing

value: pancreas

value: beta cell


title: rat pancreatic primary beta cell batch 3 under control condition

description: control


title: G1E_Mitotic_Dnase rep3


title: Young_control_3

tag: strain

value: Hippocampus

value: Wild type


title: rat pancreatic primary beta cell batch 2 with Nova1 knock-down

description: Nova1 knockdown


title: rat pancreatic primary beta cell batch 1 under control condition

description: control


title: G1E_Async_Dnase rep2

description: processed data file: G1E_Async_pooled_peaks.bed.gz; G1E_Async_pooled.bigWig

tag: cell line


title: G1E_Async_Dnase rep3


title: Young_control_5

value: 3 months


title: ER4_Mitotic_Dnase rep2


title: ER4_Async_Dnase rep3