title: pn_nic3

description: processed data file: postnatal_EdgeR_processed_counts.txt

source_name: Postnatal only nicotine received


title: pp_nic1

status: Public on May 30 2016

type: SRA

city: New Haven

first: Marina

last: Picciotto

state: CT

address: 301 Cedar Street Congess place/YPI 4th floor

department: Psychiatry

country: USA

institute: Yale University

description: processed data file: prepost_EdgeR_processed_counts.txt

library_source: genomic

data_processing:
Sequences were called from image files with the Illumina Genome Analyzer Pipeline (GApipeline) and aligned to the reference genome (UCSC mm9) using Extended Eland in the GApipeline.
The uniquely aligned reads by ELAND pipeline were considered in calling peaks. To eliminate noise and account for unequal total numbers, we used a defined analysis model (Model-based Analysis of ChIP-Seq, MACS) with default parameters to find peaks, which were called “peaks” of H3K4me3.
For comparison analysis, a customized genomic interval file was created for EdgeR  analysis.
Genome_build: mm9
Supplementary_files_format_and_content: tab-delimited text files include EdgeR processed pseudo count for each sample.

molecule: genomic DNA

organism: Mus musculus

source_name: Pre and postnatal nicotine received

growth_protocol: NIH Guide for Care and Use of Laboratory Animals. C57BL/6J mice (Jackson Laboratories, Bar Harbor, ME) used in the developmental nicotine exposure studies acclimated for 1 week, had ad libitum food and water and a 12-h light–dark cycle (lights on at 7AM).

extract_protocol:
Mouse tissue samples (50 mg) were cut into slices less than 1 mm in thickness, and fixed in 3 ml of 1% formaldehyde/PBS solution. the tissue samples were homogenized in a glass-Teflon homogenizer. Following homogenization, chromatin was isolated using the Magna ChIP G kit (Millipore, Temecula, CA) according to the Millipore protocol. Cells were lysed in Cell Lysis Buffer in the presence of protease inhibitor cocktail.
Sample preparation and sequencing on the Genome Analyzer (Illumina, San Diego, CA) were carried out according to the Illumina protocols with some modifications. Briefly, the double stranded cDNA and ChIP-enriched genomic DNA were treated with T4 DNA polymerase and the Klenow fragment for end repair. The 5’ ends of the DNA fragments were then phosphorylated by T4 polynucleotide kinase, and an adenosine base was added to the 3’ end of the fragments by Klenow (3’-5’ exo). A universal adaptor was then added to both ends of the DNA fragments by A-T ligation. Following 18 cycles of PCR with the Phusion DNA polymerase, the DNA library was then purified on a 2% agarose gel, and fragments of 170 – 300 base-pair in size were recovered. Around 15 ng of the DNA library was then used for cluster generation on a grafted GAII Flow Cell, and then sequenced on the Genome Analyzer for 36 cycles using the “Sequencing-by-synthesis” method.

treatment_protocol: Saccharin or Nicotine soultion delivered to pregnant mice from the conception. Nicotine and saccharin water weaned from offspring 21 day old. The animal let live undisturbed by 90 day old

tag: tissue

value: brain

tag: tissue compartment

value: cerebral cortex

tag: strain

tag: age

value: 3 month old

tag: chip antibody

value: H3K4me3 (Millipore, 06-598, lot JBC940052)


title: pn_nic4


title: pp_sac2

source_name: Pre and postnatal saccharin received


title: pp_sac1


title: pp_sac3


title: carcass_WT_i9

status: Public on May 31 2016

city: Cambridge

first: Dario

last: Beraldi

address: Robinson Way

country: United Kingdom

institute: Cambridge Research Institute

data_processing:
Raw sequence reads were trimmed to remove poor quality reads, adapter contamination, and the first 6 base pairs, using Trim Galore http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/). Trimmed sequencing reads were aligned to mouse genome assembly NCBIM37 (mm9) using Bowtie2. Peaks were called in the SeqMonk software (www.bioinformatics.babraham.ac.uk/projects/seqmonk/) using the MACS peak caller, with a p-value cutoff of 1x10-5 and a fragment size of 200 base pairs
Genome_build: NCBIM37
Supplementary_files_format_and_content: Bed files of positions of 5fC peaks.

source_name: carcass

extract_protocol:
Embryonic tissues were lysed in RLT plus buffer (Qiagen), homogenised via centrifugation through a QIAshreddred column (Qiagen) at full speed for two minutes in a microcentrifuge. DNA and RNA were isolated using AllPrep DNA/RNA kit (Qiagen) following the manufacturer’s instructions.
Sequencing libraries were generated using the NEBNext library preparation modules. 16 cycles of PCR amplification were performed with the Illumina PCR Master Mix and PCR Primer Cocktail. Prior to sequencing, libraries were quantified by qPCR (KAPA Biosystems) and their fragment size profile analysed by TapeStation (Agilent). Sequencing was performed on an Illumina Nextseq 500, run in paired-end mode with 100 read cycles.

value: wild-type


title: WT2 [L2553]

city: Dresden

first: Min Ae

last: Lee-Kirsch

address: Fetscherstr. 74

department: Klinik für Kinder- und Jugendmedizin

country: Germany

institute: Universitätsklinikum Carl Gustav Carus an der Technischen Universität Dresden

library_source: transcriptomic

data_processing:
For data analyses a splice junction library with a length of 120 nucleotides (60+60) per splice junction was created based on known exon-exon junctions according to the Ensembl Genes annotation (v. 67, May 2012)
Alignment of the reads to the hg19 transcriptome was performed with pBWA
A counts-per-gene table was created based on the overlap of the uniquely mapped reads with the Ensembl Genes annotation for hg19, using BEDtools (v. 2.11)
RPKM (reads per kilobase per million reads) values were calculated based on the raw read counts and used as a measure of absolute expression levels.
Genome_build: hg19
Supplementary_files_format_and_content: excel file with RPKM counts table

molecule: total RNA

organism: Homo sapiens

source_name: fibroblast

extract_protocol:
Total RNA from fibroblasts was extracted with the RNeasy Mini Kit (Qiagen) followed by DNase I digestion.
RNA libraries were prepared for sequencing using standard Illumina protocols. Strand specific RNA-Seq (dUTP method)

treatment_protocol: Oxidative stress was induced by adding hydrogen peroxide (400 µM) to the medium followed by incubation for 10 min at 37°C.

tag: genotype

value: wildtype

tag: passages

value: passages 4-11

value: skin biopsies

tag: agent


title: peritoneal macrophages_Dnmt3a conditional knockout

city: shang hai

first: Qian

last: Zhang

address: xiangyin road 800

country: China

institute: Second military medical university

data_processing:
Illumina Casava1.6 software used for basecalling.
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence, then mapped to SOAPaligner/soap2. Mismatches no more than 2 bases were allowed in the alignment.
The gene expression level is calculated by using FPKM- Fragments Per Kilobase of transcript per Million fragments mapped.
Using cuffdiff to identify differentially expressed genes.
Genome_build: GRCm38
Supplementary_files_format_and_content: tab-delimited text files include FPKM values for each Sample.

extract_protocol:
RNA was harvested using Trizol reagent，and isolated using an RNA isolation kit (QIAGEN).
The library products are ready for sequencing via Illumina HiSeq 2500.

value: 129sv and C57BL/6

tag: cell type

value: peritoneal macrophages

tag: genotype/variation

value: Dnmt3afl/fl lyz2Cre+


title: peritoneal macrophages_control

source_name: peritoneal macrophages_control

value: Dnmt3afl/fl lyz2Cre-


title: pn_nic2


title: pn_nic1


title: WT1 [L2545]


title: pn_sac4

source_name: Postnatal only saccharin received


title: SLE1 [L2550]

value: SLE


title: AGS1 [L2549_H2O2]

value: AGS

value: hydrogen peroxide (400 µM)


title: SLE1 [L2551_H2O2]


title: AGS1 [L2548]


title: WT2 [L2547_H2O2]


title: WT1 [L2546_H2O2]


title: WT2 [L2248]


title: pn_sac2


title: pn_sac1


title: WT1 [L2244]


title: fen1_RNA_rep1

status: Public on May 28 2016

city: Beijing

first: Jinkui

last: Cheng

address: No.2 Yuanmingyuan West Road

institute: China Agricultural University

data_processing:
For ChIP-seq analysis, reads were mapped to the Arabidopsis genome (TAIR10) using bowtie1 (version 0.12.9) allowing two nucleotide mismatches using following parameters: bowtie -q -k 1 -n 2 -l 36 --best -S -p 2. Mapped reads were de-duplicated and sorted by samtools (version 0.1.19-44428cd)  Peaks were found using MACS software (version 1.4.2) using H3 as a background. For H3K27me3 peaks finding in the wild type as an example, the parameters were as follow: macs14  -t WT_K27ME3.sorted.bam –c TWT_H3.sorted.bam –f  BAM -g 1.30e+8  -n WT_K27ME3 --shiftsize 73 --pvalue 1e-5 --bw=300 --mfold=10,30.TDF files were generated using igvtools count for IGV visualization.
For RNA-seq process, paired-end reads were selected and aligned to the Arabidopsis reference genome (version: TAIR10), using TopHat (v2.1.1) and Cuffdiff (v2.1.1). The output accepted_hits.bam files were used to generate TDF files by igvtools count for IGV visualization.
Genome_build: tair10

organism: Arabidopsis thaliana

source_name: young seedlings

growth_protocol: Seeds were grown on MS medium plates supplemented with 20 g/L sucrose and 0.8% agar at 22°C under long-day (23 h) illumination

extract_protocol:
For RNA-seq, total RNAs extracted from seedlings using the RNeasy Plant Mini Kit (QIAGEN). For ChIP-seq,A 2-g sample of the 10-day-old seedlings was crosslinked with 1% formaldehyde. Chromatin were isolated using nuclei extraction buffer (1 M hexylene glycol, 20 mM Tris-HCl (pH 8.0), 0.15 mM spermine, 5 mM 2-mercaptoethanol, 1% Triton X-100, 0.1 mM PMSF, and cocktail) and precipitated by centrifuging at 2,000 × g for 10 min at 4°C. After resuspension in 300 μL of nuclei lysis buffer (50 mM Tris-HCl (pH 8.0), 10 mM EDTA, 1% sodium dodecyl sulfate [SDS] and cocktail) at 4°C, the chromatin was sonicated into fragments of approximately 300 bp using a Bioruptor (15 cycles of 30 s on and 30 s off at high intensity)
For ChIP-seq library, a 10-ng quantity of ChIP or input DNA was used to prepare a high-throughput sequencing library. End repair, dA-tailing, adapter ligation, and amplification were carried out using the NEBNext® ChIP-Seq Library Prep Master Mix Set for Illumina (E6240, NEB) according to the manufacturer’s protocol. Index primers used for each sample were as follows: AGTCAA for TWT-H3K27me3; GTCCGC for TWT-H3K4me3; AGTTCC for TWT-H3K9me2; ATGTCA for TWT-H3; CCGTCC for TWT-Input; GTGGCC for fen-H3K27me3; GTGAAA for fen-H3K4me3; GTTTCG for fen-H3K9me2; CGTACG for fen-H3; and GAGTGG for fen-Input.

treatment_protocol: No treatment

tag: developmental stage

value: 10-day old whole seedlings

tag: ecotype

value: fen1 mutant


title: fen1_RNA_rep2


title: TWT_RNA_rep1

value: transgenic wild type


title: TWT_RNA_rep2


title: pn_sac3

value: brain

value: cerebral cortex


title: RNA_L2

city: Daejeon

first: Yujin

last: Jeong

address: 291 Daehak-ro

country: South Korea

institute: KAIST

data_processing:
CLC Genomics workbench 6.5 (CLC bio) was used for data trimming and read mapping.
The linker sequence was trimmed from reads of the Ribo-seq libraries before being aligned to the genome. Reads which were shorter than 25 bp after trimming or did not contain the linker sequence were discarded. Also, the random 3’-overhanging (N9) sequences in reads of dRNA-seq library were trimmed. Reads shorter than 25 bp after trimming were discarded.
The reads were aligned to the S. coelicolor genome (NC_003888) with the following parameters: mismatch cost 2, deletion cost 3, insertion cost 3, length fraction 0.9, and similarity fraction 0.9.
Only uniquely mapped reads were retained.
The expression of the genes was normalized using the DESeq package in R.
Because the sequences of the first 20 genes and the last 20 genes of S. coelicolor are duplications, those 40 genes were excluded from all analysis.
Genomic positions of the 5’-ends of uniquely aligned dRNA-seq reads were considered to be potential TSSs. Only TSSs present in both TEX+ and TEX- libraries (±5 bp) were retained. Potential TSSs within 100 bp were clustered together; they were then sub-clustered with standard deviation <10. Potential TSS with maximum reads in a sub-cluster was selected as the primary TSS. If a cluster had several TSSs, standard deviation of two adjacent TSSs was calculated. If calculated standard deviation was less than 10, the one with lower reads was removed.
Genome_build: NC_003888
Supplementary_files_format_and_content: tab-delimited text files include TSS or normalized expression values for each sample

organism: Streptomyces coelicolor

source_name: cell culture

extract_protocol:
The mycelium was resuspended in 500 μl lysis buffer composed of 20 mM Tris-HCl (pH 7.4), 140 mM NaCl, 5 mM MgCl2, and 1% Triton X-100. Resuspended cells were dripped into liquid nitrogen and then ground with pestle and mortar. The powdered cells were thawed and centrifuged at 4 °C for 5 min at 3,000 g to remove cell debris. The supernatant was recovered and centrifuged at 16,000 g for 10 min to clarify supernatant. total RNA was isolated using miRNeasy Mini kit (Qiagen) in accordance with manufacturer’s instructions.
Genomic DNA was removed by using DNA-freeTM Kit (Ambion), and rRNA was removed by using Ribo-ZeroTM rRNA Removal Kit for Meta-bacteria (Epicentre). 200 ng mRNA was then fragmented by 10× Fragmentation buffer (Ambion). First strand cDNA was synthesized with random primers (Invitrogen), then second strand cDNA was synthesized using dUTP instead of dTTP. The library for Illumina sequencing was constructed using TruSeqTM DNA Sample Prep Kit (Illumina) according to the manufacturer’s instructions with a modification: degradation of the second strand which contains dUTP by treating USER enzyme (NEB) to the library.


title: RNA_T2


title: Ribo_L

data_processing:
Library strategy: Ribo-seq
CLC Genomics workbench 6.5 (CLC bio) was used for data trimming and read mapping.
The linker sequence was trimmed from reads of the Ribo-seq libraries before being aligned to the genome. Reads which were shorter than 25 bp after trimming or did not contain the linker sequence were discarded. Also, the random 3’-overhanging (N9) sequences in reads of dRNA-seq library were trimmed. Reads shorter than 25 bp after trimming were discarded.
The reads were aligned to the S. coelicolor genome (NC_003888) with the following parameters: mismatch cost 2, deletion cost 3, insertion cost 3, length fraction 0.9, and similarity fraction 0.9.
Only uniquely mapped reads were retained.
The expression of the genes was normalized using the DESeq package in R.
Because the sequences of the first 20 genes and the last 20 genes of S. coelicolor are duplications, those 40 genes were excluded from all analysis.
Genomic positions of the 5’-ends of uniquely aligned dRNA-seq reads were considered to be potential TSSs. Only TSSs present in both TEX+ and TEX- libraries (±5 bp) were retained. Potential TSSs within 100 bp were clustered together; they were then sub-clustered with standard deviation <10. Potential TSS with maximum reads in a sub-cluster was selected as the primary TSS. If a cluster had several TSSs, standard deviation of two adjacent TSSs was calculated. If calculated standard deviation was less than 10, the one with lower reads was removed.
Genome_build: NC_003888
Supplementary_files_format_and_content: tab-delimited text files include TSS or normalized expression values for each sample

extract_protocol:
The mycelium was washed in 500 μl polysome buffer composed of 20 mM Tris-HCl (pH 7.4), 140 mM NaCl, 5 mM MgCl2, and 20 μM thiostrepton. The cells were then resuspended in the lysis buffer with 20 μM thiostrepton and dripped into liquid nitrogen and then ground with pestle and mortar. The powdered cells were thawed and centrifuged at 4 °C for 5 min at 3,000 g to remove cell debris. The supernatant was recovered and centrifuged at 16,000 g for 10 min to clarify supernatant. To digest RNA, 750 U of RNase I (Ambion) was added to the cell lysate containing 50 μg total RNA in 300 μl of 10 mM Tris-HCl (pH7.6). The samples were incubated at 37 °C for 45 min with gentle rotation, followed by addition of 200 U of SUPERase-InTM (Invitrogen). The digested samples were carefully pipetted onto 900 μl of 1 M sucrose cushion (34% (w/v) sucrose) in a polycarbonate ultracentrifuge tube (13 mm × 51 mm, Beckman). Ribosomes were pelleted by centrifugation with TLA100.2 rotor (Beckman) at 215,000 g for 4 hrs at 4 °C, and subsequently resuspended in 700 μl of RLT buffer (Qiagen). RNA was purified by phenol-chloroform extraction and ethanol precipitation.
rRNA was removed by using Ribo-ZeroTM rRNA Removal Kit for Meta-bacteria (Epicentre). The ribosome-protected RNA fragments were separated by electrophoresis using 15% polyacrylamide TBE-urea gel (Invitrogen). A fraction of the fragment between 26 bp and 32 bp was size-selected. Size-selected RNA was then dephosphorylated with T4 PNK (NEB). Universal miRNA Cloning Linker (NEB) was ligated to the RNA. Linker-ligated RNA was reverse transcribed with modified reverse transcription primer. cDNA was then purified by ethanol precipitation and separated from the unextended primer by polyacrylamide gel electrophoresis. Purified cDNA was circularized by using CircLigaseTM ssDNA Ligase (Epicentre). The sequencing library was enriched from the circulated DNA by PCR. PCR amplification was performed with varying numbers of cycles and the optimal cycle was determined from the result. The amplified library was separated from the unextended primers by electrophoresis using 8% polyacrylamide gel.


title: Ribo_M

molecule: other


title: Ribo_S


title: Ribo_T


title: RNA_M1


title: TSS_TEX

data_processing:
Library strategy: differential RNA-seq
CLC Genomics workbench 6.5 (CLC bio) was used for data trimming and read mapping.
The linker sequence was trimmed from reads of the Ribo-seq libraries before being aligned to the genome. Reads which were shorter than 25 bp after trimming or did not contain the linker sequence were discarded. Also, the random 3’-overhanging (N9) sequences in reads of dRNA-seq library were trimmed. Reads shorter than 25 bp after trimming were discarded.
The reads were aligned to the S. coelicolor genome (NC_003888) with the following parameters: mismatch cost 2, deletion cost 3, insertion cost 3, length fraction 0.9, and similarity fraction 0.9.
Only uniquely mapped reads were retained.
The expression of the genes was normalized using the DESeq package in R.
Because the sequences of the first 20 genes and the last 20 genes of S. coelicolor are duplications, those 40 genes were excluded from all analysis.
Genomic positions of the 5’-ends of uniquely aligned dRNA-seq reads were considered to be potential TSSs. Only TSSs present in both TEX+ and TEX- libraries (±5 bp) were retained. Potential TSSs within 100 bp were clustered together; they were then sub-clustered with standard deviation <10. Potential TSS with maximum reads in a sub-cluster was selected as the primary TSS. If a cluster had several TSSs, standard deviation of two adjacent TSSs was calculated. If calculated standard deviation was less than 10, the one with lower reads was removed.
Genome_build: NC_003888
Supplementary_files_format_and_content: tab-delimited text files include TSS or normalized expression values for each sample

extract_protocol:
The mycelium was resuspended in 500 μl lysis buffer composed of 20 mM Tris-HCl (pH 7.4), 140 mM NaCl, 5 mM MgCl2, and 1% Triton X-100. Resuspended cells were dripped into liquid nitrogen and then ground with pestle and mortar. The powdered cells were thawed and centrifuged at 4 °C for 5 min at 3,000 g to remove cell debris. The supernatant was recovered and centrifuged at 16,000 g for 10 min to clarify supernatant. total RNA was isolated using miRNeasy Mini kit (Qiagen) in accordance with manufacturer’s instructions.
RNA samples from various conditions were pooled to make 10 μg of total RNA. Genomic DNA was removed by using DNA-freeTM Kit (Ambion), and rRNA was removed by using Ribo-ZeroTM rRNA Removal Kit for Meta-bacteria (Epicentre). Total mRNA was split into two samples for two different libraries: the library of the primary transcriptome and the library of whole transcriptome. TerminatorTM 5’-Phosphate-Dependent Exonuclease (TEX, Epicentre) was used to treat one of the samples to enrich primary transcripts which have triphosphate at their 5’-ends, resulting in the preparation of the TEX-treated (TEX+) and non-treated (TEX-) samples. The triphosphates at the 5’-ends of mRNA were converted to monophosphate by treating RNA 5’-polyphosphatase (Epicentre). The mRNA was then purified by phenol-chloroform extraction and ethanol precipitation. 5’-RNA adaptor was ligated to the purified mRNA, then cDNA was synthesized using random 3’ overhanging primer (N9). Synthesized cDNA was purified using QIAquick PCR Purification Kit (Qiagen). The cDNA was purified again by ethanol precipitation. Purified cDNA was selected at a size range between 100 bp and 350 bp on a 2% agarose gel by Pippin Prep (Sage Science). Then the purified sequencing library was amplified by PCR with indexed primers for Illumina sequencing platform. After the enriched library was purified by ethanol precipitation, the library of size between 150 bp and 400 bp was extracted. The size-selected library was purified by ethanol precipitation. A second PCR amplification was carried out with a few PCR cycles to produce enough amount of library for Illumina sequencing. The final amplified library was purified by ethanol precipitation and those of size 150 bp to 400 bp were extracted from 2% agarose gel after electrophoresis. The final library was then purified using MinElute Gel Extraction Kit.


title: fen1_H3

tag: chip-antibodies


title: RNA_L1


title: RNA_T1


title: RNA_S1


title: TWT_H3


title: fen1_H3K27me3


title: fen1_H3K4me3


title: RNA_S2


title: RNA_M2


title: ago4-1

first: Xiuying

last: Liu

address: Yard 28, Science Park Road, Changping District

institute: Changping Laboratory

description:
ago4-1
Bisulfite conversion with EpiTect Kit (Qiagen)

data_processing:
Reads alignment with Bismark version 0.14.3
Deduplicate for alignment results
Extract methylation information with bismark_methylation_extractor
Generated wig files through swDMR version 1.0.0
wig files are UCSC specified, which can be visualized in IGV (Integrative Genomics Viewer) browser (http://www.broadinstitute.org/igv/)
Genome_build: TAIR10
Supplementary_files_format_and_content: Wig files in AGOs_BS-Seq folder are DMRs fragment loci on genome, containing Chromosome  Start End information.

source_name: 4-week plants

growth_protocol: Surface sterilized seeds were plated in MS media + 1% Sucrose, and grown under short day conditions (16h dark/8h light) for 11 days.

extract_protocol:
The plants were collected in liquid nitrogen, ground with morthat and pestle, and DNA extracted in CTAB, Rnase treated and purified with phenol:chloroform followed by Ethanol precipitation.
Bisulfite conversion with EpiTect Kit (Qiagen); Approximately 1 ug of genomic DNA were used to generate libraries using premethylated adapters (NEXTFlex Bisulfite-Seq Adapters, Bioo Scientific #511911). The adaptor-ligated fragments were purified by QIAQuick column (QIAGEN) and bisulfite converted using the EpiTect Kit (Qiagen) following the manufacturer’s instructions. The converted DNA was later enriched by 15 cycles of PCR using the Pfu Turbo Cx Polymerase (Agilent), using the specific primers provided by Bios Scientific for enrichment. The library was finally purified with Agencourt AMPure XP beads (Beckman Coulter) according to manufacturer’s instruction.

value: Landsberg erecta

value: 4 weeks old

value: aerial part


title: TSS_TAP


title: fen1_INPUT


title: fen1_H3K9me2


title: TWT_INPUT


title: TWT_H3K27me3


title: Bisulfite-Seq analysis of WGBS_Lib 41 derived from human substantia nigra cells; WGBS_Lib 41

status: Public on May 29 2016

first: BROAD

last: INSTITUTE

state: MA

institute: Broad Institute

description:
sample_term_id: UBERON_0002038
assay_term_id: OBI_0001863
nucleic_acid_term_id: SO_0000352
Design description: WGBS REMC Sequencing on Illumina
Library name: WGBS_Lib 41
EDACC Genboree Experiment Page:
http://genboree.org/java-bin/project.jsp?projectName=XML%20Submissions%2FBroad%2FEXPERIMENT%2FEDACC.16340
EDACC Genboree Sample Page:
http://genboree.org/java-bin/project.jsp?projectName=XML%20Submissions%2FBroad%2FSAMPLE%2FEDACC.5938
****************
For data usage terms and conditions, please refer to:
http://www.drugabuse.gov/funding/funding-opportunities/nih-common-fund/epigenomics-data-access-policies
****************

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

source_name: human post mortem brain region from a non Alzheimer's donor; WGBS_Lib 41

extract_protocol: Library construction protocol: Genomic DNA (1.5~5µg) was fragmented to 100-500 bp using a Covaris S2. Purified DNA fragments were end-repaired. After A-tailing, the DNA fragments were ligated with methylated paired-end adapters. Adapter-attached DNA fragments of 300-400 bp, which contain 150-250 bp genomic DNA inserts, were gel-purified. The purified DNA fragments were subjected to two cycles of sodium bisulfite conversion using the EpiTect Bisulfite kit (Qiagen). Adapter-attached, bisulfite-converted DNA molecules were enriched by PCR using PfuTurboCx Hotstart DNA polymerase (Stratagene). The PCR amplified DNA fragments were subjected to a second gel size selection to remove PCR primers and adapter dimers. The enriched library was quantified using a Qubit fluorometer and Quant-iT dsDNA HS Assay Kit. Library sequencing was performed using 101 BP PE Illumina technology.

tag: sra sample accession

value: post-mortem

tag: disease

value: Substantia Nigra

value: Female

value: Primary Tissue

value: Rush University Medical Center

value: 2x5hrs Epitect Kit

value: 5 ug

value: Covaris shearing, duty cycle 5%, intensity 5, cycles / burst 200, duration 9 minutes

value: T4 Ligase (2,000U/ul) 16C o/n

value: Illumina paired end adapter

value: Standard Protocol (Smith et al., Methods 48, 226-232)

value: DNA Methylation

value: Smith et al., Methods 48, 226-232

value: SPRI Beads

value: Pfu Turbo Cx hot start

value: Covaris S2 (TM)


title: TWT_H3K4me3

value: 10-day old whole seedlings

value: transgenic wild type


title: H295R_G2

city: Budapest

first: Vince

middle: Kornél

last: Grolmusz

address: Szentkirályi 46.

country: Hungary

institute: Semmelweis University

description:
PO3
processed_data.xlsx

data_processing:
Filtering low quality tags after that,  are used on samples FASTX-toolkit and Collapse tool in GALAXY workbench for collapsing identical sequences in FASTA files
Getting rid of low quality reads   Getting rid of reads without 3' primer   Getting rid of reads with 5' primer contaminants   Getting rid of reads with poly A   Getting rid of reads shorter than 18nt
Alignment to the genome(with Bowtie) and to mature miRna databases (with Suremir) of three samples in G1,S and G2 phases
Bowtie and Suremir
Running LCS algorithm for aligning the reads from PO1, PO2,PO3,L4,L5,L6,L25,L26,L27 samples with max 1 mismatch to mature miRNA 21 database
(It is executed also align to Rfam database with blast/bowtie, screen and remove rRNA, scRNA, snoRNA, snRNA, tRNA associated tags
Filtering the result from Suremir for unique alignment
preprocess_mismatch_matching_result
Calculating correlation between samples and between the results in Bowtie and Suremir.
R scripts (ex:plots_cross_corr.r), Venny 2.0.2 online tool
edgeR analyis on G1,S and G2 phases for differentially expressed miRna
R scripts using R version.3.1.2, edgeR_3.8.6, limma_3_22_7
Genome_build: mature mirbase v21,Genbank
Supplementary_files_format_and_content: .xls file (raw counts) for differently expressed miRNAs

extract_protocol:
Total RNA was isolated using miRNesay Mini Kit (Qiagen), accordig to the manufacturer's instructions
For library preparation TruSeq Small RNA library preparation kit (Illumina, San Diego, California) was used.

tag: cell line

tag: cell cycle phase


title: H295R_S

description:
PO2
processed_data.xlsx

value: S


title: HeLa_G2_2

description:
L27
processed_data.xlsx

source_name: HeLa

value: HeLa


title: HeLa_S_2

description:
L26
processed_data.xlsx


title: H295R_G1

description:
PO1
processed_data.xlsx


title: HeLa_S_1

description:
L5
processed_data.xlsx


title: TWT_H3K9me2


title: HeLa_G1_1

description:
L4
processed_data.xlsx


title: HeLa_G2_1

description:
L6
processed_data.xlsx


title: DPRX rep 3

status: Public on May 27 2016

city: Oxford

first: Thomas

last: Dunwell

address: South Parks Road

department: Zoology

institute: University of Oxford

description: Libraries for all samples were combined in a 15 samples multiplex which were then split across two lanes.

data_processing:
Reads for each sample were combined and aligned to the reference hg38 genome using the STAR genome aligner with default commands with the addition of --outFilterMultimapNmax 15 --outSAMstrandField intronMotif
Raw read counts for refseq annotated genes were generated using featurecounts using gene annotations from GRCh38.p2
Genome_build: hg38
Supplementary_files_format_and_content: tab-delimited text file including default output from featurecounts

growth_protocol: All cells were grown at 37oC in a 5% CO2 humidified incubator on poly-l-lysine coated plastic in Fibroblast Medium (Sciencell #2301)

extract_protocol:
RNA was extracted using a RNeasy Mini Kit following the manufactures instructions with the addition of an on-column DNAse treatment.
RNA libraries were prepared for sequencing using standard Illumina protocols
RNA-Seq; each library split across two lanes HiSeq4000, 15 half-libraries per lane

value: Human Adult Dermal Fibroblasts (HDF-a)

tag: transfected with

value: DPRX


title: TPRX1 rep 1


title: LEUTX rep 2

value: LEUTX


title: ARGFX rep 2

value: ARGFX


title: ARGFX rep 3


title: DPRX rep 2


title: DPRX rep 1


title: ARGFX rep 1


title: TPRX1 rep 2


title: shMyb

city: Mobile

first: Ajay

middle: P

last: Singh

state: Alabama

address: 1660 Springhill Ave

department: Oncologic Sciences

institute: University of South Alabama Mitchell Cancer Institute

data_processing:
RNA-sequencing and bioinformatics analysis were performed at the Genomics Core Facility at the Heflin Center for Genetics, University of Alabama at Birmingham as described by Jiang et al., PlosOne (2013)
Illumina Casava v1.8.2  software used for basecalling.
TopHat was used to align the raw RNA-Seq fastq reads to the human hg19 genome using the short read aligner Bowtie.  Cufflinks was used to align the reads from TopHat to assemble transcripts, estimate their abundances and test for differential expression and regulation.
Genome_build: hg19
Supplementary_files_format_and_content: .txt file including Scr and shMyb abundance measurements, Scr vs shMyb fold change, log2 (fold_change) and p-value values.

source_name: MiaPaCa

growth_protocol: MiaPaCa cells were maintained as monolayer cultures in RPMI 1640 media supplemented with 10% fetal bovine serum and 100 µM each of penicillin and streptomycin in a humidified atmosphere of 5% CO2 at 370C.

extract_protocol:
Total RNA was isolated using RNeasy Purification Kit.
RNA libraries were prepared and sequencing was performed using standard Illumina protocols.

treatment_protocol: MiaPaCa cells were transfected with short hairpin RNA (shRNA) expression constructs for Myb (pGFP-V-RS-shMYB) along with control plasmids (pGFP-V-RS-Scr), using FuGENE as a transfection reagent. Stable pooled population of transfected cells were selected in RPMI-media containing Puromycin (2 μg/ml), expanded and examined for stable MYB silencing.

tag: cancer type

value: Pancreatic cancer

value: MiaPaCa

value: MYB-silenced


title: Control rep 3

value: empty control vector


title: Control rep 2


title: LEUTX rep 3


title: Scr

value: MYB-overexpression


title: HeLa_G1_2

description:
L25
processed_data.xlsx


title: TPRX1 rep 3


title: LEUTX rep 1


title: S. aureus Mu50 in single species biofilms_replicate M

city: Ghent

first: Ilse

last: Vandecandelaere

address: Ottergemsesteenweg 460

department: Pharmaceutical Sciences

country: Belgium

institute: Ghent University

description: M vs. EM.xls

data_processing:
illumina base-calling
all data were analysed according to the manufacturer's instructions of the CLC Genomic Workbench 8.5.1
reads were mapped to the entire gene with 100 % similarity and no mapping to the flanking regions was allowed
RPKM values were calculated and the p value was determined using edgeR statistics
Supplementary_files_format_and_content: M vs. EM.xls and E vs. EM.xls summarized the gene expression levels in dual species biofilms compared to single species biofilms

organism: Staphylococcus aureus

source_name: single species biofilms

growth_protocol: Single and dual species biofilms of S. epidermidis ET-024 and S. aureus Mu50 were grown in mueller hinton broth for 24 hours at 37°C.

extract_protocol:
RiboPure RNA purification Bacteria Kit
TruSeq stranded library preparation Kit (Illumina)

tag: sample type

value: S. aureus Mu50


title: RAS2 H4 ChIP-seq

city: Glasgow

first: Peter

last: Adams

address: Switchback Rd, Bearsden

institute: University of Glasgow, Beatson Institute for Cancer Research

data_processing:
Basecalls performed using CASAVA 1.8.2
Sequenced reads were trimmed using trim galore v0.3.0 with parameter --quality 20 and remaining default settings
Sequenced reads were mapped to hg19 whole genome using bowtie v2.1.0 under default settings
Duplicate reads removed using picard tools v1.98 mark duplicates
Peak finding performed by SICER v1.1 (normalised to histone H4) using redundancy threshold of 1, window size of 200bp, fragment size of 150bp, effective genome fraction of 0.75, gap size of 200bp and R of 0.01.
Peaks from both antibodies were intersected.
Differential peaks were called using Diffbind (v1.8.3)
Genome_build: hg19
Supplementary_files_format_and_content: Peak files (bed format) were generated using SICER, Differential region file (bed format) was generated using DESeq

source_name: IMR90 fibroblasts

growth_protocol: IMR90 cells were cultured in DMEM supplemented with 20% (v/v) FBS, 2 mM L-Glutamine, 50 U/ml Pen-Strep and incubated at 37°C in a humidified 5% CO2 and 3% O2 atmosphere. Every 3-4 days the cells were passaged to maintain 40%–90% confluence level until they stopped proliferating through replicative senescence.

extract_protocol:
Cross-linked cells were pre-extracted with (1:1) modified nuclear lysis buffer (mNLB):IP dilution buffer (IPDB) [35 mM Tris-HCl pH 8.0, 75 mM NaCl, 5.5 mM EDTA pH 8.0, 3 mM EGTA pH 8.0, 0.5% SDS, 0.5% Triton X-100] supplemented with 10 μg/ml aprotinin, 5 μg/ml leupeptin, 50 μg/ml PMSF and sonicated at a density of 2 x 107 cells per 1 ml cold (1:1) mNLB:IPDB plus inhibitors. Sonicated chromatin solutions were cleared by centrifugation, diluted with IPDB [20 mM Tris-HCl pH 8.1, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.01% SDS] to a final mNLB:IPDB ratio of 1:10, transferred to microcentrifuge tubes containing antibodies (from Millipore (04-079) and Cell Signaling Technology (5737)) pre-bound to Dynabeads M-280 Sheep anti-Rabbit IgG magnetic beads (Life Technologies), and incubated overnight at 4° C with rotation. The ChIP reactions were washed twice with IPDB, once with High Salt Wash Buffer [20 mM Tris-HCl pH 8.0, 500 mM NaCl, 2 mM EDTA, 0.1% SDS, 1% Triton X- 100], once with LiCl Wash Buffer [10 mM Tris-HCl pH 8.1, 250 mM LiCl, 1 mM EDTA, 1% NP-40, 1% deoxycholic acid] and twice with 1X TE. Beads were aspirated to dryness, resuspended in 500 μl IP Elution Buffer [50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM EDTA pH 8.0, 1% SDS] and 0.5 μl 100 mg/ml RNase A and incubated at 65° C for 4-6 hours. To each tube, 6 μl 20 mg/ml proteinase K was added and the tubes were incubated at 45° C for 12 hours. ChIP DNA was purified usingQIAquick PCR purification kit (50), resuspended with 30 μl buffer (TE) and quantified using the Qubit dsDNA HS Assay Kit and a Qubit fluorometer (Life Technologies).
Libraries were prepared according to NEB's instructions accompanying the NEBNext® Ultra DNA library prep kit for Illumina®(E7370S/L). Libraries were sequenced on the Genome Analyzer IIx following the manufacturer's protocols.

treatment_protocol: Proliferating IMR90 cells were infected with retroviruses containing empty pBABE-puro vectors (for proliferating control) and pBABE-H-RAS-G12V (to induce OIS). Cells were cultured and passaged for 12 days post infection, while under puromycin drug selection in complete growth medium. Cells were cultured to approximately 95% confluence in 100 mm cell culture dishes containing 10 ml growth medium. This corresponds to approximately 4-5 million proliferating cells or 2 x million senescent cells per 100 mm culture dish. Cells were subjected to cross- linking in the presence of 1% formaldehyde by the addition of 625 μl of 16% formaldehyde (Thermoscientific) to 10 ml of growth medium. Cross-linking was conducted for 10 minutes at room temperature, with gentle agitation on a rocking platform. The cross-linking reaction was quenched for 5 minutes at room temperature by the addition of 500 μl 2.5 M glycine to a final concentration of 125 mM. Cells were detached from the culture dishes using plastic cell scrapers and collected in 50 ml centrifuge tubes kept on ice. Cells were subjected to centrifugation for 5 minutes at 200 x g and 4° C. The supernatant was removed by aspiration, the cell pellets were washed with cold 1X PBS and subjected to centrifugation for an additional 5 minutes at 200 x g and 4° C. The supernatant was removed by aspiration, cell pellets were snap frozen with dry ice and ethanol and stored at -80° C.

value: anti-H4. Millipore, 05-858, rabbit mAb


title: RAS1 H4 ChIP-seq


title: Control2 H4 ChIP-seq


title: RAS2 H4K20me3 ChIP-seq

value: anti-H4K20me3, Millipore, 04-079, rabbit mAb


title: Control rep 1


title: Control1 H4 ChIP-seq


title: RAS1 H4K20me3 ChIP-seq


title: Control2 H4K20me3 ChIP-seq


title: S. aureus Mu50 in single species biofilms_replicate M2

description: M vs. EM.xls


title: S. aureus Mu50 in single species biofilms_replicate M1

description: M vs. EM.xls


title: Term_H3K4me3_rep1_input

city: Iowa City

first: Jennifer

middle: Rachelle

last: Bermick

state: IA

address: 431 Newton Road, 505 EMRB

department: Pediatrics

institute: University of Iowa

description: sample comprised of 3 combined patient samples (38 week male, 37.14 week female, 41 week male)

data_processing:
Adapter sequences were trimmed using Scythe version 1
ChIP-seq reads were aligned to the hg19 genome using Bowtie2 version 2.0.0 using default parameters
Peak calling was performed by comparing the ChIP samples to input samples with MACS2 version 2.0.10.2 using default parameters or Homer v3.12.
Genome_build: hg19
Supplementary_files_format_and_content: Processed data files were generated with MACS2 version 2.0.10.2 or Homer v3.12.  All peak files are available on ChIP samples.

source_name: umbilical cord blood

extract_protocol:
Lysates were clarified from sonicated nuclei and histone-DNA complexes were isolated with antibody.
DNA was end-repaired using a combination of T4 polynucleotide kinase and T4 DNA polymerase. The blunt phosphorylated ends were treated with Klenow fragment (3' to 5' exo-) and dATP to yield a single 3' A to blunt DNA for ligation of Illumina adapters with a single 3' T overhang. After adapter ligation DNA was PCR amplified with Illumina adapters for 19 cycles and library fragments of ~250 bp (insert plus adapter and PCR primer) were selected by bead purification. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Genome Analyzer following the manufacturer's instructions.

tag: tissue

value: umbilical cord blood

value: CD14+ monocytes

tag: isolation

value: Miltenyi Biotec CD14+ magnetic bead isolation

tag: age

value: over 37 weeks term neonate


title: Adult_H3K4me3_rep2_input

description: 24 year old female

source_name: peripheral blood

value: peripheral blood

value: Adult


title: Control1 H4K20me3 ChIP-seq

tag: chip antibody


title: Adult_H3K4me3_rep1

description: 33 year old female


title: Term_H3K4me3_rep3

description: sample comprised of 3 combined patient samples (40.43 week female, 41.14 week male, 39.43 week male)


title: Adult_H3K4me3_rep2

description: 24 year old female


title: O30_H3K4me3_rep3_input

description: sample comprised of 3 combined patient samples (33.43 week female, 34 week female, 31.71 week male)

value: 30-36 weeks gestation preterm neonate


title: Adult_H3K4me3_rep1_input

description: 33 year old female


title: Term_H3K4me3_rep3_input


title: Term_H3K4me3_rep2_input

description: sample comprised of 3 combined patient samples (41 week male, 39.86 week female, 41.14 week male)


title: Adult_H3K4me3_rep3_input

description: 44 year old male


title: O30_H3K4me3_rep2_input

description: sample comprised of 3 combined patient samples (33.43 week male, 34.29 week female, 32.43 week male)


title: Term_H3K4me3_rep2


title: pp_nic3

value: brain

tag: tissue compartment

value: cerebral cortex

tag: strain

value: 3 month old

value: H3K4me3 (Millipore, 06-598, lot JBC940052)


title: Adult_H3K4me3_rep3

description: 44 year old male

tag: cell type


title: Term_H3K4me3_rep1


title: O30_H3K4me3_rep2


title: O30_H3K4me3_rep1_input

description: sample comprised of 3 combined patient samples (31.43 week female, 31.43 week female, 30.71 week male)


title: U30_H3K4me3_rep3_input

description: sample comprised of 3 combined patient samples (29.29 week male, 28.14 week male, 28.14 week female)

value: under 30 weeks gestation preterm neonate


title: U30_H3K4me3_rep2_input

description: sample comprised of 3 combined patient samples (29.29 week male, 29.14 week female, 28.14 week female)


title: U30_H3K4me3_rep2


title: U30_H3K4me3_rep1_input

description: sample comprised of 3 combined patient samples (24.86 week female, 24 week male, 29.14 week male)


title: U30_H3K4me3_rep1


title: O30_H3K4me3_rep3


title: abl vehicle total input

city: Boston

first: Anna

middle: C

last: Groner

address: 450 Brookline Ave

department: Medical Oncology

institute: Dana-Farber Cancer Institute

data_processing:
Basecalls were performed with FastQC version 0.10.0.
ChIP-seq reads were aligned to the hg19 genome assembly using Bowtie version 0.12.7.
Data were filtered according to mappable reads with 2 mismatches at most allowed.
Peaks were called using MACS version 2.0 with the following settings: band width (300), model fold (5, 50), qvalue cut-off (1*10e-2), range for calculating regional lambda (1000bps and 10000bps)
Genome_build: hg19
Supplementary_files_format_and_content: bed files were generated using MACS version 2.0, scores represent -LOG10(qvalue)

source_name: LNCaP-abl

growth_protocol: Routinely, LNCaP cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), penicillin (100 μg/ml), streptomycin (100 units/ml) and L-glutamine (2 mM), at 37 °C with an atmosphere of 5% CO2. For experimental conditions, cells were grown for 72h under hormone-starvation conditions using the media conditions described above but subsituting FBS for charcoal/dextran-treated FBS (CDT). Cells were then treated with vehicle (ETOH) or 10 nM DHT for 4h. LNCaP-abl cells were always cultured with CDT.

extract_protocol:
Lysates were clarified from sonicated cells and chromatin-DNA complexes were isolated with the indicated antibody.
Library preparation was done with the ThruPLEX-FD Prep Kit from Rubicon Genomics (R40048), which results in ChIP-seq libraries compatible with Illumina NGS Platforms. The Illumina Hiseq-2000 platform was used for sequencing.

treatment_protocol: LNCaP cells were stimulated with 10nM DHT or vehicle for 4h, while LNCaP-abl cells were only treated with vehicle for 4h.

value: LNCaP-abl

tag: treatment

value: vehicle (ETOH)


title: abl vehicle TRIM24 ChIP-seq

value: TRIM24 (Bethyl, A300-815A)


title: LNCaP vehicle total input

source_name: LNCaP

value: LNCaP


title: LNCaP DHT total input

value: 10nM DHT


city: San Francisco

first: UCSF-UBC

last: CENTER

state: CA

address: UCSF-UBC

institute: UCSF-UBC

description:
design description: miRNA-Seq analysis of breast myoepithelial cell from RM084 (m08315) using Illumina HiSeq 2000
library name: m08315
EXPERIMENT_TYPE: smRNA-Seq
EXTRACTION_PROTOCOL: Total RNA was extracted using Trizol from Invitrogen as per manufacturer's instructions.
LIBRARY_GENERATION_PCR_POLYMERASE_TYPE: Phusion DNA Polymerase (Hot Start), 2U/ul (from NEB)
LIBRARY_GENERATION_PCR_THERMOCYCLING_PROGRAM: step1: 98C for 30sec, step2: 98C for 10sec, step3: 60C for 30sec, step4: 72C for 15sec.  Repeat step2-4 for 15 cycles, 72C for 5min.
LIBRARY_GENERATION_PCR_NUMBER_CYCLES: 15
LIBRARY_GENERATION_PCR_F_PRIMER_SEQUENCE: 5' AATGATACGGCGACCACCGACAGNNNNNNGTTCAGAGTTCTACAGTCCGA 3'
LIBRARY_GENERATION_PCR_R_PRIMER_SEQUENCE: 5' CAAGCAGAAGACGGCATACGAGAT 3'
LIBRARY_GENERATION_PCR_PRIMER_CONC: 25 uM
LIBRARY_GENERATION_PCR_PRODUCT_ISOLATION_PROTOCOL: 28uL of PCR products containing unique index sequences are pooled together and the 115bp miRNA containing fraction is isolated for each pool either manually:  12% PAGE gel 200V 6hours followed by gel elution, or robotically using Baraccuda size selection robot.  Fractions are Et0H preciptated, QCed on Agilent, and submitted for indexing single end (ISE) Illumina sequencing.
EXTRACTION_PROTOCOL_SMRNA_ENRICHMENT: Flowthrough of polyA+ MultiMACS 96 Separation Unit
SMRNA_PREPARATION_INITIAL_SMRNA_QLTY: RIN 6.8
SMRNA_PREPARATION_INITIAL_SMRNA_QNTY: 1.209 ug
RNA_PREPARATION_5'_RNA_ADAPTER_SEQUENCE: 5' GTTCAGAGTTCTACAGTCCGACGATCTGGTCAA 3'
RNA_PREPARATION_3'_RNA_ADAPTER_SEQUENCE: 5' ATCTCGTATGCCGTCTTCTGCTTGT 3'
RNA_PREPARATION_REVERSE_TRANSCRIPTION_PRIMER_SEQUENCE: 5' CAAGCAGAAGACGGCATACGAGAT 3'
RNA_PREPARATION_3'_RNA_ADAPTER_LIGATION_PROTOCOL: 1ug of total RNA (RN =>7.0) in a 4uL volume and 2uL of 3' Adenylated Adapter was heat denatured at 70C for 2min then snap chilled on ice for 1min.  Then 1uL of 10X T4 RNL2 truncated buffer, 0.8uL 100mM MgCl2, 0.2uL DEPC water, 0.5uL Rnase Out, and 1.5uL T4 RNA Ligase 2 trancated was added.  Ligation incubated at 22C in a Tetrad thermocycler for 60min.
RNA_PREPARATION_5'_RNA_ADAPTER_LIGATION_PROTOCOL: 5' RNA adapter was heat denatured at 70C for 2min then snap chilled on ice.  2uL of denatured 5' RNA Adapter was added to the 3' DNA Adapter Ligation reaction product from previous step and mixed.   Then 1uL of 10mM ATP and 1uL of Ambion T4 RNA Ligase were added and ligation reaction was incubated at 37C in a Tetrad thermocycler for 60min.
RNA_PREPARATION_REVERSE_TRANSCRIPTION_PROTOCOL: To the 14uL of double adaptered miRNA product from previous step 2ul of RT-Primer was added and the mixture was heat denatured at 65C for 10 mins then quenched on ice.  The following reagents were added immediately to the sample (6ul of 5X First Strand Buffer, 2ul of 10mM dNTPs , 3ul of 100mM DTT, 1ul of RNaseOUT, and 2uL of SuperScript II RT.  Reaction was heated at 44C in a Thetrad thermocycler for 60 minutes.
LIBRARY_GENERATION_PCR_TEMPLATE: 15ul of the first strand cDNA product was used in the PCR
****************
For data usage terms and conditions, please refer to:
http://www.drugabuse.gov/funding/funding-opportunities/nih-common-fund/epigenomics-data-access-policies
****************

extract_protocol: library construction protocol: Refer to document 'miRNA3 - Plate Format miRNA Library Construction' from BC at the Roadmap Epigenomics Project site, Experimental Protocols page (URL: http://www.roadmapepigenomics.org/protocols/type/experimental/)

tag: submitted sample id

tag: Sex

value: female

tag: body site

value: Breast

tag: histological type

value: Myoepithelial cell

tag: is tumor

value: No

value: primary cell

value: breast myoepithelial cell


description:
design description: miRNA-Seq analysis of breast variant human mammary epithelial cell from RM071 (m08323) using Illumina HiSeq 2000
library name: m08323
EXPERIMENT_TYPE: smRNA-Seq
EXTRACTION_PROTOCOL: Total RNA was extracted using Trizol from Invitrogen as per manufacturer's instructions.
LIBRARY_GENERATION_PCR_POLYMERASE_TYPE: Phusion DNA Polymerase (Hot Start), 2U/ul (from NEB)
LIBRARY_GENERATION_PCR_THERMOCYCLING_PROGRAM: step1: 98C for 30sec, step2: 98C for 10sec, step3: 60C for 30sec, step4: 72C for 15sec.  Repeat step2-4 for 15 cycles, 72C for 5min.
LIBRARY_GENERATION_PCR_NUMBER_CYCLES: 15
LIBRARY_GENERATION_PCR_F_PRIMER_SEQUENCE: 5' AATGATACGGCGACCACCGACAGNNNNNNGTTCAGAGTTCTACAGTCCGA 3'
LIBRARY_GENERATION_PCR_R_PRIMER_SEQUENCE: 5' CAAGCAGAAGACGGCATACGAGAT 3'
LIBRARY_GENERATION_PCR_PRIMER_CONC: 25 uM
LIBRARY_GENERATION_PCR_PRODUCT_ISOLATION_PROTOCOL: 28uL of PCR products containing unique index sequences are pooled together and the 115bp miRNA containing fraction is isolated for each pool either manually:  12% PAGE gel 200V 6hours followed by gel elution, or robotically using Baraccuda size selection robot.  Fractions are Et0H preciptated, QCed on Agilent, and submitted for indexing single end (ISE) Illumina sequencing.
EXTRACTION_PROTOCOL_SMRNA_ENRICHMENT: Flowthrough of polyA+ MultiMACS 96 Separation Unit
SMRNA_PREPARATION_INITIAL_SMRNA_QLTY: RIN 8.2
SMRNA_PREPARATION_INITIAL_SMRNA_QNTY: 2.108 ug
RNA_PREPARATION_5'_RNA_ADAPTER_SEQUENCE: 5' GTTCAGAGTTCTACAGTCCGACGATCTGGTCAA 3'
RNA_PREPARATION_3'_RNA_ADAPTER_SEQUENCE: 5' ATCTCGTATGCCGTCTTCTGCTTGT 3'
RNA_PREPARATION_REVERSE_TRANSCRIPTION_PRIMER_SEQUENCE: 5' CAAGCAGAAGACGGCATACGAGAT 3'
RNA_PREPARATION_3'_RNA_ADAPTER_LIGATION_PROTOCOL: 1ug of total RNA (RN =>7.0) in a 4uL volume and 2uL of 3' Adenylated Adapter was heat denatured at 70C for 2min then snap chilled on ice for 1min.  Then 1uL of 10X T4 RNL2 truncated buffer, 0.8uL 100mM MgCl2, 0.2uL DEPC water, 0.5uL Rnase Out, and 1.5uL T4 RNA Ligase 2 trancated was added.  Ligation incubated at 22C in a Tetrad thermocycler for 60min.
RNA_PREPARATION_5'_RNA_ADAPTER_LIGATION_PROTOCOL: 5' RNA adapter was heat denatured at 70C for 2min then snap chilled on ice.  2uL of denatured 5' RNA Adapter was added to the 3' DNA Adapter Ligation reaction product from previous step and mixed.   Then 1uL of 10mM ATP and 1uL of Ambion T4 RNA Ligase were added and ligation reaction was incubated at 37C in a Tetrad thermocycler for 60min.
RNA_PREPARATION_REVERSE_TRANSCRIPTION_PROTOCOL: To the 14uL of double adaptered miRNA product from previous step 2ul of RT-Primer was added and the mixture was heat denatured at 65C for 10 mins then quenched on ice.  The following reagents were added immediately to the sample (6ul of 5X First Strand Buffer, 2ul of 10mM dNTPs , 3ul of 100mM DTT, 1ul of RNaseOUT, and 2uL of SuperScript II RT.  Reaction was heated at 44C in a Thetrad thermocycler for 60 minutes.
LIBRARY_GENERATION_PCR_TEMPLATE: 15ul of the first strand cDNA product was used in the PCR
****************
For data usage terms and conditions, please refer to:
http://www.drugabuse.gov/funding/funding-opportunities/nih-common-fund/epigenomics-data-access-policies
****************

value: Variant human mammary epithelial cell culture

value: primary cell culture


description:
design description: miRNA-Seq analysis of breast fibroblast from RM071 (m08317) using Illumina HiSeq 2000
library name: m08317
EXPERIMENT_TYPE: smRNA-Seq
EXTRACTION_PROTOCOL: Total RNA was extracted using Trizol from Invitrogen as per manufacturer's instructions.
LIBRARY_GENERATION_PCR_POLYMERASE_TYPE: Phusion DNA Polymerase (Hot Start), 2U/ul (from NEB)
LIBRARY_GENERATION_PCR_THERMOCYCLING_PROGRAM: step1: 98C for 30sec, step2: 98C for 10sec, step3: 60C for 30sec, step4: 72C for 15sec.  Repeat step2-4 for 15 cycles, 72C for 5min.
LIBRARY_GENERATION_PCR_NUMBER_CYCLES: 15
LIBRARY_GENERATION_PCR_F_PRIMER_SEQUENCE: 5' AATGATACGGCGACCACCGACAGNNNNNNGTTCAGAGTTCTACAGTCCGA 3'
LIBRARY_GENERATION_PCR_R_PRIMER_SEQUENCE: 5' CAAGCAGAAGACGGCATACGAGAT 3'
LIBRARY_GENERATION_PCR_PRIMER_CONC: 25 uM
LIBRARY_GENERATION_PCR_PRODUCT_ISOLATION_PROTOCOL: 28uL of PCR products containing unique index sequences are pooled together and the 115bp miRNA containing fraction is isolated for each pool either manually:  12% PAGE gel 200V 6hours followed by gel elution, or robotically using Baraccuda size selection robot.  Fractions are Et0H preciptated, QCed on Agilent, and submitted for indexing single end (ISE) Illumina sequencing.
EXTRACTION_PROTOCOL_SMRNA_ENRICHMENT: Flowthrough of polyA+ MultiMACS 96 Separation Unit
SMRNA_PREPARATION_INITIAL_SMRNA_QLTY: RIN 9
SMRNA_PREPARATION_INITIAL_SMRNA_QNTY: 1.320 ug
RNA_PREPARATION_5'_RNA_ADAPTER_SEQUENCE: 5' GTTCAGAGTTCTACAGTCCGACGATCTGGTCAA 3'
RNA_PREPARATION_3'_RNA_ADAPTER_SEQUENCE: 5' ATCTCGTATGCCGTCTTCTGCTTGT 3'
RNA_PREPARATION_REVERSE_TRANSCRIPTION_PRIMER_SEQUENCE: 5' CAAGCAGAAGACGGCATACGAGAT 3'
RNA_PREPARATION_3'_RNA_ADAPTER_LIGATION_PROTOCOL: 1ug of total RNA (RN =>7.0) in a 4uL volume and 2uL of 3' Adenylated Adapter was heat denatured at 70C for 2min then snap chilled on ice for 1min.  Then 1uL of 10X T4 RNL2 truncated buffer, 0.8uL 100mM MgCl2, 0.2uL DEPC water, 0.5uL Rnase Out, and 1.5uL T4 RNA Ligase 2 trancated was added.  Ligation incubated at 22C in a Tetrad thermocycler for 60min.
RNA_PREPARATION_5'_RNA_ADAPTER_LIGATION_PROTOCOL: 5' RNA adapter was heat denatured at 70C for 2min then snap chilled on ice.  2uL of denatured 5' RNA Adapter was added to the 3' DNA Adapter Ligation reaction product from previous step and mixed.   Then 1uL of 10mM ATP and 1uL of Ambion T4 RNA Ligase were added and ligation reaction was incubated at 37C in a Tetrad thermocycler for 60min.
RNA_PREPARATION_REVERSE_TRANSCRIPTION_PROTOCOL: To the 14uL of double adaptered miRNA product from previous step 2ul of RT-Primer was added and the mixture was heat denatured at 65C for 10 mins then quenched on ice.  The following reagents were added immediately to the sample (6ul of 5X First Strand Buffer, 2ul of 10mM dNTPs , 3ul of 100mM DTT, 1ul of RNaseOUT, and 2uL of SuperScript II RT.  Reaction was heated at 44C in a Thetrad thermocycler for 60 minutes.
LIBRARY_GENERATION_PCR_TEMPLATE: 15ul of the first strand cDNA product was used in the PCR
****************
For data usage terms and conditions, please refer to:
http://www.drugabuse.gov/funding/funding-opportunities/nih-common-fund/epigenomics-data-access-policies
****************

value: Fibroblast cultured cell

value: breast fibroblast


description:
design description: miRNA-Seq analysis of breast fibroblast from RM070 (m08321) using Illumina HiSeq 2000
library name: m08321
EXPERIMENT_TYPE: smRNA-Seq
EXTRACTION_PROTOCOL: Total RNA was extracted using Trizol from Invitrogen as per manufacturer's instructions.
LIBRARY_GENERATION_PCR_POLYMERASE_TYPE: Phusion DNA Polymerase (Hot Start), 2U/ul (from NEB)
LIBRARY_GENERATION_PCR_THERMOCYCLING_PROGRAM: step1: 98C for 30sec, step2: 98C for 10sec, step3: 60C for 30sec, step4: 72C for 15sec.  Repeat step2-4 for 15 cycles, 72C for 5min.
LIBRARY_GENERATION_PCR_NUMBER_CYCLES: 15
LIBRARY_GENERATION_PCR_F_PRIMER_SEQUENCE: 5' AATGATACGGCGACCACCGACAGNNNNNNGTTCAGAGTTCTACAGTCCGA 3'
LIBRARY_GENERATION_PCR_R_PRIMER_SEQUENCE: 5' CAAGCAGAAGACGGCATACGAGAT 3'
LIBRARY_GENERATION_PCR_PRIMER_CONC: 25 uM
LIBRARY_GENERATION_PCR_PRODUCT_ISOLATION_PROTOCOL: 28uL of PCR products containing unique index sequences are pooled together and the 115bp miRNA containing fraction is isolated for each pool either manually:  12% PAGE gel 200V 6hours followed by gel elution, or robotically using Baraccuda size selection robot.  Fractions are Et0H preciptated, QCed on Agilent, and submitted for indexing single end (ISE) Illumina sequencing.
EXTRACTION_PROTOCOL_SMRNA_ENRICHMENT: Flowthrough of polyA+ MultiMACS 96 Separation Unit
SMRNA_PREPARATION_INITIAL_SMRNA_QLTY: RIN 3
SMRNA_PREPARATION_INITIAL_SMRNA_QNTY: 1.340 ug
RNA_PREPARATION_5'_RNA_ADAPTER_SEQUENCE: 5' GTTCAGAGTTCTACAGTCCGACGATCTGGTCAA 3'
RNA_PREPARATION_3'_RNA_ADAPTER_SEQUENCE: 5' ATCTCGTATGCCGTCTTCTGCTTGT 3'
RNA_PREPARATION_REVERSE_TRANSCRIPTION_PRIMER_SEQUENCE: 5' CAAGCAGAAGACGGCATACGAGAT 3'
RNA_PREPARATION_3'_RNA_ADAPTER_LIGATION_PROTOCOL: 1ug of total RNA (RN =>7.0) in a 4uL volume and 2uL of 3' Adenylated Adapter was heat denatured at 70C for 2min then snap chilled on ice for 1min.  Then 1uL of 10X T4 RNL2 truncated buffer, 0.8uL 100mM MgCl2, 0.2uL DEPC water, 0.5uL Rnase Out, and 1.5uL T4 RNA Ligase 2 trancated was added.  Ligation incubated at 22C in a Tetrad thermocycler for 60min.
RNA_PREPARATION_5'_RNA_ADAPTER_LIGATION_PROTOCOL: 5' RNA adapter was heat denatured at 70C for 2min then snap chilled on ice.  2uL of denatured 5' RNA Adapter was added to the 3' DNA Adapter Ligation reaction product from previous step and mixed.   Then 1uL of 10mM ATP and 1uL of Ambion T4 RNA Ligase were added and ligation reaction was incubated at 37C in a Tetrad thermocycler for 60min.
RNA_PREPARATION_REVERSE_TRANSCRIPTION_PROTOCOL: To the 14uL of double adaptered miRNA product from previous step 2ul of RT-Primer was added and the mixture was heat denatured at 65C for 10 mins then quenched on ice.  The following reagents were added immediately to the sample (6ul of 5X First Strand Buffer, 2ul of 10mM dNTPs , 3ul of 100mM DTT, 1ul of RNaseOUT, and 2uL of SuperScript II RT.  Reaction was heated at 44C in a Thetrad thermocycler for 60 minutes.
LIBRARY_GENERATION_PCR_TEMPLATE: 15ul of the first strand cDNA product was used in the PCR
****************
For data usage terms and conditions, please refer to:
http://www.drugabuse.gov/funding/funding-opportunities/nih-common-fund/epigenomics-data-access-policies
****************


description:
design description: miRNA-Seq analysis of breast progenitor enriched cell from RM084 (m08316) using Illumina HiSeq 2000
library name: m08316
EXPERIMENT_TYPE: smRNA-Seq
EXTRACTION_PROTOCOL: Total RNA was extracted using Trizol from Invitrogen as per manufacturer's instructions.
LIBRARY_GENERATION_PCR_POLYMERASE_TYPE: Phusion DNA Polymerase (Hot Start), 2U/ul (from NEB)
LIBRARY_GENERATION_PCR_THERMOCYCLING_PROGRAM: step1: 98C for 30sec, step2: 98C for 10sec, step3: 60C for 30sec, step4: 72C for 15sec.  Repeat step2-4 for 15 cycles, 72C for 5min.
LIBRARY_GENERATION_PCR_NUMBER_CYCLES: 15
LIBRARY_GENERATION_PCR_F_PRIMER_SEQUENCE: 5' AATGATACGGCGACCACCGACAGNNNNNNGTTCAGAGTTCTACAGTCCGA 3'
LIBRARY_GENERATION_PCR_R_PRIMER_SEQUENCE: 5' CAAGCAGAAGACGGCATACGAGAT 3'
LIBRARY_GENERATION_PCR_PRIMER_CONC: 25 uM
LIBRARY_GENERATION_PCR_PRODUCT_ISOLATION_PROTOCOL: 28uL of PCR products containing unique index sequences are pooled together and the 115bp miRNA containing fraction is isolated for each pool either manually:  12% PAGE gel 200V 6hours followed by gel elution, or robotically using Baraccuda size selection robot.  Fractions are Et0H preciptated, QCed on Agilent, and submitted for indexing single end (ISE) Illumina sequencing.
EXTRACTION_PROTOCOL_SMRNA_ENRICHMENT: Flowthrough of polyA+ MultiMACS 96 Separation Unit
SMRNA_PREPARATION_INITIAL_SMRNA_QLTY: RIN 6.2
SMRNA_PREPARATION_INITIAL_SMRNA_QNTY: 1.438 ug
RNA_PREPARATION_5'_RNA_ADAPTER_SEQUENCE: 5' GTTCAGAGTTCTACAGTCCGACGATCTGGTCAA 3'
RNA_PREPARATION_3'_RNA_ADAPTER_SEQUENCE: 5' ATCTCGTATGCCGTCTTCTGCTTGT 3'
RNA_PREPARATION_REVERSE_TRANSCRIPTION_PRIMER_SEQUENCE: 5' CAAGCAGAAGACGGCATACGAGAT 3'
RNA_PREPARATION_3'_RNA_ADAPTER_LIGATION_PROTOCOL: 1ug of total RNA (RN =>7.0) in a 4uL volume and 2uL of 3' Adenylated Adapter was heat denatured at 70C for 2min then snap chilled on ice for 1min.  Then 1uL of 10X T4 RNL2 truncated buffer, 0.8uL 100mM MgCl2, 0.2uL DEPC water, 0.5uL Rnase Out, and 1.5uL T4 RNA Ligase 2 trancated was added.  Ligation incubated at 22C in a Tetrad thermocycler for 60min.
RNA_PREPARATION_5'_RNA_ADAPTER_LIGATION_PROTOCOL: 5' RNA adapter was heat denatured at 70C for 2min then snap chilled on ice.  2uL of denatured 5' RNA Adapter was added to the 3' DNA Adapter Ligation reaction product from previous step and mixed.   Then 1uL of 10mM ATP and 1uL of Ambion T4 RNA Ligase were added and ligation reaction was incubated at 37C in a Tetrad thermocycler for 60min.
RNA_PREPARATION_REVERSE_TRANSCRIPTION_PROTOCOL: To the 14uL of double adaptered miRNA product from previous step 2ul of RT-Primer was added and the mixture was heat denatured at 65C for 10 mins then quenched on ice.  The following reagents were added immediately to the sample (6ul of 5X First Strand Buffer, 2ul of 10mM dNTPs , 3ul of 100mM DTT, 1ul of RNaseOUT, and 2uL of SuperScript II RT.  Reaction was heated at 44C in a Thetrad thermocycler for 60 minutes.
LIBRARY_GENERATION_PCR_TEMPLATE: 15ul of the first strand cDNA product was used in the PCR
****************
For data usage terms and conditions, please refer to:
http://www.drugabuse.gov/funding/funding-opportunities/nih-common-fund/epigenomics-data-access-policies
****************

value: Progenitor enriched cell

value: breast progenitor enriched cell


title: O30_H3K4me3_rep1

value: umbilical cord blood

value: CD14+ monocytes

value: Miltenyi Biotec CD14+ magnetic bead isolation

value: 30-36 weeks gestation preterm neonate


description:
design description: miRNA-Seq analysis of skin (nevus) tissue from Nevus03 (m09676) using Illumina HiSeq 2000
library name: m09676
EXPERIMENT_TYPE: smRNA-Seq
EXTRACTION_PROTOCOL: Total RNA was extracted using Trizol from Invitrogen as per manufacturer's instructions.
LIBRARY_GENERATION_PCR_POLYMERASE_TYPE: Phusion DNA Polymerase (Hot Start), 2U/ul (from NEB)
LIBRARY_GENERATION_PCR_THERMOCYCLING_PROGRAM: step1: 98C for 30sec, step2: 98C for 10sec, step3: 60C for 30sec, step4: 72C for 15sec.  Repeat step2-4 for 15 cycles, 72C for 5min.
LIBRARY_GENERATION_PCR_NUMBER_CYCLES: 15
LIBRARY_GENERATION_PCR_F_PRIMER_SEQUENCE: 5' AATGATACGGCGACCACCGACAGNNNNNNGTTCAGAGTTCTACAGTCCGA 3'
LIBRARY_GENERATION_PCR_R_PRIMER_SEQUENCE: 5' CAAGCAGAAGACGGCATACGAGAT 3'
LIBRARY_GENERATION_PCR_PRIMER_CONC: 25 uM
LIBRARY_GENERATION_PCR_PRODUCT_ISOLATION_PROTOCOL: 28uL of PCR products containing unique index sequences are pooled together and the 115bp miRNA containing fraction is isolated for each pool either manually:  12% PAGE gel 200V 6hours followed by gel elution, or robotically using Baraccuda size selection robot.  Fractions are Et0H preciptated, QCed on Agilent, and submitted for indexing single end (ISE) Illumina sequencing.
EXTRACTION_PROTOCOL_SMRNA_ENRICHMENT: Flowthrough of polyA+ MultiMACS 96 Separation Unit
SMRNA_PREPARATION_INITIAL_SMRNA_QLTY: RQS 6.0
SMRNA_PREPARATION_INITIAL_SMRNA_QNTY: 0.8328 ug
RNA_PREPARATION_5'_RNA_ADAPTER_SEQUENCE: 5' GTTCAGAGTTCTACAGTCCGACGATCTGGTCAA 3'
RNA_PREPARATION_3'_RNA_ADAPTER_SEQUENCE: 5' ATCTCGTATGCCGTCTTCTGCTTGT 3'
RNA_PREPARATION_REVERSE_TRANSCRIPTION_PRIMER_SEQUENCE: 5' CAAGCAGAAGACGGCATACGAGAT 3'
RNA_PREPARATION_3'_RNA_ADAPTER_LIGATION_PROTOCOL: 1ug of total RNA (RN =>7.0) in a 4uL volume and 2uL of 3' Adenylated Adapter was heat denatured at 70C for 2min then snap chilled on ice for 1min.  Then 1uL of 10X T4 RNL2 truncated buffer, 0.8uL 100mM MgCl2, 0.2uL DEPC water, 0.5uL Rnase Out, and 1.5uL T4 RNA Ligase 2 trancated was added.  Ligation incubated at 22C in a Tetrad thermocycler for 60min.
RNA_PREPARATION_5'_RNA_ADAPTER_LIGATION_PROTOCOL: 5' RNA adapter was heat denatured at 70C for 2min then snap chilled on ice.  2uL of denatured 5' RNA Adapter was added to the 3' DNA Adapter Ligation reaction product from previous step and mixed.   Then 1uL of 10mM ATP and 1uL of Ambion T4 RNA Ligase were added and ligation reaction was incubated at 37C in a Tetrad thermocycler for 60min.
RNA_PREPARATION_REVERSE_TRANSCRIPTION_PROTOCOL: To the 14uL of double adaptered miRNA product from previous step 2ul of RT-Primer was added and the mixture was heat denatured at 65C for 10 mins then quenched on ice.  The following reagents were added immediately to the sample (6ul of 5X First Strand Buffer, 2ul of 10mM dNTPs , 3ul of 100mM DTT, 1ul of RNaseOUT, and 2uL of SuperScript II RT.  Reaction was heated at 44C in a Thetrad thermocycler for 60 minutes.
LIBRARY_GENERATION_PCR_TEMPLATE: 15ul of the first strand cDNA product was used in the PCR
****************
For data usage terms and conditions, please refer to:
http://www.drugabuse.gov/funding/funding-opportunities/nih-common-fund/epigenomics-data-access-policies
****************

value: Skin

value: Nevus tissue

value: primary tissue

value: skin (nevus) tissue


description:
design description: miRNA-Seq analysis of brain (cerebellum) tissue from HuFGM01 (m08313) using Illumina HiSeq 2000
library name: m08313
EXPERIMENT_TYPE: smRNA-Seq
EXTRACTION_PROTOCOL: Total RNA was extracted using Trizol from Invitrogen as per manufacturer's instructions.
LIBRARY_GENERATION_PCR_POLYMERASE_TYPE: Phusion DNA Polymerase (Hot Start), 2U/ul (from NEB)
LIBRARY_GENERATION_PCR_THERMOCYCLING_PROGRAM: step1: 98C for 30sec, step2: 98C for 10sec, step3: 60C for 30sec, step4: 72C for 15sec.  Repeat step2-4 for 15 cycles, 72C for 5min.
LIBRARY_GENERATION_PCR_NUMBER_CYCLES: 15
LIBRARY_GENERATION_PCR_F_PRIMER_SEQUENCE: 5' AATGATACGGCGACCACCGACAGNNNNNNGTTCAGAGTTCTACAGTCCGA 3'
LIBRARY_GENERATION_PCR_R_PRIMER_SEQUENCE: 5' CAAGCAGAAGACGGCATACGAGAT 3'
LIBRARY_GENERATION_PCR_PRIMER_CONC: 25 uM
LIBRARY_GENERATION_PCR_PRODUCT_ISOLATION_PROTOCOL: 28uL of PCR products containing unique index sequences are pooled together and the 115bp miRNA containing fraction is isolated for each pool either manually:  12% PAGE gel 200V 6hours followed by gel elution, or robotically using Baraccuda size selection robot.  Fractions are Et0H preciptated, QCed on Agilent, and submitted for indexing single end (ISE) Illumina sequencing.
EXTRACTION_PROTOCOL_SMRNA_ENRICHMENT: Flowthrough of polyA+ MultiMACS 96 Separation Unit
SMRNA_PREPARATION_INITIAL_SMRNA_QLTY: RIN 6.3
SMRNA_PREPARATION_INITIAL_SMRNA_QNTY: 0.9 ug
RNA_PREPARATION_5'_RNA_ADAPTER_SEQUENCE: 5' GTTCAGAGTTCTACAGTCCGACGATCTGGTCAA 3'
RNA_PREPARATION_3'_RNA_ADAPTER_SEQUENCE: 5' ATCTCGTATGCCGTCTTCTGCTTGT 3'
RNA_PREPARATION_REVERSE_TRANSCRIPTION_PRIMER_SEQUENCE: 5' CAAGCAGAAGACGGCATACGAGAT 3'
RNA_PREPARATION_3'_RNA_ADAPTER_LIGATION_PROTOCOL: 1ug of total RNA (RN =>7.0) in a 4uL volume and 2uL of 3' Adenylated Adapter was heat denatured at 70C for 2min then snap chilled on ice for 1min.  Then 1uL of 10X T4 RNL2 truncated buffer, 0.8uL 100mM MgCl2, 0.2uL DEPC water, 0.5uL Rnase Out, and 1.5uL T4 RNA Ligase 2 trancated was added.  Ligation incubated at 22C in a Tetrad thermocycler for 60min.
RNA_PREPARATION_5'_RNA_ADAPTER_LIGATION_PROTOCOL: 5' RNA adapter was heat denatured at 70C for 2min then snap chilled on ice.  2uL of denatured 5' RNA Adapter was added to the 3' DNA Adapter Ligation reaction product from previous step and mixed.   Then 1uL of 10mM ATP and 1uL of Ambion T4 RNA Ligase were added and ligation reaction was incubated at 37C in a Tetrad thermocycler for 60min.
RNA_PREPARATION_REVERSE_TRANSCRIPTION_PROTOCOL: To the 14uL of double adaptered miRNA product from previous step 2ul of RT-Primer was added and the mixture was heat denatured at 65C for 10 mins then quenched on ice.  The following reagents were added immediately to the sample (6ul of 5X First Strand Buffer, 2ul of 10mM dNTPs , 3ul of 100mM DTT, 1ul of RNaseOUT, and 2uL of SuperScript II RT.  Reaction was heated at 44C in a Thetrad thermocycler for 60 minutes.
LIBRARY_GENERATION_PCR_TEMPLATE: 15ul of the first strand cDNA product was used in the PCR
****************
For data usage terms and conditions, please refer to:
http://www.drugabuse.gov/funding/funding-opportunities/nih-common-fund/epigenomics-data-access-policies
****************

value: male

value: Brain

value: Cerebellum tissue

value: No

value: brain (cerebellum) tissue


title: U30_H3K4me3_rep3

value: under 30 weeks gestation preterm neonate


description:
design description: miRNA-Seq analysis of placenta (smooth chorionic membrane) tissue from CTL01 (m08327) using Illumina HiSeq 2000
library name: m08327
EXPERIMENT_TYPE: smRNA-Seq
EXTRACTION_PROTOCOL: Total RNA was extracted using Trizol from Invitrogen as per manufacturer's instructions.
LIBRARY_GENERATION_PCR_POLYMERASE_TYPE: Phusion DNA Polymerase (Hot Start), 2U/ul (from NEB)
LIBRARY_GENERATION_PCR_THERMOCYCLING_PROGRAM: step1: 98C for 30sec, step2: 98C for 10sec, step3: 60C for 30sec, step4: 72C for 15sec.  Repeat step2-4 for 15 cycles, 72C for 5min.
LIBRARY_GENERATION_PCR_NUMBER_CYCLES: 15
LIBRARY_GENERATION_PCR_F_PRIMER_SEQUENCE: 5' AATGATACGGCGACCACCGACAGNNNNNNGTTCAGAGTTCTACAGTCCGA 3'
LIBRARY_GENERATION_PCR_R_PRIMER_SEQUENCE: 5' CAAGCAGAAGACGGCATACGAGAT 3'
LIBRARY_GENERATION_PCR_PRIMER_CONC: 25 uM
LIBRARY_GENERATION_PCR_PRODUCT_ISOLATION_PROTOCOL: 28uL of PCR products containing unique index sequences are pooled together and the 115bp miRNA containing fraction is isolated for each pool either manually:  12% PAGE gel 200V 6hours followed by gel elution, or robotically using Baraccuda size selection robot.  Fractions are Et0H preciptated, QCed on Agilent, and submitted for indexing single end (ISE) Illumina sequencing.
EXTRACTION_PROTOCOL_SMRNA_ENRICHMENT: Flowthrough of polyA+ MultiMACS 96 Separation Unit
SMRNA_PREPARATION_INITIAL_SMRNA_QLTY: RIN 7.4
SMRNA_PREPARATION_INITIAL_SMRNA_QNTY: 0.852 ug
RNA_PREPARATION_5'_RNA_ADAPTER_SEQUENCE: 5' GTTCAGAGTTCTACAGTCCGACGATCTGGTCAA 3'
RNA_PREPARATION_3'_RNA_ADAPTER_SEQUENCE: 5' ATCTCGTATGCCGTCTTCTGCTTGT 3'
RNA_PREPARATION_REVERSE_TRANSCRIPTION_PRIMER_SEQUENCE: 5' CAAGCAGAAGACGGCATACGAGAT 3'
RNA_PREPARATION_3'_RNA_ADAPTER_LIGATION_PROTOCOL: 1ug of total RNA (RN =>7.0) in a 4uL volume and 2uL of 3' Adenylated Adapter was heat denatured at 70C for 2min then snap chilled on ice for 1min.  Then 1uL of 10X T4 RNL2 truncated buffer, 0.8uL 100mM MgCl2, 0.2uL DEPC water, 0.5uL Rnase Out, and 1.5uL T4 RNA Ligase 2 trancated was added.  Ligation incubated at 22C in a Tetrad thermocycler for 60min.
RNA_PREPARATION_5'_RNA_ADAPTER_LIGATION_PROTOCOL: 5' RNA adapter was heat denatured at 70C for 2min then snap chilled on ice.  2uL of denatured 5' RNA Adapter was added to the 3' DNA Adapter Ligation reaction product from previous step and mixed.   Then 1uL of 10mM ATP and 1uL of Ambion T4 RNA Ligase were added and ligation reaction was incubated at 37C in a Tetrad thermocycler for 60min.
RNA_PREPARATION_REVERSE_TRANSCRIPTION_PROTOCOL: To the 14uL of double adaptered miRNA product from previous step 2ul of RT-Primer was added and the mixture was heat denatured at 65C for 10 mins then quenched on ice.  The following reagents were added immediately to the sample (6ul of 5X First Strand Buffer, 2ul of 10mM dNTPs , 3ul of 100mM DTT, 1ul of RNaseOUT, and 2uL of SuperScript II RT.  Reaction was heated at 44C in a Thetrad thermocycler for 60 minutes.
LIBRARY_GENERATION_PCR_TEMPLATE: 15ul of the first strand cDNA product was used in the PCR
****************
For data usage terms and conditions, please refer to:
http://www.drugabuse.gov/funding/funding-opportunities/nih-common-fund/epigenomics-data-access-policies
****************

value: female

value: Placenta

value: Chorion smooth tissue


description:
design description: miRNA-Seq analysis of brain (insular) tissue from NB141 (m09677) using Illumina HiSeq 2000
library name: m09677
EXPERIMENT_TYPE: smRNA-Seq
EXTRACTION_PROTOCOL: Total RNA was extracted using Trizol from Invitrogen as per manufacturer's instructions.
LIBRARY_GENERATION_PCR_POLYMERASE_TYPE: Phusion DNA Polymerase (Hot Start), 2U/ul (from NEB)
LIBRARY_GENERATION_PCR_THERMOCYCLING_PROGRAM: step1: 98C for 30sec, step2: 98C for 10sec, step3: 60C for 30sec, step4: 72C for 15sec.  Repeat step2-4 for 15 cycles, 72C for 5min.
LIBRARY_GENERATION_PCR_NUMBER_CYCLES: 15
LIBRARY_GENERATION_PCR_F_PRIMER_SEQUENCE: 5' AATGATACGGCGACCACCGACAGNNNNNNGTTCAGAGTTCTACAGTCCGA 3'
LIBRARY_GENERATION_PCR_R_PRIMER_SEQUENCE: 5' CAAGCAGAAGACGGCATACGAGAT 3'
LIBRARY_GENERATION_PCR_PRIMER_CONC: 25 uM
LIBRARY_GENERATION_PCR_PRODUCT_ISOLATION_PROTOCOL: 28uL of PCR products containing unique index sequences are pooled together and the 115bp miRNA containing fraction is isolated for each pool either manually:  12% PAGE gel 200V 6hours followed by gel elution, or robotically using Baraccuda size selection robot.  Fractions are Et0H preciptated, QCed on Agilent, and submitted for indexing single end (ISE) Illumina sequencing.
EXTRACTION_PROTOCOL_SMRNA_ENRICHMENT: Flowthrough of polyA+ MultiMACS 96 Separation Unit
SMRNA_PREPARATION_INITIAL_SMRNA_QLTY: RQS 6.2
SMRNA_PREPARATION_INITIAL_SMRNA_QNTY: 1.3262 ug
RNA_PREPARATION_5'_RNA_ADAPTER_SEQUENCE: 5' GTTCAGAGTTCTACAGTCCGACGATCTGGTCAA 3'
RNA_PREPARATION_3'_RNA_ADAPTER_SEQUENCE: 5' ATCTCGTATGCCGTCTTCTGCTTGT 3'
RNA_PREPARATION_REVERSE_TRANSCRIPTION_PRIMER_SEQUENCE: 5' CAAGCAGAAGACGGCATACGAGAT 3'
RNA_PREPARATION_3'_RNA_ADAPTER_LIGATION_PROTOCOL: 1ug of total RNA (RN =>7.0) in a 4uL volume and 2uL of 3' Adenylated Adapter was heat denatured at 70C for 2min then snap chilled on ice for 1min.  Then 1uL of 10X T4 RNL2 truncated buffer, 0.8uL 100mM MgCl2, 0.2uL DEPC water, 0.5uL Rnase Out, and 1.5uL T4 RNA Ligase 2 trancated was added.  Ligation incubated at 22C in a Tetrad thermocycler for 60min.
RNA_PREPARATION_5'_RNA_ADAPTER_LIGATION_PROTOCOL: 5' RNA adapter was heat denatured at 70C for 2min then snap chilled on ice.  2uL of denatured 5' RNA Adapter was added to the 3' DNA Adapter Ligation reaction product from previous step and mixed.   Then 1uL of 10mM ATP and 1uL of Ambion T4 RNA Ligase were added and ligation reaction was incubated at 37C in a Tetrad thermocycler for 60min.
RNA_PREPARATION_REVERSE_TRANSCRIPTION_PROTOCOL: To the 14uL of double adaptered miRNA product from previous step 2ul of RT-Primer was added and the mixture was heat denatured at 65C for 10 mins then quenched on ice.  The following reagents were added immediately to the sample (6ul of 5X First Strand Buffer, 2ul of 10mM dNTPs , 3ul of 100mM DTT, 1ul of RNaseOUT, and 2uL of SuperScript II RT.  Reaction was heated at 44C in a Thetrad thermocycler for 60 minutes.
LIBRARY_GENERATION_PCR_TEMPLATE: 15ul of the first strand cDNA product was used in the PCR
****************
For data usage terms and conditions, please refer to:
http://www.drugabuse.gov/funding/funding-opportunities/nih-common-fund/epigenomics-data-access-policies
****************

value: Insular cortex tissue

value: brain (insular) tissue


title: LNCaP vehicle TRIM24 ChIP-seq

tag: cell line

value: LNCaP

value: vehicle (ETOH)

value: TRIM24 (Bethyl, A300-815A)


description:
design description: miRNA-Seq analysis of brain (brodmann 11) tissue from NB141 (m09678) using Illumina HiSeq 2000
library name: m09678
EXPERIMENT_TYPE: smRNA-Seq
EXTRACTION_PROTOCOL: Total RNA was extracted using Trizol from Invitrogen as per manufacturer's instructions.
LIBRARY_GENERATION_PCR_POLYMERASE_TYPE: Phusion DNA Polymerase (Hot Start), 2U/ul (from NEB)
LIBRARY_GENERATION_PCR_THERMOCYCLING_PROGRAM: step1: 98C for 30sec, step2: 98C for 10sec, step3: 60C for 30sec, step4: 72C for 15sec.  Repeat step2-4 for 15 cycles, 72C for 5min.
LIBRARY_GENERATION_PCR_NUMBER_CYCLES: 15
LIBRARY_GENERATION_PCR_F_PRIMER_SEQUENCE: 5' AATGATACGGCGACCACCGACAGNNNNNNGTTCAGAGTTCTACAGTCCGA 3'
LIBRARY_GENERATION_PCR_R_PRIMER_SEQUENCE: 5' CAAGCAGAAGACGGCATACGAGAT 3'
LIBRARY_GENERATION_PCR_PRIMER_CONC: 25 uM
LIBRARY_GENERATION_PCR_PRODUCT_ISOLATION_PROTOCOL: 28uL of PCR products containing unique index sequences are pooled together and the 115bp miRNA containing fraction is isolated for each pool either manually:  12% PAGE gel 200V 6hours followed by gel elution, or robotically using Baraccuda size selection robot.  Fractions are Et0H preciptated, QCed on Agilent, and submitted for indexing single end (ISE) Illumina sequencing.
EXTRACTION_PROTOCOL_SMRNA_ENRICHMENT: Flowthrough of polyA+ MultiMACS 96 Separation Unit
SMRNA_PREPARATION_INITIAL_SMRNA_QLTY: RQS 5.7
SMRNA_PREPARATION_INITIAL_SMRNA_QNTY: 1.320 ug
RNA_PREPARATION_5'_RNA_ADAPTER_SEQUENCE: 5' GTTCAGAGTTCTACAGTCCGACGATCTGGTCAA 3'
RNA_PREPARATION_3'_RNA_ADAPTER_SEQUENCE: 5' ATCTCGTATGCCGTCTTCTGCTTGT 3'
RNA_PREPARATION_REVERSE_TRANSCRIPTION_PRIMER_SEQUENCE: 5' CAAGCAGAAGACGGCATACGAGAT 3'
RNA_PREPARATION_3'_RNA_ADAPTER_LIGATION_PROTOCOL: 1ug of total RNA (RN =>7.0) in a 4uL volume and 2uL of 3' Adenylated Adapter was heat denatured at 70C for 2min then snap chilled on ice for 1min.  Then 1uL of 10X T4 RNL2 truncated buffer, 0.8uL 100mM MgCl2, 0.2uL DEPC water, 0.5uL Rnase Out, and 1.5uL T4 RNA Ligase 2 trancated was added.  Ligation incubated at 22C in a Tetrad thermocycler for 60min.
RNA_PREPARATION_5'_RNA_ADAPTER_LIGATION_PROTOCOL: 5' RNA adapter was heat denatured at 70C for 2min then snap chilled on ice.  2uL of denatured 5' RNA Adapter was added to the 3' DNA Adapter Ligation reaction product from previous step and mixed.   Then 1uL of 10mM ATP and 1uL of Ambion T4 RNA Ligase were added and ligation reaction was incubated at 37C in a Tetrad thermocycler for 60min.
RNA_PREPARATION_REVERSE_TRANSCRIPTION_PROTOCOL: To the 14uL of double adaptered miRNA product from previous step 2ul of RT-Primer was added and the mixture was heat denatured at 65C for 10 mins then quenched on ice.  The following reagents were added immediately to the sample (6ul of 5X First Strand Buffer, 2ul of 10mM dNTPs , 3ul of 100mM DTT, 1ul of RNaseOUT, and 2uL of SuperScript II RT.  Reaction was heated at 44C in a Thetrad thermocycler for 60 minutes.
LIBRARY_GENERATION_PCR_TEMPLATE: 15ul of the first strand cDNA product was used in the PCR
****************
For data usage terms and conditions, please refer to:
http://www.drugabuse.gov/funding/funding-opportunities/nih-common-fund/epigenomics-data-access-policies
****************

value: Brodmann 11 tissue

value: brain (brodmann 11) tissue


description:
design description: miRNA-Seq analysis of placenta (amniotic membrane) tissue from CTL01 (m08328) using Illumina HiSeq 2000
library name: m08328
EXPERIMENT_TYPE: smRNA-Seq
EXTRACTION_PROTOCOL: Total RNA was extracted using Trizol from Invitrogen as per manufacturer's instructions.
LIBRARY_GENERATION_PCR_POLYMERASE_TYPE: Phusion DNA Polymerase (Hot Start), 2U/ul (from NEB)
LIBRARY_GENERATION_PCR_THERMOCYCLING_PROGRAM: step1: 98C for 30sec, step2: 98C for 10sec, step3: 60C for 30sec, step4: 72C for 15sec.  Repeat step2-4 for 15 cycles, 72C for 5min.
LIBRARY_GENERATION_PCR_NUMBER_CYCLES: 15
LIBRARY_GENERATION_PCR_F_PRIMER_SEQUENCE: 5' AATGATACGGCGACCACCGACAGNNNNNNGTTCAGAGTTCTACAGTCCGA 3'
LIBRARY_GENERATION_PCR_R_PRIMER_SEQUENCE: 5' CAAGCAGAAGACGGCATACGAGAT 3'
LIBRARY_GENERATION_PCR_PRIMER_CONC: 25 uM
LIBRARY_GENERATION_PCR_PRODUCT_ISOLATION_PROTOCOL: 28uL of PCR products containing unique index sequences are pooled together and the 115bp miRNA containing fraction is isolated for each pool either manually:  12% PAGE gel 200V 6hours followed by gel elution, or robotically using Baraccuda size selection robot.  Fractions are Et0H preciptated, QCed on Agilent, and submitted for indexing single end (ISE) Illumina sequencing.
EXTRACTION_PROTOCOL_SMRNA_ENRICHMENT: Flowthrough of polyA+ MultiMACS 96 Separation Unit
SMRNA_PREPARATION_INITIAL_SMRNA_QLTY: RIN 8
SMRNA_PREPARATION_INITIAL_SMRNA_QNTY: 0.836 ug
RNA_PREPARATION_5'_RNA_ADAPTER_SEQUENCE: 5' GTTCAGAGTTCTACAGTCCGACGATCTGGTCAA 3'
RNA_PREPARATION_3'_RNA_ADAPTER_SEQUENCE: 5' ATCTCGTATGCCGTCTTCTGCTTGT 3'
RNA_PREPARATION_REVERSE_TRANSCRIPTION_PRIMER_SEQUENCE: 5' CAAGCAGAAGACGGCATACGAGAT 3'
RNA_PREPARATION_3'_RNA_ADAPTER_LIGATION_PROTOCOL: 1ug of total RNA (RN =>7.0) in a 4uL volume and 2uL of 3' Adenylated Adapter was heat denatured at 70C for 2min then snap chilled on ice for 1min.  Then 1uL of 10X T4 RNL2 truncated buffer, 0.8uL 100mM MgCl2, 0.2uL DEPC water, 0.5uL Rnase Out, and 1.5uL T4 RNA Ligase 2 trancated was added.  Ligation incubated at 22C in a Tetrad thermocycler for 60min.
RNA_PREPARATION_5'_RNA_ADAPTER_LIGATION_PROTOCOL: 5' RNA adapter was heat denatured at 70C for 2min then snap chilled on ice.  2uL of denatured 5' RNA Adapter was added to the 3' DNA Adapter Ligation reaction product from previous step and mixed.   Then 1uL of 10mM ATP and 1uL of Ambion T4 RNA Ligase were added and ligation reaction was incubated at 37C in a Tetrad thermocycler for 60min.
RNA_PREPARATION_REVERSE_TRANSCRIPTION_PROTOCOL: To the 14uL of double adaptered miRNA product from previous step 2ul of RT-Primer was added and the mixture was heat denatured at 65C for 10 mins then quenched on ice.  The following reagents were added immediately to the sample (6ul of 5X First Strand Buffer, 2ul of 10mM dNTPs , 3ul of 100mM DTT, 1ul of RNaseOUT, and 2uL of SuperScript II RT.  Reaction was heated at 44C in a Thetrad thermocycler for 60 minutes.
LIBRARY_GENERATION_PCR_TEMPLATE: 15ul of the first strand cDNA product was used in the PCR
****************
For data usage terms and conditions, please refer to:
http://www.drugabuse.gov/funding/funding-opportunities/nih-common-fund/epigenomics-data-access-policies
****************

value: Amnion tissue

value: primary tissue

value: placenta (amniotic membrane) tissue


description:
design description: miRNA-Seq analysis of brain astrocyte from NHA (m09680) using Illumina HiSeq 2000
library name: m09680
EXPERIMENT_TYPE: smRNA-Seq
EXTRACTION_PROTOCOL: Total RNA was extracted using Trizol from Invitrogen as per manufacturer's instructions.
LIBRARY_GENERATION_PCR_POLYMERASE_TYPE: Phusion DNA Polymerase (Hot Start), 2U/ul (from NEB)
LIBRARY_GENERATION_PCR_THERMOCYCLING_PROGRAM: step1: 98C for 30sec, step2: 98C for 10sec, step3: 60C for 30sec, step4: 72C for 15sec.  Repeat step2-4 for 15 cycles, 72C for 5min.
LIBRARY_GENERATION_PCR_NUMBER_CYCLES: 15
LIBRARY_GENERATION_PCR_F_PRIMER_SEQUENCE: 5' AATGATACGGCGACCACCGACAGNNNNNNGTTCAGAGTTCTACAGTCCGA 3'
LIBRARY_GENERATION_PCR_R_PRIMER_SEQUENCE: 5' CAAGCAGAAGACGGCATACGAGAT 3'
LIBRARY_GENERATION_PCR_PRIMER_CONC: 25 uM
LIBRARY_GENERATION_PCR_PRODUCT_ISOLATION_PROTOCOL: 28uL of PCR products containing unique index sequences are pooled together and the 115bp miRNA containing fraction is isolated for each pool either manually:  12% PAGE gel 200V 6hours followed by gel elution, or robotically using Baraccuda size selection robot.  Fractions are Et0H preciptated, QCed on Agilent, and submitted for indexing single end (ISE) Illumina sequencing.
EXTRACTION_PROTOCOL_SMRNA_ENRICHMENT: Flowthrough of polyA+ MultiMACS 96 Separation Unit
SMRNA_PREPARATION_INITIAL_SMRNA_QLTY: RQS 7.5
SMRNA_PREPARATION_INITIAL_SMRNA_QNTY: 1.877 ug
RNA_PREPARATION_5'_RNA_ADAPTER_SEQUENCE: 5' GTTCAGAGTTCTACAGTCCGACGATCTGGTCAA 3'
RNA_PREPARATION_3'_RNA_ADAPTER_SEQUENCE: 5' ATCTCGTATGCCGTCTTCTGCTTGT 3'
RNA_PREPARATION_REVERSE_TRANSCRIPTION_PRIMER_SEQUENCE: 5' CAAGCAGAAGACGGCATACGAGAT 3'
RNA_PREPARATION_3'_RNA_ADAPTER_LIGATION_PROTOCOL: 1ug of total RNA (RN =>7.0) in a 4uL volume and 2uL of 3' Adenylated Adapter was heat denatured at 70C for 2min then snap chilled on ice for 1min.  Then 1uL of 10X T4 RNL2 truncated buffer, 0.8uL 100mM MgCl2, 0.2uL DEPC water, 0.5uL Rnase Out, and 1.5uL T4 RNA Ligase 2 trancated was added.  Ligation incubated at 22C in a Tetrad thermocycler for 60min.
RNA_PREPARATION_5'_RNA_ADAPTER_LIGATION_PROTOCOL: 5' RNA adapter was heat denatured at 70C for 2min then snap chilled on ice.  2uL of denatured 5' RNA Adapter was added to the 3' DNA Adapter Ligation reaction product from previous step and mixed.   Then 1uL of 10mM ATP and 1uL of Ambion T4 RNA Ligase were added and ligation reaction was incubated at 37C in a Tetrad thermocycler for 60min.
RNA_PREPARATION_REVERSE_TRANSCRIPTION_PROTOCOL: To the 14uL of double adaptered miRNA product from previous step 2ul of RT-Primer was added and the mixture was heat denatured at 65C for 10 mins then quenched on ice.  The following reagents were added immediately to the sample (6ul of 5X First Strand Buffer, 2ul of 10mM dNTPs , 3ul of 100mM DTT, 1ul of RNaseOUT, and 2uL of SuperScript II RT.  Reaction was heated at 44C in a Thetrad thermocycler for 60 minutes.
LIBRARY_GENERATION_PCR_TEMPLATE: 15ul of the first strand cDNA product was used in the PCR
****************
For data usage terms and conditions, please refer to:
http://www.drugabuse.gov/funding/funding-opportunities/nih-common-fund/epigenomics-data-access-policies
****************

value: NHA

value: Brain

value: Astrocyte cell culture

value: primary cell culture

value: brain astrocyte


title: LNCaP DHT TRIM24 ChIP-seq

value: 10nM DHT


description:
design description: miRNA-Seq analysis of placenta (trophoblast) tissue from CTL02 (m08320) using Illumina HiSeq 2000
library name: m08320
EXPERIMENT_TYPE: smRNA-Seq
EXTRACTION_PROTOCOL: Total RNA was extracted using Trizol from Invitrogen as per manufacturer's instructions.
LIBRARY_GENERATION_PCR_POLYMERASE_TYPE: Phusion DNA Polymerase (Hot Start), 2U/ul (from NEB)
LIBRARY_GENERATION_PCR_THERMOCYCLING_PROGRAM: step1: 98C for 30sec, step2: 98C for 10sec, step3: 60C for 30sec, step4: 72C for 15sec.  Repeat step2-4 for 15 cycles, 72C for 5min.
LIBRARY_GENERATION_PCR_NUMBER_CYCLES: 15
LIBRARY_GENERATION_PCR_F_PRIMER_SEQUENCE: 5' AATGATACGGCGACCACCGACAGNNNNNNGTTCAGAGTTCTACAGTCCGA 3'
LIBRARY_GENERATION_PCR_R_PRIMER_SEQUENCE: 5' CAAGCAGAAGACGGCATACGAGAT 3'
LIBRARY_GENERATION_PCR_PRIMER_CONC: 25 uM
LIBRARY_GENERATION_PCR_PRODUCT_ISOLATION_PROTOCOL: 28uL of PCR products containing unique index sequences are pooled together and the 115bp miRNA containing fraction is isolated for each pool either manually:  12% PAGE gel 200V 6hours followed by gel elution, or robotically using Baraccuda size selection robot.  Fractions are Et0H preciptated, QCed on Agilent, and submitted for indexing single end (ISE) Illumina sequencing.
EXTRACTION_PROTOCOL_SMRNA_ENRICHMENT: Flowthrough of polyA+ MultiMACS 96 Separation Unit
SMRNA_PREPARATION_INITIAL_SMRNA_QLTY: RIN 8.2
SMRNA_PREPARATION_INITIAL_SMRNA_QNTY: 0.776 ug
RNA_PREPARATION_5'_RNA_ADAPTER_SEQUENCE: 5' GTTCAGAGTTCTACAGTCCGACGATCTGGTCAA 3'
RNA_PREPARATION_3'_RNA_ADAPTER_SEQUENCE: 5' ATCTCGTATGCCGTCTTCTGCTTGT 3'
RNA_PREPARATION_REVERSE_TRANSCRIPTION_PRIMER_SEQUENCE: 5' CAAGCAGAAGACGGCATACGAGAT 3'
RNA_PREPARATION_3'_RNA_ADAPTER_LIGATION_PROTOCOL: 1ug of total RNA (RN =>7.0) in a 4uL volume and 2uL of 3' Adenylated Adapter was heat denatured at 70C for 2min then snap chilled on ice for 1min.  Then 1uL of 10X T4 RNL2 truncated buffer, 0.8uL 100mM MgCl2, 0.2uL DEPC water, 0.5uL Rnase Out, and 1.5uL T4 RNA Ligase 2 trancated was added.  Ligation incubated at 22C in a Tetrad thermocycler for 60min.
RNA_PREPARATION_5'_RNA_ADAPTER_LIGATION_PROTOCOL: 5' RNA adapter was heat denatured at 70C for 2min then snap chilled on ice.  2uL of denatured 5' RNA Adapter was added to the 3' DNA Adapter Ligation reaction product from previous step and mixed.   Then 1uL of 10mM ATP and 1uL of Ambion T4 RNA Ligase were added and ligation reaction was incubated at 37C in a Tetrad thermocycler for 60min.
RNA_PREPARATION_REVERSE_TRANSCRIPTION_PROTOCOL: To the 14uL of double adaptered miRNA product from previous step 2ul of RT-Primer was added and the mixture was heat denatured at 65C for 10 mins then quenched on ice.  The following reagents were added immediately to the sample (6ul of 5X First Strand Buffer, 2ul of 10mM dNTPs , 3ul of 100mM DTT, 1ul of RNaseOUT, and 2uL of SuperScript II RT.  Reaction was heated at 44C in a Thetrad thermocycler for 60 minutes.
LIBRARY_GENERATION_PCR_TEMPLATE: 15ul of the first strand cDNA product was used in the PCR
****************
For data usage terms and conditions, please refer to:
http://www.drugabuse.gov/funding/funding-opportunities/nih-common-fund/epigenomics-data-access-policies
****************

value: male

value: Placenta

value: Trophoblast

value: No

value: placenta (trophoblast) tissue


description:
design description: miRNA-Seq analysis of placenta (trophoblast) tissue from CTL07 (m09679) using Illumina HiSeq 2000
library name: m09679
EXPERIMENT_TYPE: smRNA-Seq
EXTRACTION_PROTOCOL: Total RNA was extracted using Trizol from Invitrogen as per manufacturer's instructions.
LIBRARY_GENERATION_PCR_POLYMERASE_TYPE: Phusion DNA Polymerase (Hot Start), 2U/ul (from NEB)
LIBRARY_GENERATION_PCR_THERMOCYCLING_PROGRAM: step1: 98C for 30sec, step2: 98C for 10sec, step3: 60C for 30sec, step4: 72C for 15sec.  Repeat step2-4 for 15 cycles, 72C for 5min.
LIBRARY_GENERATION_PCR_NUMBER_CYCLES: 15
LIBRARY_GENERATION_PCR_F_PRIMER_SEQUENCE: 5' AATGATACGGCGACCACCGACAGNNNNNNGTTCAGAGTTCTACAGTCCGA 3'
LIBRARY_GENERATION_PCR_R_PRIMER_SEQUENCE: 5' CAAGCAGAAGACGGCATACGAGAT 3'
LIBRARY_GENERATION_PCR_PRIMER_CONC: 25 uM
LIBRARY_GENERATION_PCR_PRODUCT_ISOLATION_PROTOCOL: 28uL of PCR products containing unique index sequences are pooled together and the 115bp miRNA containing fraction is isolated for each pool either manually:  12% PAGE gel 200V 6hours followed by gel elution, or robotically using Baraccuda size selection robot.  Fractions are Et0H preciptated, QCed on Agilent, and submitted for indexing single end (ISE) Illumina sequencing.
EXTRACTION_PROTOCOL_SMRNA_ENRICHMENT: Flowthrough of polyA+ MultiMACS 96 Separation Unit
SMRNA_PREPARATION_INITIAL_SMRNA_QLTY: RQS 7.1
SMRNA_PREPARATION_INITIAL_SMRNA_QNTY: 1.5379 ug
RNA_PREPARATION_5'_RNA_ADAPTER_SEQUENCE: 5' GTTCAGAGTTCTACAGTCCGACGATCTGGTCAA 3'
RNA_PREPARATION_3'_RNA_ADAPTER_SEQUENCE: 5' ATCTCGTATGCCGTCTTCTGCTTGT 3'
RNA_PREPARATION_REVERSE_TRANSCRIPTION_PRIMER_SEQUENCE: 5' CAAGCAGAAGACGGCATACGAGAT 3'
RNA_PREPARATION_3'_RNA_ADAPTER_LIGATION_PROTOCOL: 1ug of total RNA (RN =>7.0) in a 4uL volume and 2uL of 3' Adenylated Adapter was heat denatured at 70C for 2min then snap chilled on ice for 1min.  Then 1uL of 10X T4 RNL2 truncated buffer, 0.8uL 100mM MgCl2, 0.2uL DEPC water, 0.5uL Rnase Out, and 1.5uL T4 RNA Ligase 2 trancated was added.  Ligation incubated at 22C in a Tetrad thermocycler for 60min.
RNA_PREPARATION_5'_RNA_ADAPTER_LIGATION_PROTOCOL: 5' RNA adapter was heat denatured at 70C for 2min then snap chilled on ice.  2uL of denatured 5' RNA Adapter was added to the 3' DNA Adapter Ligation reaction product from previous step and mixed.   Then 1uL of 10mM ATP and 1uL of Ambion T4 RNA Ligase were added and ligation reaction was incubated at 37C in a Tetrad thermocycler for 60min.
RNA_PREPARATION_REVERSE_TRANSCRIPTION_PROTOCOL: To the 14uL of double adaptered miRNA product from previous step 2ul of RT-Primer was added and the mixture was heat denatured at 65C for 10 mins then quenched on ice.  The following reagents were added immediately to the sample (6ul of 5X First Strand Buffer, 2ul of 10mM dNTPs , 3ul of 100mM DTT, 1ul of RNaseOUT, and 2uL of SuperScript II RT.  Reaction was heated at 44C in a Thetrad thermocycler for 60 minutes.
LIBRARY_GENERATION_PCR_TEMPLATE: 15ul of the first strand cDNA product was used in the PCR
****************
For data usage terms and conditions, please refer to:
http://www.drugabuse.gov/funding/funding-opportunities/nih-common-fund/epigenomics-data-access-policies
****************

value: female


description:
design description: miRNA-Seq analysis of breast luminal epithelial cell from RM084 (m08314) using Illumina HiSeq 2000
library name: m08314
EXPERIMENT_TYPE: smRNA-Seq
EXTRACTION_PROTOCOL: Total RNA was extracted using Trizol from Invitrogen as per manufacturer's instructions.
LIBRARY_GENERATION_PCR_POLYMERASE_TYPE: Phusion DNA Polymerase (Hot Start), 2U/ul (from NEB)
LIBRARY_GENERATION_PCR_THERMOCYCLING_PROGRAM: step1: 98C for 30sec, step2: 98C for 10sec, step3: 60C for 30sec, step4: 72C for 15sec.  Repeat step2-4 for 15 cycles, 72C for 5min.
LIBRARY_GENERATION_PCR_NUMBER_CYCLES: 15
LIBRARY_GENERATION_PCR_F_PRIMER_SEQUENCE: 5' AATGATACGGCGACCACCGACAGNNNNNNGTTCAGAGTTCTACAGTCCGA 3'
LIBRARY_GENERATION_PCR_R_PRIMER_SEQUENCE: 5' CAAGCAGAAGACGGCATACGAGAT 3'
LIBRARY_GENERATION_PCR_PRIMER_CONC: 25 uM
LIBRARY_GENERATION_PCR_PRODUCT_ISOLATION_PROTOCOL: 28uL of PCR products containing unique index sequences are pooled together and the 115bp miRNA containing fraction is isolated for each pool either manually:  12% PAGE gel 200V 6hours followed by gel elution, or robotically using Baraccuda size selection robot.  Fractions are Et0H preciptated, QCed on Agilent, and submitted for indexing single end (ISE) Illumina sequencing.
EXTRACTION_PROTOCOL_SMRNA_ENRICHMENT: Flowthrough of polyA+ MultiMACS 96 Separation Unit
SMRNA_PREPARATION_INITIAL_SMRNA_QLTY: RIN 7
SMRNA_PREPARATION_INITIAL_SMRNA_QNTY: 1.525 ug
RNA_PREPARATION_5'_RNA_ADAPTER_SEQUENCE: 5' GTTCAGAGTTCTACAGTCCGACGATCTGGTCAA 3'
RNA_PREPARATION_3'_RNA_ADAPTER_SEQUENCE: 5' ATCTCGTATGCCGTCTTCTGCTTGT 3'
RNA_PREPARATION_REVERSE_TRANSCRIPTION_PRIMER_SEQUENCE: 5' CAAGCAGAAGACGGCATACGAGAT 3'
RNA_PREPARATION_3'_RNA_ADAPTER_LIGATION_PROTOCOL: 1ug of total RNA (RN =>7.0) in a 4uL volume and 2uL of 3' Adenylated Adapter was heat denatured at 70C for 2min then snap chilled on ice for 1min.  Then 1uL of 10X T4 RNL2 truncated buffer, 0.8uL 100mM MgCl2, 0.2uL DEPC water, 0.5uL Rnase Out, and 1.5uL T4 RNA Ligase 2 trancated was added.  Ligation incubated at 22C in a Tetrad thermocycler for 60min.
RNA_PREPARATION_5'_RNA_ADAPTER_LIGATION_PROTOCOL: 5' RNA adapter was heat denatured at 70C for 2min then snap chilled on ice.  2uL of denatured 5' RNA Adapter was added to the 3' DNA Adapter Ligation reaction product from previous step and mixed.   Then 1uL of 10mM ATP and 1uL of Ambion T4 RNA Ligase were added and ligation reaction was incubated at 37C in a Tetrad thermocycler for 60min.
RNA_PREPARATION_REVERSE_TRANSCRIPTION_PROTOCOL: To the 14uL of double adaptered miRNA product from previous step 2ul of RT-Primer was added and the mixture was heat denatured at 65C for 10 mins then quenched on ice.  The following reagents were added immediately to the sample (6ul of 5X First Strand Buffer, 2ul of 10mM dNTPs , 3ul of 100mM DTT, 1ul of RNaseOUT, and 2uL of SuperScript II RT.  Reaction was heated at 44C in a Thetrad thermocycler for 60 minutes.
LIBRARY_GENERATION_PCR_TEMPLATE: 15ul of the first strand cDNA product was used in the PCR
****************
For data usage terms and conditions, please refer to:
http://www.drugabuse.gov/funding/funding-opportunities/nih-common-fund/epigenomics-data-access-policies
****************

value: Breast

value: Luminal epithelial cell

value: primary cell

value: breast luminal epithelial cell


description:
design description: miRNA-Seq analysis of pancreas islet from ZGI 213 (m09675) using Illumina HiSeq 2000
library name: m09675
EXPERIMENT_TYPE: smRNA-Seq
EXTRACTION_PROTOCOL: Total RNA was extracted using Trizol from Invitrogen as per manufacturer's instructions.
LIBRARY_GENERATION_PCR_POLYMERASE_TYPE: Phusion DNA Polymerase (Hot Start), 2U/ul (from NEB)
LIBRARY_GENERATION_PCR_THERMOCYCLING_PROGRAM: step1: 98C for 30sec, step2: 98C for 10sec, step3: 60C for 30sec, step4: 72C for 15sec.  Repeat step2-4 for 15 cycles, 72C for 5min.
LIBRARY_GENERATION_PCR_NUMBER_CYCLES: 15
LIBRARY_GENERATION_PCR_F_PRIMER_SEQUENCE: 5' AATGATACGGCGACCACCGACAGNNNNNNGTTCAGAGTTCTACAGTCCGA 3'
LIBRARY_GENERATION_PCR_R_PRIMER_SEQUENCE: 5' CAAGCAGAAGACGGCATACGAGAT 3'
LIBRARY_GENERATION_PCR_PRIMER_CONC: 25 uM
LIBRARY_GENERATION_PCR_PRODUCT_ISOLATION_PROTOCOL: 28uL of PCR products containing unique index sequences are pooled together and the 115bp miRNA containing fraction is isolated for each pool either manually:  12% PAGE gel 200V 6hours followed by gel elution, or robotically using Baraccuda size selection robot.  Fractions are Et0H preciptated, QCed on Agilent, and submitted for indexing single end (ISE) Illumina sequencing.
EXTRACTION_PROTOCOL_SMRNA_ENRICHMENT: Flowthrough of polyA+ MultiMACS 96 Separation Unit
SMRNA_PREPARATION_INITIAL_SMRNA_QLTY: RQS 6.7
SMRNA_PREPARATION_INITIAL_SMRNA_QNTY: 1.8371 ug
RNA_PREPARATION_5'_RNA_ADAPTER_SEQUENCE: 5' GTTCAGAGTTCTACAGTCCGACGATCTGGTCAA 3'
RNA_PREPARATION_3'_RNA_ADAPTER_SEQUENCE: 5' ATCTCGTATGCCGTCTTCTGCTTGT 3'
RNA_PREPARATION_REVERSE_TRANSCRIPTION_PRIMER_SEQUENCE: 5' CAAGCAGAAGACGGCATACGAGAT 3'
RNA_PREPARATION_3'_RNA_ADAPTER_LIGATION_PROTOCOL: 1ug of total RNA (RN =>7.0) in a 4uL volume and 2uL of 3' Adenylated Adapter was heat denatured at 70C for 2min then snap chilled on ice for 1min.  Then 1uL of 10X T4 RNL2 truncated buffer, 0.8uL 100mM MgCl2, 0.2uL DEPC water, 0.5uL Rnase Out, and 1.5uL T4 RNA Ligase 2 trancated was added.  Ligation incubated at 22C in a Tetrad thermocycler for 60min.
RNA_PREPARATION_5'_RNA_ADAPTER_LIGATION_PROTOCOL: 5' RNA adapter was heat denatured at 70C for 2min then snap chilled on ice.  2uL of denatured 5' RNA Adapter was added to the 3' DNA Adapter Ligation reaction product from previous step and mixed.   Then 1uL of 10mM ATP and 1uL of Ambion T4 RNA Ligase were added and ligation reaction was incubated at 37C in a Tetrad thermocycler for 60min.
RNA_PREPARATION_REVERSE_TRANSCRIPTION_PROTOCOL: To the 14uL of double adaptered miRNA product from previous step 2ul of RT-Primer was added and the mixture was heat denatured at 65C for 10 mins then quenched on ice.  The following reagents were added immediately to the sample (6ul of 5X First Strand Buffer, 2ul of 10mM dNTPs , 3ul of 100mM DTT, 1ul of RNaseOUT, and 2uL of SuperScript II RT.  Reaction was heated at 44C in a Thetrad thermocycler for 60 minutes.
LIBRARY_GENERATION_PCR_TEMPLATE: 15ul of the first strand cDNA product was used in the PCR
****************
For data usage terms and conditions, please refer to:
http://www.drugabuse.gov/funding/funding-opportunities/nih-common-fund/epigenomics-data-access-policies
****************

value: Pancreas

value: Islet

value: pancreas islet