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| Status |
Public on May 17, 2010 |
| Title |
miRNA-Seq analysis of breast variant human mammary epithelial cell from RM035 (HS1740) |
| Sample type |
SRA |
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| Source name |
HS1740-1
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| Organism |
Homo sapiens |
| Characteristics |
submitted sample id: MIC01-RNA
donor_id: RM035
Sex: female
body site: Breast
histological type: Variant human mammary epithelial cell culture
is tumor: No
biomaterial_type: primary cell culture
cell type: breast variant human mammary epithelial cell
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| Extracted molecule |
total RNA |
| Extraction 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/)
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| Library strategy |
miRNA-Seq |
| Library source |
transcriptomic |
| Library selection |
size fractionation |
| Instrument model |
Illumina Genome Analyzer IIx |
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| Description |
design description: miRNA-Seq analysis of breast variant human mammary epithelial cell from RM035 (HS1740) using Illumina Genome Analyzer IIx
library name: HS1740
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 8.9
SMRNA_PREPARATION_INITIAL_SMRNA_QNTY: 1.0 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
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For data usage terms and conditions, please refer to:
http://www.drugabuse.gov/funding/funding-opportunities/nih-common-fund/epigenomics-data-access-policies
****************
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| Data processing |
**********************************************************************
ANALYSIS FILE NAME: GSM543034_UCSF-UBC.Breast-vHMEC-Individual-2.smRNA-Seq.Run108066Lane7.wig
ANALYSIS ALIAS: UCSF-UBC.Breast-vHMEC-Individual-2.smRNA-Seq.Run108066Lane7
ANALYSIS TITLE: Raw Signal Density Graphs of Breast, vHMEC, Individual 2 smRNA-Seq Data
ANALYSIS DESCRIPTION: Illumina smRNA-Seq read mappings from Breast, vHMEC, Individual 2 were processed into density graphs of raw signal representing the aligned read density.
ANALYSIS TYPE: ABUNDANCE_MEASUREMENT
EDACC Genboree Analysis Page: http://genboree.org/java-bin/project.jsp?projectName=XML%20Submissions%2FEDACC%2FANALYSIS%2FEDACC.2823
DATA_ANALYSIS_LEVEL: 2
EXPERIMENT_TYPE: smRNA-Seq
SOFTWARE: In house programs and scripts
SOFTWARE_VERSION: NA
READ_EXTENSION: 0bp
GENOMIC_WINDOW: 20bp
TREATMENT_OF_REGIONS_PRONE_TO_MULTIPLE_ALIGNMENTS: None
**********************************************************************
ANALYSIS FILE NAME: GSM543034_UCSF-UBC.Breast_vHMEC.smRNA-Seq.RM035.bed
ANALYSIS CENTER: EDACC
ANALYSIS ALIAS: HS1740-1_430N8AAXX.hg19.level.1
ANALYSIS TITLE: Mapping of Breast vHMEC smRNA-Seq Data
ANALYSIS DESCRIPTION: Illumina reads produced by smRNA-Seq on Breast vHMEC, Donor RM035 were mapped to the human genome using Pash.
ANALYSIS TYPE: REFERENCE_ALIGNMENT
EDACC Genboree Analysis Page: http://genboree.org/java-bin/project.jsp?projectName=XML%20Submissions%2FEDACC%2FANALYSIS%2FEDACC.5203
DATA_ANALYSIS_LEVEL: 1
EXPERIMENT_TYPE: smRNA-Seq
GENOME_ASSEMBLY: NCBI Build GRCh37/UCSC Build hg19
SOFTWARE: Pash
SOFTWARE_VERSION: 3.0
MAXIMUM_ALIGNMENT_LENGTH: Read length
MISMATCHES_ALLOWED: 10% of read length
ALIGNMENTS_ALLOWED: 100
TREATMENT_OF_MULTIPLE_ALIGNMENTS: If a read maps to more than 100 positions it is removed from consideration.
TREATMENT_OF_IDENTICAL_ALIGNMENTS_OF_MULTIPLE_READS: None
ALIGNMENT_POSTPROCESSING: None
RELEASE_NUMBER: Human Epigenome Atlas 2
PERCENT_READS_MAPPING_TO_miRBase_v16_miRNA: 84.41
PERCENT_READS_MAPPING_TO_REPEATMASKER_RNA_REPEATS: 5.0
**********************************************************************
ANALYSIS FILE NAME: GSM543034_UCSF-UBC.Breast_vHMEC.smRNA-Seq.RM035.wig
ANALYSIS CENTER: EDACC
ANALYSIS ALIAS: HS1740-1_430N8AAXX.hg19.level.2
ANALYSIS TITLE: Raw Signal Density Graphs of Breast vHMEC smRNA-Seq Data
ANALYSIS DESCRIPTION: Illumina smRNA-Seq read mappings from Breast vHMEC, Donor RM035 were processed into density graphs of raw signal representing the aligned read density.
ANALYSIS TYPE: ABUNDANCE_MEASUREMENT
EDACC Genboree Analysis Page: http://genboree.org/java-bin/project.jsp?projectName=XML%20Submissions%2FEDACC%2FANALYSIS%2FEDACC.5205
DATA_ANALYSIS_LEVEL: 2
EXPERIMENT_TYPE: smRNA-Seq
GENOME_ASSEMBLY: NCBI Build GRCh37/UCSC Build hg19
SOFTWARE: In house programs and scripts
SOFTWARE_VERSION: NA
READ_EXTENSION: 0bp
GENOMIC_WINDOW: 20bp
TREATMENT_OF_REGIONS_PRONE_TO_MULTIPLE_ALIGNMENTS: None
RELEASE_NUMBER: Human Epigenome Atlas 2
BROWSER_TRACK_NAME: Bv smRNA 35
BROWSER_TRACK_DESCRIPTION: UCSF-UBC-UCD Breast vHMEC smRNA-Seq Donor RM035 EA Release 2
PERCENT_READS_MAPPING_TO_miRBase_v16_miRNA: 84.41
PERCENT_READS_MAPPING_TO_REPEATMASKER_RNA_REPEATS: 5.0
**********************************************************************
ANALYSIS FILE NAME: GSM543034_UCSF-UBC.Breast_vHMEC.smRNA-Seq.RM035.tag.tab
ANALYSIS CENTER: EDACC
ANALYSIS ALIAS: HS1740-1_430N8AAXX.hg19.level.2.tag
ANALYSIS TITLE: Sequence Tag Counts of Breast vHMEC smRNA-Seq Data
ANALYSIS DESCRIPTION: Illumina reads produced by smRNA-Seq on Breast vHMEC, Donor RM035 had the sequence trimmed for adapters and the count of unique sequences calculated.
ANALYSIS TYPE: ABUNDANCE_MEASUREMENT
EDACC Genboree Analysis Page: http://genboree.org/java-bin/project.jsp?projectName=XML%20Submissions%2FEDACC%2FANALYSIS%2FEDACC.5207
DATA_ANALYSIS_LEVEL: 2
EXPERIMENT_TYPE: smRNA-Seq
GENOME_ASSEMBLY: NCBI Build GRCh37/UCSC Build hg19
SOFTWARE: In house programs and scripts
SOFTWARE_VERSION: NA
RELEASE_NUMBER: Human Epigenome Atlas 2
PERCENT_READS_MAPPING_TO_miRBase_v16_miRNA: 84.41
PERCENT_READS_MAPPING_TO_REPEATMASKER_RNA_REPEATS: 5.0
**********************************************************************
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| Submission date |
May 12, 2010 |
| Last update date |
May 15, 2019 |
| Contact name |
UCSF-UBC CENTER |
| Organization name |
UCSF-UBC
|
| Street address |
UCSF-UBC
|
| City |
San Francisco |
| State/province |
CA |
| ZIP/Postal code |
94143 |
| Country |
USA |
| |
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| Platform ID |
GPL10999 |
| Series (1) |
| GSE16368 |
UCSF-UBC Human Reference Epigenome Mapping Project |
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| Relations |
| Named Annotation |
GSM543034_UCSF-UBC.Breast_vHMEC.smRNA-Seq.RM035.wig.gz |
| Named Annotation |
GSM543034_UCSF-UBC.Breast-vHMEC-Individual-2.smRNA-Seq.Run108066Lane7.wig.gz |
| SRA |
SRX1314836 |
| BioSample |
SAMN03416820 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM543034_UCSF-UBC.Breast-vHMEC-Individual-2.smRNA-Seq.Run108066Lane7.wig.gz |
12.5 Mb |
(ftp)(http) |
WIG |
| GSM543034_UCSF-UBC.Breast_vHMEC.smRNA-Seq.RM035.bed.gz |
2.8 Mb |
(ftp)(http) |
BED |
| GSM543034_UCSF-UBC.Breast_vHMEC.smRNA-Seq.RM035.tag.tab.gz |
345.3 Kb |
(ftp)(http) |
TAB |
| GSM543034_UCSF-UBC.Breast_vHMEC.smRNA-Seq.RM035.wig.gz |
850.3 Kb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Processed data provided as supplementary file |
| Raw data are available in SRA |
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