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| Status |
Public on Apr 18, 2012 |
| Title |
polyA RNA sequencing of H1 BMP4 Derived Mesendoderm Cells; polyA-RNA-seq_h1-mesendoderm_r2a |
| Sample type |
SRA |
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| Source name |
H1 BMP4 Derived Mesendoderm Cultured Cell Line, biological replicate 2 (r2); polyA-RNA-seq_h1-mesendoderm_r2a
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| Organism |
Homo sapiens |
| Characteristics |
sample alias: H1-Mesendoderm_RNA_r2
sample common name: H1 BMP4 Derived Mesendoderm Cultured Cells
disease: None
biomaterial_provider: Thomson Laboratory
biomaterial_type: Cell Line
line: H1
lineage: NA
differentiation_stage: embryonic stem cell differentiated by treatment with fibronectin and human collagen
differentiation_method: H1 cells were cultured on plates coated with fibronectin and human collagen
passage: 5
medium: 50% StemLine II serum-free HSC expansion medium (HSFEM; Sigma), 50% ESFM, GlutaMAX (1/100 dilution), Ex-Cyte supplement (1/2000 dilution), 100 mM MTG, and 10 ng/ml FGF2.
Sex: Male
batch: Replicate 2
experiment_type: mRNA-Seq
extraction_protocol: Qiagen RNeasy mini kit, performed as per manufacturer's instructions
cdna_preparation_initial_rna_qnty: 4 µg
cdna_preparation_polya_rna/: Dynabeads oligo(dt)25 (Invitrogen)
cdna_preparation_fragmentation: PolyA RNA resuspended in 2x Superscript III buffer supplemented with 10 mM DTT was incubated at 94°C for 4 min
cdna_preparation_fragment_size_range: 200-250
cdna_preparation_first_strand_synthesis_enzyme: SuperScript III (Invitrogen)
cdna_preparation_first_strand_purification: Purification with RNAClean XP beads (Agencourt)
cdna_preparation_second_strand_synthesis_enzyme: DNA Polymerase I (E. coli) (New England Biolabs)
cdna_preparation_second_strand_synthesis_dntp_mix: dNTP mix made from 10 mM dATP, dCTP, dGTP, dUTP
cdna_preparation_purification: Purification with AMPure XP beads (Agencourt)
dna_preparation_adaptor: TruSeq Multiplexed Adapters (Illumina)
dna_preparation_adaptor_ligation_protocol: 16°C for 16 hours with T4 DNA ligase (New England Biolabs)
dna_preparation_post-ligation_fragment_size_selection: Two rounds of purification with AMPure XP beads (Agencourt)
dna_preparation_uracil_dna_glycosylase_digestion: One half of adapter-ligated cDNA digested with uracil DNA glycosylase (Enzymatics) and used for subsequent steps.
library_generation_pcr_polymerase_type: TruSeq PCR Master Mix (Illumina)
library_generation_pcr_thermocycling_program: 98°C 30 sec; 10 cycles of 98°C 10 sec, 60°C 30 sec, 72°C 30 sec; 72°C 10 min
library_generation_pcr_number_cycles: 10
library_generation_pcr_primer: TruSeq PCR Primer Cocktail (Illumina)
library_generation_pcr_product_isolation_protocol: Purification with AMPure XP beads (Agencourt)
extraction_protocol_mrna_enrichment: Dynabeads oligo(dt)25 protocol
rna_preparation_reverse_transcription_primer_sequence: Random hexamers
rna_preparation_reverse_transcription_protocol: First-strand Superscript III (Invitrogen) RT protocol. RNAClean XP purification of first strand product. Second-strand synthesis using dUTP-containing dNTPS and DNA Polymerase 1 (E. coli) (New England Biolabs)
library_generation_pcr_template: cDNA
library_generation_pcr_template_conc: 5 ng/µL
library_generation_pcr_f_primer_sequence: 5' AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATC 3'
library_generation_pcr_r_primer_sequence: 5' CAAGCAGAAGACGGCATACGAGAT 3'
library_generation_pcr_primer_conc: 200nM
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| Extracted molecule |
total RNA |
| Extraction protocol |
Library construction protocol: Four µg of total RNA was extracted from frozen cell pellets using the RNeasy Mini Kit (Qiagen, Valencia, CA). PolyA RNA was isolated from total RNA using Dynabeads oligo(dt)25 (Invitrogen). PolyA selected RNA was eluted in 2x Superscript III buffer supplemented with 10mM DTT and was fragmented by incubation at 94°C for 4 minutes. First-strand cDNA was synthesized with the following reaction composition: 0.5 µL random hexamer, 0.5 µL RNase Out, 1 µL DTT (100 mM), 0.5 µL dNTP (25 mM), 0.5 µL SuperScript III (20 µL final). The reaction was incubated at 25°C for 10 min then 50°C for 50 min. Single-stranded cDNA was isolated by purification with RNAClean XP beads (Beckman Coulter Genomics, Danvers, MA). The dUTP-containing second-strand was synthesized with the following reaction: 1.5 µL NEBuffer 2 (NEB), 1 µL dNTP mix (10 mM dATP, dCTP, dGTP, and dUTP), 0.2 µL RNase H, 1 µL DNA Polymerase I (E. coli) (New England Biolabs) (15 µL final). The reaction was incubated at 16°C for 2.5 hours. The dUTP-containing cDNA was isolated by purification with AMPure XP beads (Beckman Coulter Genomics, Danvers, MA). The dUTP-containing cDNA was then end repaired and a 3' A base was added. TruSeq adapters (Illumina, San Diego, CA) were ligated to the dUTP-containing cDNA at 16°C for 16 hours with T4 DNA ligase (New England Biolabs). Adapter-ligated cDNA was isolated by two rounds of purification with AMPure XP beads. The dUTP-containing strands were digested at 37°C for 30 min with uracil DNA glycosylase (Enzymatics). Adapter-ligated, single-stranded DNA molecules were enriched by 10 cycles of PCR with the following reaction composition: 5 µL TruSeq PCR Primer Cocktail (Illumina), 25 µL TruSeq PCR Master Mix (Illumina) (50 µL final). The thermocycling parameters were: 98°C 30 sec, then 10-15 cycles of 98°C 10 sec, 60°C 30 sec and 72°C 30 sec, ending with one 72°C 5 min step. The reaction products were purified using AMPure XP beads.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
sample_term_id: NTR_0000856
assay_term_id: OBI_0001271
nucleic_acid_term_id: SO_0000871
Design description: polyA RNA sequencing of H1 BMP4 Derived Mesendoderm Cells
Library name: polyA-RNA-seq_h1-mesendoderm_r2a
EDACC Genboree Experiment Page:
http://genboree.org/java-bin/project.jsp?projectName=XML%20Submissions%2FUCSD%2FEXPERIMENT%2FEDACC.12101
EDACC Genboree Sample Page:
http://genboree.org/java-bin/project.jsp?projectName=XML%20Submissions%2FUCSD%2FSAMPLE%2FEDACC.12099
****************
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: GSM915325_UCSD.H1_BMP4_Derived_Mesendoderm_Cultured_Cells.mRNA-Seq.polyA-RNA-seq_h1-mesendoderm_r2a.bed
ANALYSIS CENTER: EDACC
ANALYSIS ALIAS: polyA-RNA-seq_h1-mesendoderm_r2a.hg19.level.1.release.8
ANALYSIS TITLE: Mapping of H1 BMP4 Derived Mesendoderm Cultured Cells mRNA-Seq Data
ANALYSIS DESCRIPTION: Illumina reads produced by mRNA-Seq on H1 BMP4 Derived Mesendoderm Cultured Cells, Library polyA-RNA-seq_h1-mesendoderm_r2a 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.13727
DATA_ANALYSIS_LEVEL: 1
EXPERIMENT_TYPE: mRNA-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: 1
TREATMENT_OF_MULTIPLE_ALIGNMENTS: If a read maps to more than 1 position it is removed from consideration.
TREATMENT_OF_IDENTICAL_ALIGNMENTS_OF_MULTIPLE_READS: None
ALIGNMENT_POSTPROCESSING: None
RELEASE_NUMBER: Human Epigenome Atlas 8
QUALITY SCORES:
NUMBER_OF_MAPPED_READS: 317,296,097
NUMBER_OF_MRNA-SEQ_EXPERIMENTS_SCORED_IN_THIS_RELEASE: 22
PERCENT_READS_MAPPING_TO_UCSC_GENES: 83.72
PERCENT_READS_MAPPING_TO_UCSC_GENES_PERCENTILE: 23
MAXIMUM_REPLICATE_CORRELATION: 0.65
**********************************************************************
ANALYSIS FILE NAME: GSM915325_UCSD.H1_BMP4_Derived_Mesendoderm_Cultured_Cells.mRNA-Seq.polyA-RNA-seq_h1-mesendoderm_r2a.wig
ANALYSIS CENTER: EDACC
ANALYSIS ALIAS: polyA-RNA-seq_h1-mesendoderm_r2a.hg19.level.2.release.8
ANALYSIS TITLE: Raw Signal Density Graphs of H1 BMP4 Derived Mesendoderm Cultured Cells mRNA-Seq Data
ANALYSIS DESCRIPTION: Illumina mRNA-Seq read mappings from H1 BMP4 Derived Mesendoderm Cultured Cells, Library polyA-RNA-seq_h1-mesendoderm_r2a 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.13737
DATA_ANALYSIS_LEVEL: 2
EXPERIMENT_TYPE: mRNA-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 8
BROWSER_TRACK_NAME: HBDM mRNA 2a
BROWSER_TRACK_DESCRIPTION: UCSD H1 BMP4 Derived Mesendoderm Cultured Cells mRNA-Seq Library polyA-RNA-seq_h1-mesendoderm_r2a EA Release 8
QUALITY SCORES:
NUMBER_OF_MAPPED_READS: 317,296,097
NUMBER_OF_MRNA-SEQ_EXPERIMENTS_SCORED_IN_THIS_RELEASE: 22
PERCENT_READS_MAPPING_TO_UCSC_GENES: 83.72
PERCENT_READS_MAPPING_TO_UCSC_GENES_PERCENTILE: 23
MAXIMUM_REPLICATE_CORRELATION: 0.65
**********************************************************************
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| Submission date |
Apr 13, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
UCSD AND SALK |
| Organization name |
University of California, San Diego
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| Street address |
Health Sciences Drive
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| City |
La Jolla |
| State/province |
CA |
| ZIP/Postal code |
92092 |
| Country |
USA |
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| Platform ID |
GPL11154 |
| Series (1) |
| GSE16256 |
UCSD Human Reference Epigenome Mapping Project |
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| Relations |
| Reanalyzed by |
GSE87112 |
| Reanalyzed by |
GSE99453 |
| SRA |
SRX142112 |
| BioSample |
SAMN00855413 |
| Named Annotation |
GSM915325_UCSD.H1_BMP4_Derived_Mesendoderm_Cultured_Cells.mRNA-Seq.polyA-RNA-seq_h1-mesendoderm_r2a.wig.gz |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM915325_UCSD.H1_BMP4_Derived_Mesendoderm_Cultured_Cells.mRNA-Seq.polyA-RNA-seq_h1-mesendoderm_r2a.bed.gz |
3.6 Gb |
(ftp)(http) |
BED |
| GSM915325_UCSD.H1_BMP4_Derived_Mesendoderm_Cultured_Cells.mRNA-Seq.polyA-RNA-seq_h1-mesendoderm_r2a.wig.gz |
21.2 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
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