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| Status |
Public on Oct 13, 2009 |
| Title |
Strand-specific, shotgun sequencing of mRNA from the IMR90 cell line; mRNA-seq_imr90_r1 |
| Sample type |
SRA |
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| Source name |
Strand-specific, shotgun sequencing of mRNA from the IMR90 cell line
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| Organism |
Homo sapiens |
| Characteristics |
disease: None
biomaterial_provider: ATCC
biomaterial_type: Cell Line
line: IMR90
lineage: NA
batch: 1
differentiation_stage: Fetal lung fibroblast
differentiation_method: NA
passage: 4
medium: Minimum Essential Medium, Eagle with Earle's Balanced Salt Solution supplemented with 10% Fetal Bovine Serum (FBS), 1% Pen/Strep (penicillin, streptomycin), 1% GlutaMax (stable form of glutamine)
Sex: Female
experiment_type: mRNA-Seq
extraction_protocol: mirVana miRNA isolation kit (Applied Biosystems), performed as per manufacturer's instructions to isolate total RNA, followed by treatment with DNaseI (Qiagen) for 30 min at room temperature
extraction_protocol_mrna_enrichment: 2 x RiboMinus (Life Technologies) rRNA depletion (5S, 5.8S, 12S, 18S, 28S)
extraction_protocol_fragmentation: 15 min at 70 ˚C with RNA Fragmentation kit (Ambion/Applied Biosystems) as per manufacturer's instructions
mrna_preparation_initial_mrna_qnty: 200 ng
mrna_preparation_fragment_size_range: 50-400 bp
rna_preparation_5'_rna_adapter_sequence: 5' GUUCAGAGUUCUACAGUCCGACGAUC
rna_preparation_3'_rna_adapter_sequence: 5' UCGUAUGCCGUCUUCUGCUUGidT, 5' adenylated (Illumina)
rna_preparation_reverse_transcription_primer_sequence: 5' CAAGCAGAAGACGGCATACGA
rna_preparation_5'_dephosphorylation: Fragmented RNA was treated with 5 U Antarctic phosphatase (New England Biolabs) for 40 min at 37˚C in the presence of 40 U RNaseOut followed by phosphatase heat inactivation at 65˚C for 5 min. The RNA was purified using 66 µl SPRI beads (Agencourt) and eluted in 11 µl 10 mM Tris buffer pH 8.0.
rna_preparation_5'_phosphorylation: Phosphorylation was performed by addition of 10 U PNK (New England Biolabs), 1 mM ATP, and 20 U RNaseOut (Life Technologies) and incubation at 37˚C for 1 h.
rna_preparation_3'_rna adapter_ligation_protocol: 1 µl of 1:10 diluted adenylated 3’ RNA adapter oligonucleotide was added to the phosphorylated RNA and incubated at 70˚C for 2 min followed by placement on ice. The 3’ RNA adapter ligation reaction was performed by addition of 2 µl 10x T4 RNA ligase 2 truncated ligation buffer, 1.6 µl 100 mM MgCl2, 20 U RNaseOut and 300 U T4 RNA ligase 2 truncated (New England Biolabs) and incubation at 22˚C for 1 h.
rna_preparation_5'_rna_adapter_ligation_protocol: Ligation of the 5’ RNA adapter was performed by addition to the 3’ adapter ligated reaction of 1 µl 1:1 diluted, heat denatured (70˚C 2 min) 5’ RNA adapter oligonucleotide, 1 µl 10 mM ATP, and 10 U T4 RNA ligase (Promega), and incubation at 20˚C for 1 h. RNA was purified using 66 µl SPRI beads and eluted in 10 µl 10 mM Tris buffer pH 8.0.
rna_preparation_reverse_transcription_protocol: To the RNA ligation products, 2 µl 1:5 diluted RT primer was added and heat denatured (70˚C 2 min), followed by incubation on ice. Added to the denatured RNA/primer solution was 4 µl 5x first strand buffer, 1 µl 12.5 mM dNTPs, 2 µl 100 mM DTT, and 40 U RNaseOut, followed by incubation at 48˚C for 1 min. To this, 200 U Superscript II reverse transcriptase (Life Technologies) was added, followed by incubation at 44˚C for 1 h.
library_generation_pcr_template: The entire reverse transcription reaction was used in the PCR enrichment of the library.
library_generation_pcr_polymerase_type: Phusion hot-start high fidelity DNA polymerase (New England Biolabs). 1x Phusion polymerase buffer and 4 U Phusion hot-start high fidelity DNA polymerase was used in a 100 µl reaction.
library_generation_pcr_thermocycling_program: 98˚C 30 sec; 98˚C 10 sec, 60˚C 30 sec, 72˚C 15 sec; 72˚C 10 min
library_generation_pcr_number_cycles: 15
library_generation_pcr_f_primer_sequence: 5' AATGATACGGCGACCACCGACAGGTTCAGAGTTCTACAGTCCGA
library_generation_pcr_r_primer_sequence: 5' CAAGCAGAAGACGGCATACGA
library_generation_pcr_primer_conc: 0.25 µM
library_generation_pcr_product_isolation_protocol: PCR products were purified in two steps, first by purification using 180 µl SPRI beads and elution in 30 µl 10 mM Tris buffer pH 8.0, followed by purification with 39 µl SPRI beads and elution in 10 µl 10 mM Tris buffer pH 8.0.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Library construction protocol: Total RNA was isolated from IMR90 cells and mRNA isolated by RiboMinus depletion of rRNA. mRNA was fragmented, dephosphorylated, 5' phosphorylated, then sequentially ligated to the adenylated 3' RNA adapter then to the 5' RNA adapter. Following reverse transcription of the adapter ligated RNA, the library was enriched by 15 cycles of PCR.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina Genome Analyzer II |
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| Description |
sample_term_id: EFO_0001196
assay_term_id: OBI_0001271
nucleic_acid_term_id: SO_0000871
Library name: mRNA-seq_imr90_r1
EDACC Genboree Sample Page:
http://genboree.org/java-bin/project.jsp?projectName=XML%20Submissions%2FUCSD%2FSAMPLE%2FEDACC.1121
EDACC Genboree Experiment Page:
http://genboree.org/java-bin/project.jsp?projectName=XML%20Submissions%2FUCSD%2FEXPERIMENT%2FEDACC.1440
****************
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: GSM438363_UCSD.IMR90.mRNA-Seq.mRNA-seq_imr90_r1.bed
ANALYSIS CENTER: EDACC
ANALYSIS ALIAS: mRNA-seq_imr90_r1.hg19.level.1.release.4
ANALYSIS TITLE: Mapping of IMR90 Cell Line mRNA-Seq Data
ANALYSIS DESCRIPTION: SOLiD reads produced by mRNA-Seq on the IMR90 Cell Line, Library IMR90 mRNA 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.9489
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 4
QUALITY SCORES:
NUMBER_OF_MAPPED_READS: 27,821,581
NUMBER_OF_MRNA-SEQ_EXPERIMENTS_SCORED_IN_THIS_RELEASE: 8
PERCENT_READS_MAPPING_TO_UCSC_GENES: 93.6
PERCENT_READS_MAPPING_TO_UCSC_GENES_PERCENTILE: 100
MAXIMUM_REPLICATE_CORRELATION: NA
**********************************************************************
ANALYSIS FILE NAME: GSM438363_UCSD.IMR90.mRNA-Seq.mRNA-seq_imr90_r1.wig
ANALYSIS CENTER: EDACC
ANALYSIS ALIAS: mRNA-seq_imr90_r1.hg19.level.2.release.4
ANALYSIS TITLE: Raw Signal Density Graphs of IMR90 Cell Line mRNA-Seq Data
ANALYSIS DESCRIPTION: SOLiD mRNA-Seq read mappings from the IMR90 Cell Line, Library IMR90 mRNA 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.9497
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 4
BROWSER_TRACK_NAME: IMR90 mRNA r1
BROWSER_TRACK_DESCRIPTION: UCSD IMR90 Cell Line mRNA-Seq Library mRNA-seq_imr90_r1 EA Release 4
QUALITY SCORES:
NUMBER_OF_MAPPED_READS: 27,821,581
NUMBER_OF_MRNA-SEQ_EXPERIMENTS_SCORED: 8
PERCENT_READS_MAPPING_TO_UCSC_GENES: 93.6
PERCENT_READS_MAPPING_TO_UCSC_GENES_PERCENTILE: 100
MAXIMUM_REPLICATE_CORRELATION: NA
**********************************************************************
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| Submission date |
Aug 11, 2009 |
| Last update date |
May 15, 2019 |
| Contact name |
UCSD AND SALK |
| Organization name |
University of California, San Diego
|
| Street address |
Health Sciences Drive
|
| City |
La Jolla |
| State/province |
CA |
| ZIP/Postal code |
92092 |
| Country |
USA |
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| Platform ID |
GPL9115 |
| Series (1) |
| GSE16256 |
UCSD Human Reference Epigenome Mapping Project |
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| Relations |
| Reanalyzed by |
GSE99453 |
| SRA |
SRX007167 |
| BioSample |
SAMN00004463 |
| Named Annotation |
GSM438363_UCSD.IMR90.mRNA-Seq.mRNA-seq_imr90_r1.wig.gz |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM438363_UCSD.IMR90.mRNA-Seq.mRNA-seq_imr90_r1.bed.gz |
207.2 Mb |
(ftp)(http) |
BED |
| GSM438363_UCSD.IMR90.mRNA-Seq.mRNA-seq_imr90_r1.wig.gz |
5.2 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
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