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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Oct 17, 2014 |
| Title |
aphneg0h1 |
| Sample type |
SRA |
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| Source name |
090 hTERT-immortalized fibroblasts
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| Organism |
Homo sapiens |
| Characteristics |
Sex: female
cell type: primary adult skin fibroblast
cell line: 090
merged_sample: 090mrgs0h1
treatment: none
replicate: 1
sequenced molecule: nascent total RNA
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| Treatment protocol |
Bromouridine (Bru, Aldrich) was added to the media at a final concentration of 2 mM and cells were incubated at 37°C for 30 min. Cells were then washed 3 times in PBS and harvested. When indicated, cells were pre-treated for 24 hours with 0.4 uM aphidicolin prior to Bru labeling.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated by using TRIzol reagent (Invitrogen), and Bru-labeled RNA was isolated from the total RNA by incubation with anti-BrdU antibodies (BD Biosciences) conjugated to magnetic beads (Dynabeads, Goat anti-Mouse IgG; Invitrogen) under gentle agitation at room temperature for 1 h.
Bru-labeled RNA was mixed with first strand buffer and random primers and fragmented by heating at 85 ̊C for 10 minutes. The first strand cDNA was then synthesized in the presence of Actinomycin D to result in strand specific reads. After purifying the first strand cDNA using AMPure RNAclean beads (Beckman Coulter), the second strand cDNA was synthesized. The resulting cDNA was purified with AMPure XP beads, after which the Illumina TruSeq RNA Sample Prep Kit was used to repair the cDNA ends, adenylate and ligate adaptors to the cDNA. The samples were then run on a 3% agarose gel and size-selected by excising gel slices in the 300bp region. These gel slices were purified using the QIAEX II Gel Extraction Kit (Qiagen) and then the Illumina TruSeq Kit PCR reagents were used to enrich the DNA fragments. After a final purification using AMPure XP beads, the quality and concentration of the DNA libraries were determined using an Agilent Bioanalyzer.
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
genome_build: hg19
supplementary_files_format_and_content: segments.bed = extended BED format file, one record for each HMM transcription segment, where column 4 is a unique identifier for the corresponding fused and revised transcription unit in format chrom:start-end(strand), column 5 is always 0, column 7 is the segment’s HMM state (0..9), column 8 is segment RPKM, and columns 9 is the transcription unit RPKM.
supplementary_files_format_and_content: genes.bed = BED format file, one record per gene, where column 4 is the gene name and column 5 is its RPKM
Library strategy: Bru-Seq (variant of RNA-Seq)
Call bases with Illumina Casava v1.8.2.
Subtract reads that align to the reference ribosomal DNA complete repeating unit using bowtie v.0.12.8 and parameters -n 3 -k 1 -m 1.
Align remaining reads to the reference genome using TopHat v1.4.1 and parameters –min-isoform-fraction 0, --max-multihits 1, --no-closure-search, --no-coverage-search, --bowtie-n, --initial-read-mismatches 3.
Determine fractional read coverage for every genome base using Bedtools v.2.16.2 such that a base covered by one read is recorded as having a coverage of 1/read_length.
Calculate fractional RPKM values for annotated genes, where a gene is the span of its various transcript isoforms.
Separately calculate RPKM values for 1 Kb genome bins.
Merge all samples from each cell line by summing their read counts per 1 Kb bin and recalculating bin RPKM values.
Subject merged bin RPKM values to a strand-specific Hidden Markov model (HMM) segmentation algorithm with ten logarithmically distributed output transcription states (indices 0 to 9, bed column 7).
Calculate aggregate RPKM values for each called transcription segment (bed column 8).
Empirically assign HMM transcription states of 0 to 2 a Boolean transcription state of “not transcribed” and HMM states 3 to 9 a Boolean transcription state of “transcribed”.
Fuse adjacent transcribed segments into preliminary transcription units.
Revise transcription units by comparing them to the Ensembl gene annotation such that transcription units covering at least 80% of any transcript isoform of each of two adjacent genes on the same strand were split at the most proximal transcription start site of all matching transcripts of the downstream gene.
Calculate aggregate RPKM for each fused and revised transcription unit (bed column 9).
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| Submission date |
Mar 13, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Thomas E. Wilson |
| Organization name |
University of Michigan
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| Department |
Department of Pathology
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| Street address |
2065 BSRB, 109 Zina Pitcher
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| City |
Ann Arbor |
| State/province |
MI |
| ZIP/Postal code |
48109-2200 |
| Country |
USA |
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| Platform ID |
GPL11154 |
| Series (1) |
| GSE55862 |
Bru-seq nascent RNA sequencing of human and mouse cells: correlation to replication timing and genome instability |
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| Relations |
| BioSample |
SAMN02688755 |
| SRA |
SRX487506 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1347653_GSM1347654_090mrgs0h1.segments.bed.gz |
909.7 Kb |
(ftp)(http) |
BED |
| GSM1347653_aphneg0h1.genes.bed.gz |
680.8 Kb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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