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Status |
Public on Jun 13, 2013 |
Title |
Cytoplasm DMSO 1 |
Sample type |
SRA |
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Source name |
HEK293 Cytoplasm DMSO 1
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Organism |
Homo sapiens |
Characteristics |
cell line: HEK293 cell type: human embryonic kidney
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Treatment protocol |
HEK cells starved for glutamine for 12h. 30 minutes proior to feeding with complete media, 100nM rapamycin was added to the starvation media. After 30 minutes, complete media -/+ rapamycin was added and cells were cultured for 7h.
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Extracted molecule |
polyA RNA |
Extraction protocol |
Cytosolic extracts (detergent lysis) were prepared from control and rapa treated cells. 10% of cytosolic extract was used to prepare RNA with TrireagentLS. The remainder was fractionated across 15-45% sucrose gradients centriguged at 100,000xg in Beckman SW41 rotor. Polyribosome peaks were purified using a Gradient Station by Biocomp Instruments. TruSeq mRNA (Illumina)
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Library strategy |
OTHER |
Library source |
transcriptomic |
Library selection |
other |
Instrument model |
Illumina HiSeq 2000 |
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Description |
Cyd01
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Data processing |
Library strategy: Frac-Seq Illumina Casava1.8.2 software used for basecalling. All paired end reads were trimmed at the 3' end to a final length of 75x58bp Reads were mapped with bowtie2 to the set of human repeatMasker elements and these reads were eliminated from further processing. The trimmed, repeatMasker filtered, sequenced reads (75bp read1, 58bp read2) were mapped to hg19 with tophat (2.0.4) using bowtie2 (2.0.0-beta7), with an estimated mean distance between the read pairs of 5bp (100bp std dev). A gtf file of the hg19 UCSC Known Genes browser track was supplied to TopHat. In the case of multiply mapped reads, the single best hit as scored by bowtie2 was kept. Single-end mappings were discarded. Potential PCR duplicates at each mapping position were removed using Samtools rmdup. Genome_build: hg19 Transcript levels in HEK293 cytosolic and polyribosomal fractions was determined using HiSeq2000. We used the Mixture of Isoforms (MISO) Bayesian Inference model v1.0 for quantification of alternative RNA processing events (Katz et al. 2010). The MISO algorithm counts the numbers of reads that are common to both events and the reads that are exclusive to one isoform or the other, in order to estimate the %-spliced-in (Y) in a given sample. We employed an “event-centric” approach, considering annotated alternative mRNA processing events with at least 20 exclusive reads and a maximum confidence interval width of 0.45 for the MISO estimated posterior distribution of percent-spliced-in (“Psi”, Y, Supplemental Table 5 lists all of the MISO outputs). The MISO output is described in the linked supplementary files (*MISO.txt). Additionally the tables include annotations of cis-regulatory elements that overlap either the Y or 1-Y isoform.
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Submission date |
Mar 18, 2013 |
Last update date |
May 15, 2019 |
Contact name |
Jeremy Sanford |
E-mail(s) |
jsanfor2@ucsc.edu
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Organization name |
UC Santa Cruz
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Department |
MCD Biology
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Street address |
1156 High Street
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City |
Santa Cruz |
State/province |
CA |
ZIP/Postal code |
95064 |
Country |
USA |
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Platform ID |
GPL11154 |
Series (1) |
GSE45237 |
Frac-seq reveals preferential polyribosome association of minor mRNA isoforms |
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Relations |
SRA |
SRX251916 |
BioSample |
SAMN01983866 |
Supplementary data files not provided |
SRA Run Selector |
Raw data are available in SRA |
Processed data are available on Series record |
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