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Sample GSM1099616

Query DataSets for GSM1099616

Status

Public on Jun 13, 2013

Title

Cytoplasm DMSO 2

Sample type

SRA

Source name

HEK293 Cytoplasm DMSO 2

Organism

Homo sapiens

Characteristics

cell line: HEK293
cell type: human embryonic kidney

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.

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)

Library strategy

OTHER

Library source

transcriptomic

Library selection

other

Instrument model

Illumina HiSeq 2000

Description

Cyd05

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.

Submission date

Mar 18, 2013

Last update date

May 15, 2019

Contact name

Jeremy Sanford

E-mail(s)

jsanfor2@ucsc.edu

Organization name

UC Santa Cruz

Department

MCD Biology