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Sample GSM1371407

Query DataSets for GSM1371407

Status

Public on Aug 19, 2015

Title

WT_total_RNA_CT00_rep2

Sample type

SRA

Source name

U2-OS Cells

Organism

Homo sapiens

Characteristics

cell type: osteosarcoma
cell line: U2-OS
replicate: 2
sirna treatment: None

Treatment protocol

To synchronize cells grown overnight, their media was replaced with phenol red free DMEM containing 0.1 mM luciferin and 0.1 μM dexamethasone. Plates were then sealed with sterile vacuum grease (Corning) and incubated in a 36˚C, non-humidified incubator at 5% CO2

Growth protocol

A U2OS human osteosarcoma cell line expressing luciferase under the control of the Per2 promoter was used. Cells were maintained in a humidified 37˚C incubator at 5% CO2 in DMEM (Life Technologies), supplemented with 10% FBS, 4 mM L-glutamine, 50 units/mL penicillin, and 50 μg/mL streptomycin. 2.25x10^6 cells were seeded per 15 cm plate. These cells were grown overnight.

Extracted molecule

total RNA

Extraction protocol

Cells in 15 cm plates were treated with 100 μg/mL cycloheximide for 3 minutes at 36˚C, 24.5 hours post-synchronization when luciferase levels were at their nadir. Cells were 80% confluent at the time of collection. All subsequent steps were done on ice. Spent media was aspirated from the plates and the cells were washed with 10 mL of cold PBS before treatment with 790 μL of cold lysis buffer [20 mM Tris, pH 7.5, 150 mM NaCl, 5 mM MgCl2, 1 mM dithiothreitol, 100 μg/mL cycloheximide, 1% Triton X-100, and 0.02 units/μL Turbo DNase]. Cells were recovered from the plate using a cell scraper, triturated with a 1.0 mL pipette, and incubated on ice for 10 minutes with occasional mixing. The lysate was then triturated 10 times through a 26 gauge needle and centrifuged at 20,000 g for 10 minutes at 4˚C. 150 μL of the cleared lysate was added to 700 μL of QIAzol lysis reagent and stored at -80˚C. Collection of cells was repeated every 2 hours for an entire 24 hour time course. Cleared lysate frozen with QIAzol lysis reagent was processed according to the Qiagen miRNeasy Quick-Start Protocol.
RNA integrity was verified by Bioanalyzer (Agilent Technologies) before library preparation as described according to the TruSeq Stranded Total RNA Sample Prep Kit (Illumina).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Data processing

Raw data was aligned to GRCh37/hg19 genome using STAR (v2.3.0e) with default parameters.
RPF reads mapping to gene coding sequences (CDSs) were quantified by running HTSeq (v0.5.4p1) in stranded mode, with default parameters. To assign a single CDS to each gene, the isoforms with the longest CDSs were used for quantification. RNA-seq reads were similarly quantified across the entire length of the transcript. Gene models were taken from the UCSC Known Genes annotation.
Read counts were normalized with DESeq2, using default parameters.
Genome_build: GRCh37/hg19
Supplementary_files_format_and_content: txt files contain normalized, gene-level read counts

Submission date

Apr 18, 2014

Last update date

May 15, 2019

Contact name

Nicholas Lahens

Organization name

University of Pennsylvania

Department

ITMAT

Street address

Smilow Center for Translational Research 10-110 3400 Civic Center Blvd, Bldg 421