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Sample GSM1371469

Query DataSets for GSM1371469

Status

Public on Aug 19, 2015

Title

Bmal1_kd_ribo_profile_CT04_rep1

Sample type

SRA

Source name

U2-OS Cells

Organism

Homo sapiens

Characteristics

cell type: osteosarcoma
cell line: U2-OS
replicate: 1
sirna treatment: Bmal1 siRNA

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. Cleared lysate was flash frozen in a dry ice/ethanol bath before storage at -80˚C. Collection of cells was repeated every 2 hours for an entire 24 hour time course.
Frozen cleared lysate was thawed on ice, and RNase I (Life Technologies) was added to 400 μL of cleared lysate to a final activity of 0.75 units/μL. This mixture was incubated for 45 minutes at room temperature with gentle mixing on a tube rotator, and the nuclease was subsequently inactivated by the addition of 20 μL of SUPERase•In (Life Technologies). The digestion was carefully layered onto a 0.9 mL sucrose cushion [20 mM Tris, pH 7.5, 150 mM NaCl, 5 mM MgCl2, 1 mM dithiothreitol, 100 μg/mL cycloheximide, 20 units/mL SUPERase•In, 34% sucrose, w/v] in an 11  34 mm polycarbonate ultracentrifuge tube (Beckman Coulter), and centrifuged for 4 hours at 78,000 rpm in a prechilled TLA-100.2 rotor. The ribosome pellet was resuspended in 700 μL of QIAzol lysis reagent and processed according to the Qiagen miRNeasy Quick-Start Protocol. RNA was then isopropanol precipitated from the elution, and resuspended in 5 μL of 10 mM Tris, pH 8.0. RNA samples were run on a 15% TBE-Urea gel (Life Technologies), stained with SYBR Gold, and visualized with a Dark Reader blue light transilluminator (Clare Chemical Research). The 26-34 nt region demarcated by the size selection RNA oligos NI-NI-19 and NI-NI-20 was excised from the gel, and the RPFs were extracted and isopropanol precipitated from the gel fragment. Library generation was performed as described previously, with some modifications. Briefly, RPFs were resuspended in 10 μL of 10 mM Tris, pH 8.0, and dephosphorylated with T4 polynucleotide kinase (New England Biolabs) in the presence of 20 units of SUPERase•In in a final volume of 40 μL. The reaction was incubated for 1 hour at 37˚C, and inactivated for 10 minutes at 70˚C. RNAs were isopropanol precipitated, and ligated to the preadenylated and 3′ blocked cloning Linker-1 (IDT) by incubation with truncated T4 RNA ligase 2 (New England Biolabs) for 2.5 hours at room temperature. Ligated RPFs were isopropanol precipitated and gel purified from a 15% TBE-Urea gel before being reverse transcribed with SuperScript III (Life Technologies), using primer NI-NI-9. The RNA template was then removed by alkaline hydrolysis, and the reverse transcription products were isopropanol precipitated and gel purified. DNA fragments were circularized with CircLigase (Epicentre) in a final volume of 20 μL and reverse transcription products corresponding to rRNAs were removed by subtractive hybridization using MyOne Streptavidin C1 Dynabeads (Life Technologies) and the three biotinylated DNA oligos NI-NI-21, NI-NI-23, and NI-NI-24, which are complementary to the rRNAs with accession numbers NR_003285.2:124-155, NR_003287.1:182-213, and NR_003287.1:1435-1461, respectively. The resulting DNA was precipitated and used as a template for PCR amplification with Phusion polymerase (New England Biolabs) using NI-NI-2 and one of the index amplification primers CJ-NI-4, CJ-NI-6, CJ-NI-12, or CJ-NI-23.

Library strategy

OTHER

Library source

transcriptomic

Library selection

other

Instrument model

Illumina HiSeq 2000

Description

In order to knockdown Bmal1 expression, 3.0x10^4 cells were seeded per 35 mm plate for the purposes of monitoring. For collection, 5.6x10^5 cells were seeded per 15 cm plate. These cells were grown overnight in complete DMEM without antibiotics, and transfected using Lipofectamine RNAiMAX (Life Technologies) according to the manufacturer’s protocols. A mixture of siRNAs that target Bmal1 (siBMAL1-1 thru siBMAL1-5) was utilized, and 12 picomoles of this mixture was added to each 35 mm plate, and 120 picomoles of siRNA mixture was added to each 15 cm plate. Cells were then synchronized with dexamethasone following transfection as described in the treatment protocol.
library selection: Ribosome profiling (Ribo-seq)

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