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Sample GSM2579690 Query DataSets for GSM2579690
Status Public on Jun 15, 2017
Title Rpl10a siRNA_combined_heavy_RNA-seq_rep2
Sample type SRA
Source name Embryonic stem cells (ESCs)
Organism Mus musculus
Characteristics cell line: E14 ESC
genotype: Wild type mESCs treated with Rpl10a siRNA
Treatment protocol Gene knockdown was achieved by transfecting mESCs with small double-stranded interfering RNAs (siRNA) with Lipofectamine 2000 (Invitrogen, 11668). siRNA used for targeting Rpl10a: SASI_Mm01_00200342 (Sigma). MISSION siRNA Universal Negative Control 2 (Sigma, SIC002) was used as a control. Cells were briefly treated with 100 μg/ml cycloheximide (Sigma) in mESC media for 1 min prior to harvest.
Growth protocol E14 mouse embryonic stem cells (ESCs) were grown in knockout DMEM (Invitrogen 10829-018) with 15% ESC grade FBS (EMD Millipore ES-009-B), 1x L-glutamine (EMD Millipore TMS-002-C), 1x ES-grade Penicillin/streptomycin (EMD Millipore TMS-AB2-C), 1x non-essential amino acid (EMD Millipore TMS-001-C), 0.1% 2-mercaptoethanol, 1x Leukemia Inhibitory Factor (LIF, EMD Millipore ESG1107) in 37 °C, 5% CO2 incubator. 1 hr prior to harvest, cells were incubated with fresh mESC media and were at ~60-70% confluency.
Extracted molecule total RNA
Extraction protocol The RNA-Seq libraries following gradient fractionation were prepared using the TruSeq Stranded mRNA Library Prep Kit (Illumina) by the Stanford Functional Genomics Facility and sequenced on a NextSeq sequencer (Illumina) with 2 x 75 bp read length.
The media was then aspirated and cells were scraped from the plate in cold PBS with 100 μg/ml cycloheximide. After centrifugation, the cell pellet was re-suspended in cold lysis buffer (20 mM Tris pH 7.5, 150 mM NaCl, 15 mM MgCl2, 100 μg/ml cycloheximide, 1 mM DTT, 0.5% Triton X-100, 8% glycerol, 20 U/ml TURBO™ DNase, 1x Combined Protease and Phosphatase Inhibitor (Thermo 78443)) and incubated for 30 min at 4°C with occasional vortex. The lysate was clarified by sequential centrifugation for 5 min at 1,800 x g and 10,000 x g at 4°C to remove nucleus and mitochondria. A 250 μl of lysate (with RNA concentration of ~ 1 μg/μl) was then treated with RNase A/T1 mix (0.5 mg RNase A (Life Technologies AM2272) and 1,000 U RNase T1 (Life Technologies 2280)) for 30 min at room temperature to digest mRNAs not protected by the ribosome. The digestion was stopped by adding 4.5 μl of SUPERase-In™ RNase Inhibitor (20 U/μl, Ambion AM2696). For the total Ribo-Seq, lysate was then loaded onto a 1 M sucrose cushion. Ribosomes were pelleted by centrifugation at 70, 000 rpm for 4 hr at 4°C. Pellet were re-suspended in TRIzol® for RNA extraction. For the RPL10A-Ribo-Seq and RPS25-Ribo-Seq, ribosomes containing RPS25 or RPL10A were enriched by IP using anti-FLAG® M2 magnetic beads (Sigma) on a head-over-tail rotator at 4 °C for 2 hrs. For RPL22-Ribo-Seq, ribosomes containing RPL22 were enriched by IP by incubating with anti-HA antibody (ab9110) on a head-over-tail rotator at 4 °C for 4 hrs and and then Dynabeads Protein A (Life Technologies, 10001D) at 4 °C for 1 hr. Beads were washed 5 min for 3 times in washing buffer (20 mM Tris pH 7.5, 200 mM NaCl, 15 mM MgCl2, 100 μg/ml cycloheximide, 0.5% Triton X-100). After the final wash, liquid was aspirated and immunoprecipitated ribosomes were eluted in TRIzol® for RNA extraction. Polysome profiling was performed comparing the control siRNA and Rpl10a knockdown. After polysome fractionation, RNA was extracted using Acid-Phenol:Chloroform, pH 4.5 (with IAA, 125:24:1) (Ambion, AM9722). We collected RNA from combined light fractions containing the free RNPs, 40S/60S ribosomal free subunits, 80S/monosome as well as light polysomes (2-3 ribosomes on a mRNA molecule), and combined heavy polysome fractions containing the most actively translating ribosomes (≥4 ribosomes along a mRNA molecule).
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina NextSeq 500
Description RNA (with rRNA depletion)
Data processing For Ribo-Seq, sequencing reads were parsed by cutadapt to remove the 3’ adapter sequence and reads with good sequencing qualities (ascii quality score>33) were kept. A layered alignment employing Bowtie 2 was performed to first discard reads mapping to rRNA, tRNA or snRNA sequences. Non-rRNA/tRNA/snRNA reads were then aligned against the canonical isoform of UCSC known gene transcripts (mm10). Mapped footprints were assigned to a specific position based on the A sites. The position of A site in relative to the 5’ end of each read is calculated as follows: 29-30 nt: +15; 31-33 nt: +16 and 34-35 nt: +17. For each gene, the total number of ribosome footprints mapping to the CDS excluding the first 15 or last 5 codons were counted. The density of ribosome footprints was calculated as Reads Per Kilobase per Million mapped reads (RPKM). Genes having ≥5 RPKM were kept for further analysis.
For polysome profiling, the paired-end reads were aligned to the mouse genome using the STAR RNA-Seq aligner (Dobin et al., 2013), and the total number of reads uniquely mapped to each genes were counted. We then calculated the amount of mRNAs in the combined medium and heavy polysome fractions compared to all other fractions as an estimate of their translation activities.
Genome_build: mm10
Supplementary_files_format_and_content: Text files include normalized read values for protein-coding genes
Submission date Apr 17, 2017
Last update date May 15, 2019
Contact name Zhen Shi
Organization name Stanford
Street address 279 Campus Drive
City Stanford
State/province California
ZIP/Postal code 94305
Country USA
Platform ID GPL19057
Series (1)
GSE73357 Heterogeneous ribosomes exist and selectively translate distinct subpools of mRNAs in stem cells
BioSample SAMN06759977
SRA SRX2741996

Supplementary file Size Download File type/resource
GSM2579690_Rpl10a_siRNA_combined_heavy_2.txt.gz 46.3 Kb (ftp)(http) TXT
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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