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Sample GSM1891622 Query DataSets for GSM1891622
Status Public on Jun 15, 2017
Title RPL10A-Ribo-Seq (3xFLAG-Rpl10a cells)_rep1
Sample type SRA
 
Source name Embryonic stem cells (ESCs)
Organism Mus musculus
Characteristics cell line: E14 ESC
genotype: Bearing 1 allele of 3xFLAG-Rpl10a at endogeneous locus
ip antibody: anti-FLAG M2 magnetic beads (Sigma, Catalog# M8823, Lot# SLBL1128V)
Treatment protocol 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 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, 0.1 mg/ml heparin, 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. After a brief wash, 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. 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.
All Ribo-Seq libraries were prepared as described in Ingolia, N. T., Brar, G. A., Rouskin, S., McGeachy, A. M. & Weissman, J. S. The ribosome profiling strategy for monitoring translation in vivo by deep sequencing of ribosome-protected mRNA fragments. Nat Protoc 7, 1534–1550 (2012)
 
Library strategy OTHER
Library source transcriptomic
Library selection other
Instrument model Illumina NextSeq 500
 
Description Ribosome footprints
Data processing library strategy: 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.
Genome_build: mm10
Supplementary_files_format_and_content: Text file includes UCSC_IDs and RPKM values for each protein-coding gene
 
Submission date Sep 23, 2015
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
Relations
BioSample SAMN04112937
SRA SRX1287939

Supplementary file Size Download File type/resource
GSM1891622_Rpl10a-Ribo-Seq_1.txt.gz 47.9 Kb (ftp)(http) TXT
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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