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| Status |
Public on Jun 15, 2017 |
| Title |
RPS25-Ribo-Seq (Rps25-3xFLAG cells)_rep2 |
| Sample type |
SRA |
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| Source name |
Embryonic stem cells (ESCs)
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| Organism |
Mus musculus |
| Characteristics |
cell line: E14 ESC
genotype: Bearing 1 allele of Rps25-3xFLAG at endogeneous locus
ip antibody: anti-FLAG M2 magnetic beads (Sigma, Catalog# M8823, Lot# SLBL1128V)
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| Treatment protocol |
Cells were briefly treated with 100 μg/ml cycloheximide (Sigma) in mESC media for 1 min prior to harvest.
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| 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.
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| 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)
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
Ribosome footprints
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| 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
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| Submission date |
Sep 23, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Zhen Shi |
| Organization name |
Stanford
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| Street address |
279 Campus Drive
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| City |
Stanford |
| State/province |
California |
| ZIP/Postal code |
94305 |
| Country |
USA |
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| Platform ID |
GPL19057 |
| Series (1) |
| GSE73357 |
Heterogeneous ribosomes exist and selectively translate distinct subpools of mRNAs in stem cells |
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| Relations |
| BioSample |
SAMN04112934 |
| SRA |
SRX1287944 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1891627_Rps25-Ribo-Seq_2.txt.gz |
49.5 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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