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| Status |
Public on Feb 14, 2016 |
| Title |
MCF10A control RP |
| Sample type |
SRA |
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| Source name |
MCF10A control RP
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| Organism |
Homo sapiens |
| Characteristics |
cell line: MCF10A
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| Treatment protocol |
For inhibition of mTOR and nutrient starvation experiments, MCF10A cells were treated either with 250 nM of Torin 1 (Tocris Bioscience, Bristol, UK) or with EBSS medium (Sigma) for 2 hours, respectively.
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| Growth protocol |
MCF10A cells were cultured in DMEM/F12 1:1 medium supplemented with 5% horse serum, EGF (10 ng/ml), insulin (10 μg/ml), cholera toxin (100 ng/ml), and hydrocortisone (500 ng/ml) in 5% CO2 at 37°C.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Approximately 30e6 cells were treated with chloramphenicol (100μg/ml) for 15 minutes and cycloheximide (100μg/ml) for 5 minutes. Cells were lysed in buffer B (20 mM Tris-HCl, pH 7.8, 100mM KCl, 10mM MgCl2, 1% Triton X-100, 2mM DTT, 100 μg/ml chloramphenicol, 100 μg/ml cycloheximide, 1x Complete protease inhibitor). Lysates were centrifuged at 5000 rpm and the supernatant was treated with 2U/μl of RNase I (Ambion) for 40 minutes at room temperature. Lysates were fractionated on a linear sucrose gradient (7% - 47%) using the SW- 41Ti rotor at 36,000 rpm for 2hrs. Fractions enriched in mito-monosomes and cytosolic monosomes were identified by western blotting, pooled and treated with proteinase K (Roche) in 1% SDS. Released RPFs were purified using Trizol reagent (Invitrogen) following the manufacturer’s instructions.
RNA was gel-purified on a denaturing 10% polyacrylamide urea (7 M) gel. A section corresponding to 30-33 nucleotides was excised, eluted and ethanol precipitated. The resulting fragments were 3′-dephosphorylated using T4 polynucleotide kinase (New England Biolabs Inc. Beverly, MA, USA) for 6 h at 37°C in 2-(N-morpholino)ethanesulfonic acid (MES) buffer (100 mM MES-NaOH, pH 5.5, 10 mM MgCl2, 10 mM β-mercaptoethanol, 300 mM NaCl). 3′ adaptor was added with T4 RNA ligase 1 (New England Biolabs Inc. Beverly, MA, USA) for 2.5 h at 37°C. Ligation products were 5′-phosphorylated with T4 polynucleotide kinase for 30 min at 37°C. 5′ adaptor was added with T4 RNA ligase 1 for 18 h at 22°C.
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
adapter sequence (3'): TGGAATTCTCGGGTGCCAAGGAACTCCAGTCACATCACGATCTCGTATGCCGTCTTCTGCTTG
ribosome profiling
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| Data processing |
(Ribosome profiling data only) Adapter sequences were trimmed from raw data using cutadapt 1.1 with parameters (--quality-base=33 -O 12 -m 20 -q 5). Reads without adapter sequence were discarded.
rRNA was filtered from data by alignment to transcript database compiled from Ensembl 37.65 (gene categories “rRNA”, “rRNA_pseudogene” and “Mt_rRNA”) using bowtie (v2.0.6) with parameters (--seed 42 --local) and retention of the unmapped reads.
Analogous to rRNA filtering, tRNA was filtered by alignment to a tRNA database compiled from the GtRNAdb (Chan and Lowe, NAR, 2009).
Reads not mapped in previous steps were used in final alignment using tophat (v2.0.6) with parameters (--seed 42 -n 2 -m 1 --no-novel-juncs --no-novel-indels --no-coverage-search --segment-length 25) to hg19 and transcripts defined by GENCODE v19/BASIC.
Genome_build: hg19
Supplementary_files_format_and_content: WIG files were generated from alignment files output by tophat in the final alignment step, only using primary alignments and alignments with mapping quality >= 10. Scores indicate read coverage, normalized to reads per 10 million reads.
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| Submission date |
Jul 28, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Koos Rooijers |
| E-mail(s) |
k.rooijers@nki.nl
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| Organization name |
Netherlands Cancer Institute
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| Department |
Gene Regulation
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| Lab |
Agami Lab
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| Street address |
Plesmanlaan 121
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| City |
Amsterdam |
| ZIP/Postal code |
1066CX |
| Country |
Netherlands |
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| Platform ID |
GPL11154 |
| Series (2) |
| GSE59816 |
Ribosome profiling and RNA sequencing of MCF10A cells treated with torin or by serum starvation |
| GSE59821 |
Restrictions in amino acid availability revealed by differential ribosome codon reading |
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| Relations |
| BioSample |
SAMN02943084 |
| SRA |
SRX663255 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1446834_MCF10A_control_1_batch3_RP.normalized.minus.wig.gz |
4.0 Mb |
(ftp)(http) |
WIG |
| GSM1446834_MCF10A_control_1_batch3_RP.normalized.plus.wig.gz |
4.1 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Processed data provided as supplementary file |
| Raw data are available in SRA |
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