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GEO help: Mouse over screen elements for information. |
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Status |
Public on Jul 26, 2017 |
Title |
Ribo_seq_shABCF1_rep2 |
Sample type |
SRA |
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Source name |
mammalian cells
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Organism |
Mus musculus |
Characteristics |
cell type: mouse embryonic fibroblast strain: C57BL/6J
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Treatment protocol |
MEF cells were grown to 80% confluence on 10-cm petri dish and treated with DMSO or 250 nM Torin1 for 2 h.
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Growth protocol |
MEF wild-type and knock-down cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) with 10% fetal bovine serum (FBS).
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Extracted molecule |
total RNA |
Extraction protocol |
MEF cells were washed using ice-cold PBS containing 100 µg ml-1 cycloheximide and then lysed in polysome lysis buffer ((10 mM HEPES, pH 7.4, 100 mM KCl, 5 mM MgCl2, 100 µg ml-1 cycloheximide and 2% Triton X-100). Cell debris were removed by centrifugation at14,000 rpm for 10 min at 4°C. Fragmented RNA were dephosphorylated for 1 hr at 37°C in 15 µl reaction (1× T4 polynucleotide kinase buffer, 10 U SUPERase_In and 20 U T4 polynucleotide kinase). The products were separated on a 15% polyacrylamide TBE-urea gel (Invitrogen) and visualized using SYBR Gold (Invitrogen). Selected regions of the gel corresponding to 40-60 nt (for RNA-seq ) or 25-35 nt (for Ribo-seq) were excised. The gel slices were disrupted by using centrifugation through the holes at the bottom of the tube. RNA fragments were dissolved by soaking overnight in 400 μl gel elution buffer (300 mM NaOAc, pH 5.5, 1 mM EDTA, 0.1 U/ml SUPERase_In). The gel debris was removed using a Spin-X column (Corning), followed by ethanol precipitation.Purified RNA fragments were tailed followed by reverse transcription. Reverse transcription products were purified on a 10% polyacrylamide TBE-urea gel and circularized. Circular single-strand DNA was re-linearized and the expected 100-nt product bands were excised for purification followed by PCR amplification. PCR products were recovered and sent for deep sequencing.
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Library strategy |
OTHER |
Library source |
transcriptomic |
Library selection |
other |
Instrument model |
Illumina Genome Analyzer |
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Description |
total RNA following rRNA removal
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Data processing |
Library strategy: RIBO-seq For replicate 1, the 3' adaptor AAAAAAAAAA and low quality bases were trimmed by Cutadapt. The trimmed reads were mapped to Mouse transcriptom (GRCm38.83) by Bowtie, using parameters: -a --best -m1 --strata. Two mismatches were permitted. For replicate 2, the 3' adaptor CTGTAGGCACCATCAAT were trimmed by Cutadapt. The first 6 nucleotide and the last 4 nucleotides were also clipped after adaptor removal. The trimmed reads were mapped to Mouse transcriptom (GRCm38.83) by Bowtie, using parameters: -a --best -m1 --strata. Two mismatches were permitted. The PCR duplicates were removed from the mapped reads using the methods in Lecanda et al., 2016 (PMID: 27450428) Genome_build: GRCm38.83 Supplementary_files_format_and_content: RPKM files were generated. Each line represents one mRNAs. RPKM values were separated by Tabs.
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Submission date |
Jul 25, 2017 |
Last update date |
May 15, 2019 |
Contact name |
Yuanhui Mao |
E-mail(s) |
maoyuanhui123@gmail.com
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Organization name |
Zhejiang University
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Street address |
1369 West Wenyi Road
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City |
Hangzhou |
State/province |
Zhejiang |
ZIP/Postal code |
310000 |
Country |
China |
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Platform ID |
GPL9185 |
Series (1) |
GSE101865 |
Scope and mechanism of eIF4F-independent mRNA translation |
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Relations |
BioSample |
SAMN07414077 |
SRA |
SRX3033793 |
Supplementary file |
Size |
Download |
File type/resource |
GSM2717363_RPKM_Ribo_seq_CGG_ABCF1_KD_rep2.txt.gz |
131.1 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
Raw data are available in SRA |
Processed data provided as supplementary file |
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