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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Jan 04, 2021 |
| Title |
RNA_ME1 |
| Sample type |
SRA |
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| Source name |
cerebral cortex
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| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
tissue: cerebral cortex
age: embryonic day 15.5
genotype: Mettl3(f/f, Emx1-cre)
rna fraction: total RNA
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| Extracted molecule |
total RNA |
| Extraction protocol |
For RNA-seq, cerebral cortices of mice were removed, and RNA was harvested using Trizol reagent.NEBNext® UltraTM RNA Library Prep Kit for Illumina® (Cat#E7530L) was used with 1 ug of total RNA for the construction of sequencing libraries. For Ribo-Seq, three groups of E15.5 mouse cerebral cortices were rapidly washed twice in ultra-pure water, and grind thoroughly with liquid nitrogen to powder. Sample was treated with specific lysis buffer with cycloheximide (50mg/mL) to acquire the lysate. Concentration of the lysate was measured by the NanoDrop™ 2000 Spectrophotometer. To digest the RNA other than RPFs, cell or tissue lysate was treated with unspecific endoribonuclease RNase I. Isolation of monosomes was performed by size-exclusion chromatography with MicroSpin S-400 HR columns. The RNA samples were then treated with rRNA depletion kit to deplete the samples of as much rRNA contamination as possible before PAGE purification of the relatively short (20~38 nt) RPFs. Following PAGE purification, the both ends of RPF were phosphorylated and ligated with 5’ and 3’ adapters respectively. Then the fragments were reversely transcribed to the cDNAs and amplified by PCR. After library construction, the concentration of library was measured by The Qubit® 2.0 Fluorometer and adjusted to 1ng/uL. Agilent 2100 Bioanalyzer was deployed to examine the insert size of the acquired library. At last, the accurate concentration of cDNA library was again examined using qPCR. Once the insert size and concentration of the library was identical, the samples can then be subjected for sequencing. Raw data (raw reads) of FASTQ format were firstly processed through cut adapt.
RNA libraries were prepared for sequencing using standard Illumina protocols
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
RNA_ME1 represents the Mettl3(f/f，Emx1-cre) sample biological repeat 1 of RNA-seq
RNA-seq_ read_count.txt
RNA-seq_ RPKM.txt
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| Data processing |
For RNA-seq, In first step, clean data (clean reads) were obtained by removing reads containing adapter, reads containing ploy-N and low quality reads from raw data. At the same time, Q20, Q30 and GC content the clean data were calculated.
Reads mapping to the reference genome was performed.
Index of the reference genome was built using Hisat2 v2.0.5 and paired-end clean reads were aligned to the reference genome using Hisat2 v2.0.5. We selected Hisat2 as the mapping tool,and three sets of raw reads were obtained.
For Ribo-seq, Ribo-seq used TopHat2 for genome mapping.
Quantification of mapped results to gene level was carried out using HTSeq.
For samples with biological replicates, DESeq2 R package (1.14.1) was used for differential expression analysis. DESeq2 provide statistical routines for determining differential expression in digital gene expression data using a model based on the negative binomial distribution.
Genome_build: mm10
Supplementary_files_format_and_content: Ribo-seq_ read_count.txt include raw gene count for each Sample in Ribo-seq
RNA-seq_ read_count.txt include raw gene count for each Sample in RNA-seq
Ribo-seq_ FPKM.txt include FPKM values for each Sample in Ribo-seq
Ribo-seq_ RPKM.txt include RPKM for each Sample in RNA-seq
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| Submission date |
Jul 23, 2020 |
| Last update date |
Jan 05, 2021 |
| Contact name |
kunzhao du |
| E-mail(s) |
Dukunzhao@stu.hqu.edu.cn
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| Organization name |
huaqiao university
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| Street address |
jimei
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| City |
xiamen |
| ZIP/Postal code |
361021 |
| Country |
China |
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| Platform ID |
GPL13112 |
| Series (1) |
| GSE154992 |
Combined analysis of ribo-seq and rna-seq reveals that Mettl3 regulates embryonic cerebral cortex development in mice by controlling the proliferation and differentiation of neural progenitor cells |
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| Relations |
| BioSample |
SAMN15627342 |
| SRA |
SRX8807540 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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