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| Status |
Public on Sep 30, 2018 |
| Title |
P0 Ovary ELAVL2 RIP-Seq |
| Sample type |
SRA |
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| Source name |
P0 ovary
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| Organism |
Mus musculus |
| Characteristics |
tissue: ovary
age: P0
rip antibody: ELAVL2 (Protein tech, 14008-1-AP)
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| Treatment protocol |
Newborn ovaries were disected in ice-cold PBS and were immeadiately frozen in liquid nitrogen. Frozen tissues were stored at -80 degree until the use.
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| Growth protocol |
Mice were housed in special pathogen free room
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| Extracted molecule |
total RNA |
| Extraction protocol |
One hundred newborn ovaries were homogenized in IP buffer (20 mM HEPES/KOH, pH7.5, 150 mM NaCl, 2.5 mM MgCl2, 0.1% NP-40, 1 mM dithiothreitol, 1× protease inhibitor cocktail (Roche), 100 U ml-1 RNase inhibitor (Takara)),and the debris was removed by centrifugation (10000 × g at 4°C for 10 min).The supernatants were incubated with magnetic beads conjugated to protein G (Invitrogen), which were pre-incubated with anti-ELAVL2 antibody (5 μg, Protein tech, 14008-1-AP) at 4°C for 2 hours.The lysate-bead mixtures were then incubated at 4°C for 6 hours with gentle rotation. The bead-antibody complexes were washed thrice with IP buffer and transferred to fresh 1.5 ml tubes. The complexes were washed again with IP buffer. The precipitated proteins and RNA were eluted by incubating the beads-antibody complex with the elution buffer (IP buffer containing 0.5% SDS) at 70°C for 5 min. Input samples (5% of starting materials) were taken from the centrifuged supernatants. Input and eluted samples were dissolved in TRIzol reagent (Invitrogen), and total RNAs were extracted according to the manufacture's protocol. The quality of RNAs was checked using the 2100 Bioanalyzer (Agilent).
Anti-sense RNAs were amplified by using TargetAmp 1-Round aRNA Amplification Kit 103 (Epicentre) according to the manufacture's protocol, and sequence libraries were then generated using the KAPA Stranded mRNA-Seq Kit Illumina platform (KAPA Biosystems) according to the manufacturer's protocol. The resulting libraries were sequenced on the Illumina GAIIx (single end 36 base reads with TruSeq SBS kit v5 for GA., Illumina).
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| Library strategy |
RIP-Seq |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina Genome Analyzer IIx |
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| Data processing |
Basecalls and Demultiplexing were performed using CASAVA version 1.8.2
Quality check of RIP-Seq reads was performed using FastQC version 0.11.2 and any duplicated reads were discarded
The RIP-Seq reads were aligned to the mm10 genome assembly using TopHat2 version 2.0.8b with no-multiplehit option
Peakcalls performed using MACS2 version 2.1.0.20140616 with following options; nomodel, q-value cutoff = 0.05, broad region calling is off
The peaks were annotated using ChIPpeakAnno and biomaRt; reference database is Ensembl
Genome_build: mm10
Supplementary_files_format_and_content: tsv files were generated using MACS2 and R; the peak file can be modified to load IGV
Supplementary_files_format_and_content: Elavl2_MACS2Out_16dec2015_peaks.tsv: Result of MACS2 peakcalls
Supplementary_files_format_and_content: elavl2.rip_narrow_peak.ENSMUSG-gene_id.tsv: Annotated peaks with ENSMUSG IDs
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| Submission date |
Apr 18, 2018 |
| Last update date |
Sep 30, 2018 |
| Contact name |
Yuzuru Kato |
| E-mail(s) |
yukato@nig.ac.jp
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| Phone |
+81-55-981-6832
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| Organization name |
National Institute of Genetics
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| Department |
Division of Mammalian Development
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| Lab |
Saga lab.
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| Street address |
1111 Yata
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| City |
Mishima |
| State/province |
Shizuoka |
| ZIP/Postal code |
411-8540 |
| Country |
Japan |
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| Platform ID |
GPL11002 |
| Series (2) |
| GSE113304 |
Primordial follicle formation involves dynamic conversion of RNA regulatory proteins (RIP-seq data set) |
| GSE113305 |
Primordial follicle formation involves dynamic conversion of RNA regulatory proteins |
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| Relations |
| BioSample |
SAMN08945486 |
| SRA |
SRX3960355 |
| 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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