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| Status |
Public on Apr 13, 2021 |
| Title |
HP_ovary2 |
| Sample type |
SRA |
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| Source name |
ovary with high egg production performance
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| Organism |
Columba livia |
| Characteristics |
tisssue: ovary
age: 2 years old
development satge: sexual maturity
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| Treatment protocol |
none
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| Growth protocol |
none
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated and purified using Trizol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer's procedure. The total RNA quantity and purity were analysis of Bioanalyzer 2100 and RNA 1000 Nano LabChip Kit (Agilent, CA, USA) with RIN number >7.0.
Approximately 5 ug of total RNA was used to deplete ribosomal RNA according to the manuscript of the Ribo-Zero™ rRNA Removal Kit (Illumina, San Diego, USA). After the enriched circRNAs were fragmented into small pieces using divalent cations under high temperature. After removing ribosomal RNAs, the left RNAs were reverse-transcribed to create the cDNA,which were next used to synthesise U-labeled second-stranded DNAs with E. coli DNA polymerase I, RNase H and dUTP. An A-base is then added to the blunt ends of each strand, preparing them for ligation to the indexed adapters. Each adapter contains a T-base overhang for ligating the adapter to the A-tailed fragmented DNA. Single-or dual-index adapters are ligated to the fragments, and size selection was performed with AMPureXP beads. After the heat-labile UDG enzyme treatment of the U-labeled second-stranded DNAs.The ligated products are amplified with PCR by the following conditions: initial denaturation at 95℃ for 3 min; 8cycles of denaturation at 98℃ for 15 sec, annealing at 60℃ for 15 sec, and extension at 72℃ for 30 sec; and then final extension at 72℃ for 5 min. The average insert size for the final cDNA library was 300 bp (±50 bp). At last, we performed the paired-end sequencing on an Illumina Novaseq™ 6000 following the vendor's recommended protocol.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
ovary with high egg production performance
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| Data processing |
Firstly, cutadapt-1.9 (cutadapt.readthedocs.io/en/stable/) was used to remove the reads that contained adapter contamination, low quality bases and undetermined bases. Then sequence quality was verified using FastQC v0.10.1 (www.bioinformatics.babraham.ac.uk/projects/fastqc/).
We used hisat2-2.0.4 (ccb.jhu.edu/software/hisat2/)to map reads to the genome of Homo sapiens, Ensembl v96 (command line: hisat2 -1 R1.fastq.gz -2 R2.fastq.gz -S mapped.sam). The mapped reads of each sample were assembled using stringtie-1.3.4 (ccb.jhu.edu/software/stringtie/) with default parameters (command line: stringtie -p 2 -G genome.gtf -o output.gtf -l mapped.bam). Then, all transcriptomes from Samples were merged to reconstruct a comprehensive transcriptome using gffcompare (github.com/gpertea/gffcompare/). After the final transcriptome was generated, StringTie was used to perform expression level for mRNAs by calculating FPKM (FPKM = [total_exon_fragments / mapped_reads(millions) × exon_length(kb)]).
LncRNA identification:First of all, transcripts that overlapped with known mRNAs and transcripts shorter than 200 bp were discarded. Then we utilized CPC0.9-r2 (cpc2.cbi.pku.edu.cn/) with default parameters(cpc2 -i novel.fa -o cpc2.out) and CNCI2.0 (wwww.bioinfo.org/software/cnci) with default parameters (CNCI.py -f novel.fa -o CNCI.result -p 1 -m ve -g novel.gtf -d genome.fa) to predict transcripts with coding potential. All transcripts with CPC score <-1 and CNCI score <0 were removed and remained transcripts were considered as lncRNAs.
Different expression analysis of mRNAs and lncRNAs:StringTie was used to perform expression level for mRNAs and lncRNAs by calculating FPKM. The differentially expressed mRNAs and lncRNAs were selected with log2 (fold change) >= 1 or log2 (fold change) <= -1 and with statistical significance p value < 0.05 by R package edgeR (bioconductor.org/packages/release/bioc/html/edgeR.html/) .
Target gene prediction and functional analysis of lncRNAs:To explore the function of lncRNAs, we predicted the cis-target genes of lncRNAs. lncRNAs may play a cis role acting on neighboring target genes. In this study, coding genes in 100,000 upstream and downstream were selected by python script. Then, we showed functional analysis of the target genes for lncRNAs with GO and KEGG enrichment analysis.
Genome_build: Columba livia (assembly Cliv_1.0)
Supplementary_files_format_and_content: tab-delimited text files include FPKM values for each Sample …
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| Submission date |
Dec 08, 2020 |
| Last update date |
Apr 13, 2021 |
| Contact name |
Haiguang Mao |
| E-mail(s) |
[email protected]
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| Phone |
13989490184
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| Organization name |
Zhejiang University
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| Street address |
Yuhangtang Road 866
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| City |
Hangzhou |
| State/province |
Zhejiang |
| ZIP/Postal code |
310058 |
| Country |
China |
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| Platform ID |
GPL29485 |
| Series (1) |
| GSE162867 |
Comparative Transcriptome Profiling of mRNA and lncRNA of Ovaries in High and Low Egg Production Performance in Domestic Pigeons (Columba livia) |
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| Relations |
| BioSample |
SAMN17028955 |
| SRA |
SRX9651769 |
| 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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