 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Aug 25, 2019 |
| Title |
HHCC_M2 |
| Sample type |
SRA |
| |
|
| Source name |
Cucumis ×hytivus J.F. Chen & J.H. Kirkbride, C. ×hytivus, mature leaf
|
| Organism |
Cucumis x hytivus |
| Characteristics |
ploidy: allotetraploid
tissue: leaf
leaf developmental stage: mature
harvest stage: the first fully developed leaf
|
| Growth protocol |
The young leaf (first leaf counting from the top) and the mature leaf (the first fully developed leaf) of self-cross plants (S10) of synthesized new species, Cucumis ×hytivus J. F. Chen & J. H. Kirkbr. and the mature leaves of the diploid parents, C. hystrix Chakr. and C. sativus ‘BeijingJietou’ were the plant material for sequencing. Single seeds were sown in a plug tray after soaking and pre-germination and then cultured in climate chambers (RDN-1000E-4, Dongnan Instrument Co, Ltd, Ningbo, China) at an air temperature of 25/20°C (14 h/10 h, day/night), a light intensity of 280±20 µmol m-2 s-1 and 60±10% relative humidity. On the 20th day after sowing, healthy and uniform seedlings were transferred to plastic plots (11 cm diameter, 9 cm height) and cultured in the climate chambers with the same climate setting. The seedlings were irrigated by flooding the bench for 10 min two times per day with 1/2 nutrient solution, according to the Japanese Garden test formula.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using the TRIzol Reagent. The mRNA was isolated using oligo (dT)-attached magnetic beads. The four libraries were sequenced using IlluminaHiSeqTM 2500.
RNA libraries were prepared for sequencing using standard Illumina protocols
|
| |
|
| Library strategy |
miRNA-Seq |
| Library source |
transcriptomic |
| Library selection |
size fractionation |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
mature leaf
|
| Data processing |
After sequencing, raw reads were further analysed using the ACGT101-miR program (LC Sciences, Houston, Texas, USA). After removing junk reads and sequences < 18 nt and > 25 nt, remaining sequences were aligned using Rfam (http://rfam.janelia.org), Cucumber Genome Network database (http://www.icugi.org/cgi-bin/ICuGI/index.cgi) and repeat database (http://www.girinst.org/repbase). The sequences that matched Rfam, mRNA, rRNA, tRNA, snoRNA, snRNA, other non-coding RNAs and repeat sequences were filtered out. The valid sequences were blasted against the reported pre-miRNAs and mature miRNAs in plants from miRBase 21.0 (ftp://mirbase.org/pub/mirbase/21/). A maximum of one mismatch between target and known miRNAs from miRBase database was allowed. Moreover, only the pre-miRNAs with stable hairpin structures were considered and secondary structures of the pre-miRNAs were predicted using RNAfold software (http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi).. miRNAs that are conserved among plants as compared with miRBase 21.0 and with stable hairpin structures were divided to conserved miRNAs. Minimal folding free energy index (MFEI) is another important factor for miRNA identification52. Novel miRNAs were only considered when minimal folding free energy index (MFEI) of their pre-miRNAs were ≥ 0.80 in this study. Moreover, the normalized copy number of the novel miRNAs was required to be ≥ 10 in at least one sample.
Target genes of miRNAs identified in Cucumis were predicted with Targetfinder. To clarify the functions of the target genes, GO functional annotation and KEGG pathway analysis of the miRNA targets were performed using GO database (http://www.geneontology.org/) and KEGG database (http://www.genome.jp/kegg/),
The alignment software, bowtie 0.12.8, was used to map the reads back to the transcriptome. Then, the number of mapped clean reads for each unigene was counted and normalized into a RPKM value (Reads Per kb per Million reads), which is widely used to calculate the unigene expression (Mortazavi et al., 2008). To detect differentially expressed genes, statistical analyses among libraries were performed following the formula as described (Shen et al., 2012), where FDR (false discovery rate) were used to determine the threshold the P value in multiple tests and analyses using the q-value package (Benjamini et al., 2001). In our work, the differentially expressed unigenes between two samples were screened with the threshold of FDR ≤ 0.001 and the absolute value of log2Ratio ≥ 1. Furthermore, the GO classifications and the KEGG pathway enrichments were compared between up-regulated and down-regulated unigenes.
Genome_build: http://www.icugi.org/cgi-bin/ICuGI/index.cgi, genome v2
Supplementary_files_format_and_content: expression data of miRNA (raw reads and norm reads)
|
| |
|
| Submission date |
Mar 31, 2016 |
| Last update date |
Aug 27, 2019 |
| Contact name |
Xiaqing Yu |
| E-mail(s) |
yuxiaqing2014@163.com
|
| Organization name |
Nanjing Agricultural University
|
| Street address |
Weigang No.1
|
| City |
Nanjing |
| ZIP/Postal code |
210095 |
| Country |
China |
| |
|
| Platform ID |
GPL21679 |
| Series (1) |
| GSE79757 |
High-throughput sequencing reveals change of microRNA expression caused by allopolyploidization in Cucumis |
|
| Relations |
| BioSample |
SAMN04592651 |
| SRA |
SRX1671425 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data provided as supplementary file |
| Processed data are available on Series record |
|
|
|
|
 |
