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Sample GSM1649562

Query DataSets for GSM1649562

Status

Public on Mar 25, 2018

Title

Reken501, 1 h cold treatment (A1)

Sample type

SRA

Source name

leaf

Organism

Hevea brasiliensis

Characteristics

clone: Reken501
treatment: 1 h cold treatment (A1)

Treatment protocol

These two cultivals in two extension units stages with three independent repeat plants were treated without cold stress as controls or subjected to 4°C treatment for 1 h and 24 h respectively.

Growth protocol

Hevea cultivated variety ‘Reken501’ and ‘93-114’ were grown at the experimental station of the Rubber Research Institute, Chinese Tropical Academy of Agricultural Sciences, Danzhou, China.

Extracted molecule

total RNA

Extraction protocol

Total RNA was extracted from the collected mixed leaves using the CTAB extraction and LiCl precipitation methods according to previous reports with slight modification (Chang et al.,1993;Reid et al.,2006).Total RNA concentration was quantified using a NanoDrop ND-1000 spectrophotometer. The quality of RNA was analyzed using an Agilent 2100 Bioanalyzer and further verified on 1.0% denaturing agarose gels.
Approximately 20 ug of total RNA from each sample pool of the two clones ‘Reken501’ and ‘93-114’ were used for the cDNA library construction via the standard protocols. The detailed process was described at the previous research paper(Wei et al.,2011).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Description

A1
Two extension units stages of one-year-old with three independent repeat plants

Data processing

Filter Dirty Raw Reads: Raw reads that only have 3' adaptor fragments should be removed before data analysis
Clean Reads:Reads after filtering dirty raw reads, on which all following analysis are based
Assembly:The methods of the data filtering and de novo assembly of the present Hevea transcriptome were performed in the BGI (Beijing Genomics Institute, Shenzhen, China) and were highly similar with the previous research paper(Wei et al.,2011).The raw reads generated by the Illumina HiSeq2000 genome analyzer was cleaned by removing adaptor sequences and the low-quality sequences including the empty reads, reads with unknown bases 'N' or with more than 10% Q < 20 bases). Then de novo assembly of the filtered clean reads was carried out by using SOAPdenovo (http://soap.genomics.org.cn/soapdenovo.html) with the default settings and an adjusted K-mer value (K =25) to generate the best assembled contigs. The scaffolds were generated by connecting the contigs between each pair of contigs using ‘N’ to represent unknown bases with the SOAPdenovo method. Paired-end reads were used again for scaffold gap filling to generate the unigenes with the sequences containing the least Ns and could not be further extended at either end. Moreover, the overlapping unigenes from six libraries were assembled into a continuous sequence using the overlapping ends of the clustered homologous unigenes, and redundant sequences were removed to yield nonredundant unigenes with the maximum length using the TGICL (TIGR Gene Indices Clustering) tools. The parameters of the TIGR tools were set at a sequence similarity of 94% and an overlap length of 100 bp among the clustered homologous unigenes.The sets of unigenes from the six distinct samples were further integrated as a reference transcriptome. To obtain a global overview of the reference Heave transcriptome, all the assembled unigenes were used for BLASTx searches against the public available databases including NR (non-redundant protein sequences in NCBI), NT and Swiss-Prot protein database with a cut-off E-value of 10-5. In addition, to get the functional annotation of the above sequences, Blast2GO software was used to get the GO (gene ontology) terms (http://www.geneontology.org). Also, the COG (Clusters of Orthologous Groups) (http://www.ncbi.nlm.nih.gov/COG) and KEGG (Kyoto Encyclopedia of Gene and Genomones Pathyway) (http://www.genome.jp/kegg) annotation was performed respectively against these databases using BLASTx program (E-value threshold 10-5).
Supplementary_files_format_and_content: difference expression unigenes between two clones in distinct stages, with excel format

Submission date

Apr 03, 2015

Last update date

May 15, 2019

Contact name

Wei-Min Tian

Organization name

Rubber Research Institute

Department

Chinese Academy of Tropical Agricultural Sciences

Street address

Baodao Village