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| Status |
Public on Jul 01, 2016 |
| Title |
L_japonica_Shoot_apex |
| Sample type |
SRA |
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| Source name |
Shoot apex
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| Organism |
Lonicera japonica |
| Characteristics |
tissues: Shoot apex
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| Treatment protocol |
Not applicable
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| Growth protocol |
All nine tissues for L. japonica, namely, shoot apex, stem, leaf-1 (youngest leaf near shoot apex), leaf-2 (second leaf), leaf-3 (mature leaf), green floral bud, white floral bud, white flower, and yellow flower were harvested in June 2014. L. japonica plants were cultivated in the natural environment of Chiba University pharmaceutical garden, Chiba (located at 35˚36’17.7’’N; 140˚08’06.9’’E). All tissues from L. japonica were harvested on the ice, cut into small pieces, and were snap freezed by liquid N2 before storing at -80˚C prior to RNA extraction.
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| Extracted molecule |
total RNA |
| Extraction protocol |
The frozen tissues from L. japonica were powdered using a Multi beads shocker (Yasui Kikai, Japan), and were used for subsequent extraction of total RNA using RNeasy Plant Mini Kit (Qiagen, USA) according to manufacturer’s instructions. RNA quality was assessed using Agilent Bioanalyzer 2100 (Agilent Technology, USA), and RNA samples with RNA integrity number (RIN) above 8 were used for cDNA library preparation.
mRNA for each samples were isolated from the total RNA by using beads with oligo (dT), and were added with fragmentation buffer to shear mRNA into short fragments, which were then used as template for the synthesis of first strand of cDNA using random hexamer primers. cDNA library for Illumina sequencing was prepared using SureSelect Strand specific RNA library kit (Agilent Technology, USA) according to manufacturer’s instructions.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
All nine tissues for L. japonica, namely, shoot apex, stem, leaf-1 (youngest leaf near shoot apex), leaf-2 (second leaf), leaf-3 (mature leaf), green floral bud, white floral bud, white flower, and yellow flower were harvested in June 2014. L. japonica plants were cultivated in the natural environment of Chiba University pharmaceutical garden, Chiba (located at 35˚36’17.7’’N; 140˚08’06.9’’E).
Lonicera_japonica_de_novo_transcriptome_assembly.fasta
Lonicera_japonica_FPKM_annotation_description.xlsx
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| Data processing |
For de novo transcriptome assembly, we pre-processed raw reads, and removed adapter sequences, empty reads, reads with ambiguous ‘N’ bases > 5%, raw reads of low quality (Q<20), and raw reads with an average length less than 30 bases. Filtering of raw reads was performed using Trimmomatic program (Bolger et al., 2014), resulting into paired end raw reads, and unpaired reads which lost its corresponding sequence partner due to quality control.
De novo transcriptome assembly for L. japonica was obtained by merging three popular assemblers, namely, SOAPdenovo-Trans, Trinity v 2.0.6, and CLC Genomics workbench v8.0.3 (https://www.qiagenbioinformatics.com/) (Qiagen, USA). For SOAPdenovo-Trans, we performed six independent de novo transcriptome assemblies using kmer-sizes as 31, 41, 51, 63, 71, and 91, and resultant assemblies were analysed using perl script from assemblathon_2 to obtain N50-values and other assembly related stats . De novo transcriptome assembly using Trinity, and CLC Genomics workbench were performed using default kmer size and default parameters. Resultant transcriptome assemblies from SOAPdenovo-Trans using kmer size as 31 emerged as the best assembly on the basis of different assembly parameters, which was then pooled together with assemblies from Trinity , and CLC Genomics workbench into one merged assembly, and were processed by CD-HIT-EST v 4.6 (built on Mar 5, 2015) with parameters used as “-c 0.95 –n 8” to remove sequence redundancy. Sequences with a length less than 200 bps were dropped, and resulted de novo transcriptome assembly were used for further characterization.
For transcriptome expression analysis, clean pair reads for each tissue were used for alignment over L. japonica transcriptome assembly using Bowtie 2.0 program , and RSEM program was used for abundance estimation. To calculate unigene expression, we used Fragments per Kilobase exon per Million mapped fragments (FPKM) method. Unsupervised principal component analysis for all nine tissues was performed by DESeq 2 program using count data for unigenes obtained from RSEM program.
Supplementary_files_format_and_content: .fasta file as De novo transcriptome assembly obtained using transcripts from nine tissues of L. japonica.
Supplementary_files_format_and_content: .results files were generated using RSEM program. Clean reads for each tissues were alligned on Lonicera japonicus de novo transcriptome assembly, and abundace were measured using RSEM program. Content of each file contains transcript_id (which is contig), gene_id (which is unigene), length, effective_length, expected_count, TPM, FPKM, and IsoPct for each tissues. FPKM values for each tissues were used for furthur analysis.
Supplementary_files_format_and_content: .xlsx files include Transcript_id, Gene_id, Given_Unigene_id_for_manuscript, FPKM values for transcripts from all nine tissues of L. japonica, and blastx based annotation and top hits associated with each transcript. This processed dataset combines top-hit blastx annotaion of transcripts and their abundance across different tissues of L. japonica.
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| Submission date |
May 26, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Mami Yamazaki |
| E-mail(s) |
mamiy@faculty.chiba-u.jp
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| Organization name |
Chiba University
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| Department |
Graduate School of Pharmaceutical Sciences
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| Lab |
Department of Molecular Biology and Biotechnology
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| Street address |
Inohana 1-8-1, Chuo-ku
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| City |
Chiba |
| ZIP/Postal code |
2608675 |
| Country |
Japan |
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| Platform ID |
GPL21949 |
| Series (1) |
| GSE81949 |
De novo transcriptome assembly and characterization of nine tissues of Lonicera japonica to identify potential candidate genes involved in chlorogenic acid, luteolosides, and secoiridoid biosynthesis pathways |
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| Relations |
| BioSample |
SAMN05180087 |
| SRA |
SRX1802149 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2178339_L_japonica_Shoot_apex.isoforms.results.txt.gz |
3.3 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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