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| Status |
Public on Nov 10, 2022 |
| Title |
B3 |
| Sample type |
SRA |
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| Source name |
whole seedlings
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| Organism |
Dendrobium huoshanense |
| Characteristics |
tissue: whole seedlings
treatment: cold stress treatments
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| Treatment protocol |
Annual plants were randomly selected as experimental materials for freezing stress, and the total plants were then collected and frozen in liquid nitrogen at different time points (0 and 7 d) after treatment. All samples had three biological repeats.
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| Growth protocol |
Annual plants were randomly selected as experimental materials for freezing stress, and were cultured at a temperature of 4 ° C, light cycle of 11 h· d-1, light intensity of 1500～ 2000 Lx.
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| Extracted molecule |
total RNA |
| Extraction protocol |
75μL EB（0.25M, NaCl; 0.05M, Tris-HCl (pH = 7.5); 20mM, EDTA; 1%(w/v) sodium dodecyl sulphate）, 750μL CI and 30μL β-mercaptoethanol were mixed in a 2.0mL RNase-free micro-centrifuge tube.Grinded tissue samples of the whole D. huoshanense seedlings at about 80 mg with liquid nitrogen into powder and transferred the milled powder samples to 2 mL tubes. Vortex vigorously. Place at 20˚C for 5 minutes before centrifuging the samples at 4˚C for 5 min at 12000×g.1/10 volume of 3M sodium acetate (pH = 5.2) and 2.5 volume of absolute ethanol were added to the supernatant. Mix gently and incubate at 4˚C for 30 minutes.Centrifuge at 4˚C for 10 min at 12000×g to recover the nucleic acids.200 μL of DEPC-treated water was added to dissolve the nucleic acids and 500μL of 10M LiCl was added to the solution. Mix gently and then place on ice for 60 minutes.Centrifuge at 4˚C for 10 min at 12000×g. Discard the supernatant and wash the pellet with 800μL of 75% (V/V) ethanol. Air-dry in a ventilator for 5-10min.50 μL of DEPC-treated water was added to dissolve RNA.RNA integrity and quality were evaluated by 1% agarose gel electrophoresis and spectrophotometric analysis using a Nano Drop and Agilent 2100 bioanalyzer (Thermo Fisher Scientific, MA, USA).
Oligo(dT) attached magnetic beads were used to purified mRNA with eliminating rRNA and tRNA in the total RNA. Purified mRNA of control sample and cold treated samples of the whole D. huoshanense seedlings were fragmented into small pieces with fragment buffer at appropriate temperature. Then, First-strand cDNA was generated by random hexamer-primed reverse transcription, followed by a second-strand cDNA synthesis. After that, RNA Index Adapters and A-Tailing Mix were added by incubating to end repair. The fragments of cDNA obtained from previous steps were amplified by PCR, and the PCR products were purified by Ampure XP Beads firstly, dissolved in EB solution. The products were validated on the Agilent Technologies 2100 bioanalyzer for quality control. The double stranded PCR products obtained from previous steps were heated to denature and circularized by the splint oligo sequence to get the final library, and the single stranded circular DNA (ssCir DNA) was formatted as the final library.
The constructional final library was further amplified with phi29 to make DNA nanoball (DNB) which had more than 300 copies of each molecular, DNBs were loaded into the patterned nano-array and single end of 50 bases reads were generated on MGI2000 platform (BGI-Shenzhen, China).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
MGISEQ-2000RS |
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| Data processing |
Raw reads were filtered via SOAPnuke (v1.4.0) by removing reads containing adaptors, poly-N or low quality, and then clean reads were obtained.
Trinity (v2.0.6) was used to assemble the clean reads and Tgicl (v2.0.6) to perform clustering and eliminate redundant data in the assembled transcripts to obtain unique genes. The assembled transcripts were processed for further expression analysis and functional annotation.
Clean reads were mapped to the assembled unique genes by Bowtie2 (v2.2.5)and the expression level of genes were calculated by RSEM (v1.2.8) and normalized to FPKM (Fragments per kilo base of transcript per million mapped reads). Functional annotation of genes was achieved by mapping genes to different databases (NT, NR, KOG, KEGG) using the software BLAST (v2.2.23). GO annotation was performed by Blast2GO (v 2.5.0) with NR annotations. DEseq2 was used to detect DEGs (Different expressed genes), and DEGs with fold change > 2 or <-2 and adjusted P value <= 0.001 were considered to be significantly different expressed genes. GO enrichment analysis and KEGG enrichment analysis were performed using Phyper, a function of R. The significant levels of terms and pathways were corrected by Q value with a rigorous threshold (Q value < 0.05).
Assembly: transcript.fa
Supplementary files format and content: tab-delimited text files include FPKM values for each Sample
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| Submission date |
Nov 07, 2022 |
| Last update date |
Nov 11, 2022 |
| Contact name |
Shihai Xing |
| E-mail(s) |
xshshihai@ahtcm.edu.cn
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| Phone |
15800731683
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| Organization name |
Anhui University of Chinese Medicine
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| Street address |
No.1 Qianjiang Road, Xinzhan District
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| City |
Hefei |
| State/province |
Anhui |
| ZIP/Postal code |
230012 |
| Country |
China |
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| Platform ID |
GPL32838 |
| Series (1) |
| GSE217439 |
Transcriptomic responses to cold stress in Dendrobium huoshanense C.Z. Tang et S.J. Cheng |
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| Relations |
| BioSample |
SAMN31636244 |
| SRA |
SRX18192659 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6718812_B3.txt.gz |
2.5 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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