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Sample GSM3312852

Query DataSets for GSM3312852

Status

Public on Jul 31, 2018

Title

cucumber miRNA HS-3

Sample type

SRA

Source name

shoot apical tissue

Organism

Cucumis sativus

Characteristics

common name: Chinese long 9930
cultivar: 9930
tissue: shoot apical tissue
developmental stage: four-week-seedling
treatment: grown in 8 h light at 28°C /16 h dark at 25°C

Treatment protocol

conditions set as follows: 16 h light in 28 °C /8 h dark in 25 °C (high temperature, long day, HL), 8 h light in 28 °C /16 h dark in 25 °C (high temperature, short day, HS), 16 h light in 20 °C /8 h dark in 15 °C (low temperature, long day, LL), and 8 h light in 20 °C /16 h dark in 15 °C (low temperature, short day, LS). A 70% humidity was used for all of these plants.

Growth protocol

Seedlings were grown in artificial climate chambers

Extracted molecule

total RNA

Extraction protocol

When four true-leaves were unfolded, shoot apical tissues (1 mm) were dissected under a microscope and snap-frozen in liquid nitrogen and kept at −80 °C for further use. In each experiment, more than 500 shoot apices and three biological replicates were used. Total RNA was extracted using the TRIzol reagent (Invitrogen, USA). DNase (Promega, USA) was used to remove potential DNA contamination.
1. For RNA seq, Total RNAs (10 μg) were subjected to poly-A selection, fragmentation, random priming and cDNA synthesis with the Illumina Gene Expression Sample Prep kit (CA, USA). The cDNA fragments were end repaired, ligated to adapters and then enriched by PCR. The fragments were purified with 6% TBE PAGE gel electrophoresis. After denaturation, the single-chain fragments were fixed onto the Solexa Sequencing Chip (Flowcell) and consequently grown into single-molecule cluster sequencing templates through in situ amplification.
2. For miRNA seq, A total of 30 μg of RNA was resolved on denatured polyacrylamide gels. Gel fragments with the size range of 18-30 nt were excised and recovered. These small RNAs were ligated with 5’ and 3’RNA adapters using T4 RNA ligase. The adapter-ligated small RNAs were subsequently transcribed into cDNA by Super-Script II Reverse Transcriptase (Invitrogen) and amplified using primers specific for the ends of the adapters.
3. For degradome seq, Approximately 200 μg of total RNA was polyadenylated using the Oligotex mRNA mini kit (Qiagen). A 5′ RNA adapter was added to the cleavage products (which possessed a free 5′-monophosphate at their 3’ termini) using the T4 RNA ligase (Takara). Then, the ligated products were purified using the Oligotex mRNA mini kit (Qiagen) for reverse transcription to generate the first strand of cDNA using an oligo dT primer via SuperScript II RT (Invitrogen). After the cDNA library was amplified for 6 cycles (94 °C for 30 s, 60 °C for 20 s, and 72 °C for 3 min) using Phusion Taq (NEB), the PCR products were digested with restriction enzymes and further ligated to double-stranded DNA adapters. The ligated products were subjected to PCR amplification and gel purification and finally used for high-throughput sequencing with the Illumina HiSeq 2000.

Library strategy

miRNA-Seq

Library source

transcriptomic

Library selection

size fractionation

Instrument model

Illumina HiSeq 2000

Description

miRNA.txt

Data processing

BaseCall V1.6
SOAP
BLAST
Bowtie
RSEM
Genome_build: Cucumis sativus L. var. sativus cv. 9930 _v20; ftp://cucurbitgenomics.org/pub/cucurbit/genome/cucumber/Chinese_long/
Supplementary_files_format_and_content: tab-delimited text files include RPKM values for each Sample

Submission date

Jul 30, 2018

Last update date

Jul 31, 2018

Contact name

Xiaohui Zhang

E-mail(s)

[email protected]

Phone

86-10-82105947

Organization name

Institute of Vegetables and Flowers, Chinese Academy of Agricultural Science