GEO Accession viewer

NCBI > GEO > Accession Display

Not logged in | Login

GEO help: Mouse over screen elements for information.

Sample GSM4423407

Query DataSets for GSM4423407

Status

Public on Mar 30, 2020

Title

T3-3

Sample type

SRA

Source name

Ovary tissue

Organism

Cucumis sativus

Characteristics

tissue: Ovary
time: the embryos were cultured for 20d

Extracted molecule

total RNA

Extraction protocol

Total RNA was extracted using Trizol reagent (Invitrogen, CA, USA) following the manufacturer's procedure. Total RNA was isolated using TRIzol (Invitrogen), and DNase I (Fermentas) digestion was performed for 30 min at 25 oC to remove DNA, according to the manufacturer’s instructions. The integrity and quality of the total RNA were checked using a NanoDrop 1000 spectrophotometer and formaldehyde-agarose gel electrophoresis. RNA was used only when the OD260: OD280 ratio was above 1.8. Approximately 10 ug of total RNA representing a specific adipose type was subjected to isolate Poly (A) mRNA with poly-T oligoattached magnetic beads (Invitrogen). Following purification, the poly(A)- or poly(A)+ RNA fractions is fragmented into small pieces using divalent cations under elevated temperature. Then the cleaved RNA fragments were reverse-transcribed to create the final cDNA library in accordance with the protocol for the mRNA-Seq sample preparation kit (Illumina, San Diego, USA), the average insert size for the paired-end libraries was 300 bp (±50 bp). And then we performed the paired-end sequencing on an Illumina HiSeqTM 2000 at the (Personal-bio, China) following the vendor's recommended protocol.
RNA libraries were prepared for sequencing using standard Illumina protocols

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Data processing

Illumina HiSeq 2000 software used for basecalling.
The reads with more than 10% unknown bases and those of low quality were removed from the raw sequencing data using the software Illumina GA Pipeline (v1.6).
The filtered reads were aligned to the cucumber Chinese long v2 genome using TopHat2 (http://tophat.cbcb.umd.edu/) software.
Use HTSeq 0.6.1p2 (http://www-huber.embl.de/users/anders/HTSeq) to compare the read count value of each gene as the original expression amount of the gene
perform expression level for mRNAs by calculating RPKM
Genome_build: Cucumis_sativus_v 20
Supplementary_files_format_and_content: tab-delimited text files include RPKM values for each Sample

Submission date

Mar 20, 2020

Last update date

Mar 30, 2020

Contact name

LU YUXI

E-mail(s)

lyxrun@sina.com

Organization name

Institute of Horticulture

Street address

Guizhou Academy of Agricultural Sciences