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Sample GSM7157700

Query DataSets for GSM7157700

Status

Public on Apr 09, 2024

Title

S48h3

Sample type

SRA

Source name

Leaf tissue

Organism

Apocynum venetum

Characteristics

tissue: Leaf tissue
cell line: wild P. apocinae
treatment: nutrient solution (containing 300 mmol/L NaCl)
time point: 48h

Extracted molecule

total RNA

Extraction protocol

A. Add 600 uL Lysis/Binding Buffer to the cell or tissue lysate. Then add 30 uL miRNA Homogenate Additive to homogenate, and mix well by vortexing or inverting the tube several times. Leave the mixture on ice for 10 min. B. Add a volume of Acid-Phenol: Chloroform that is equal to the lysate volume before addition of the miRNA Homogenate Additive. Centrifuge for 5 min at maximum speed (10,000 x g) to separate the aqueous and organic phases. Carefully remove the aqueous (upper) phase without disturbing the lower phase, and transfer it to a fresh tube. C. Add 1.25 volumes of room temperature 100% ethanol to the aqueous phase. D. Pipet the lysate/ethanol mixture (from the previous step) onto the Filter Cartridge (Note: Up to 700 uL can be applied to a Filter Cartridge at a time.). Centrifuge for 30 sec at 13,000 rpm to pass the mixture through the filter. Discard the flow-through.E. Apply 350 uL miRNA Wash Solution 1 to the Filter Cartridge and centrifuge for 30 sec at 13,000 rpm. Discard the flow-through from the Collection Tube, and replace the Filter Cartridge into the same Collection Tube. F. The 10 uL DNase I and 70 uL Buffer RDD QIAGEN (#79254) were mixed. Then the mixture was added to the Filter Cartridge. Leave it at the room temperature for 15 min. G. Apply 350 uL miRNA Wash Solution 1 to the Filter Cartridge and centrifuge for 30 sec at 13,000 rpm. Discard the flow-through from the Collection Tube, and replace the Filter Cartridge into the same Collection Tube. H. Apply 500 uL Wash Solution 2/3 and centrifuge for 30 sec at 13,000 rpm. Draw it through the Filter Cartridge as in the previous step. Repeat with a second 500 uL aliquot of Wash Solution 2/3. I. Spin the assembly for 1 min to remove residual fluid from the filter. Transfer the Filter Cartridge into a fresh Collection Tube. Apply 100 uL of pre-heated (95°C) Elution Solution to the center of the filter. Leave it at the room temperature for 2 min. Spin for ~20-30 sec at maximum speed to recover the RNA. Collect the eluate (which contains the RNA) and store it at -70°C.
1. Purify and Fragment mRNA;2.Synthesize First Strand cDNA;3. Synthesize Second Strand cDNA;4. Adenylate 3’Ends;5. Ligate Adapters;6. Enrich DNA Fragments;7.Purification;8.Validate library

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Description

Centrifuf (Eppendorf, Centrifuge 5418, Germany); PCR (Bio-rad, MyCycler, Beijing); Ultraviolet spectrophotometer (Thermo, NanoDrop2000, USA); Quantifier (Invitrogen, Qubit2.0, USA); Invitrogen, Magnetic Stand9, USA; Bioanalyzer (Aglient, 2100, USA).

Data processing

1. Quality control and De novo assembly
2. Functional annotation
3. Unigene quantification, analysis of differentially expressed unigenes (DEGs), cluster analysis, GO and KEGG enrichment
Assembly: No reference genome
Supplementary files format and content: tab-delimited text files include FPKM values for each Sample

Submission date

Apr 08, 2023

Last update date

Apr 09, 2024

Contact name

xi zhen

E-mail(s)

[email protected]

Phone

13474916990

Organization name

College of Grassland Resources and Environment, Inner Mongolia Agricultural University