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Sample GSM8306421

Query DataSets for GSM8306421

Status

Public on Jun 11, 2024

Title

CK_24_3h_3

Sample type

SRA

Source name

root

Organism

Chrysanthemum x morifolium

Characteristics

tissue: root
genotype: CK
treatment: 24_3h

Treatment protocol

8-week-old seedlings of EWT and CK were subjected to waterlogging stress by submerging them in water up to 2 cm above the upper leaves

Growth protocol

The experiment was conducted in a greenhouse with a 16 h of light (25℃) and 8 h of darkness (20℃)

Extracted molecule

total RNA

Extraction protocol

Total RNA was extracted using the Tiangen RNA Extraction Kit with the RNase-Free DNase Set (Tiangen Biotech, Beijing, China), according to the manufacturer’s instructions.
Libraries for mRNA-seq were constructed and sequenced by Novogene (Beijing).
Total RNA was used as input material for the RNA sample preparations. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in First Strand Synthesis Reaction Buffer(5X). First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase(RNase H-). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3’ ends of DNA fragments, Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 370~420 bp in length, the library fragments were purified with AMPure XP system. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Description

WT_24_3h_3

Data processing

Raw data (raw reads) of fastq format were firstly processed through fastp software. In this step, clean data (clean reads) were obtained by removing reads containing adapter, reads containing ploy-N and low quality reads from raw data. At the same time, Q20, Q30 and GC content the clean data were calculated. All the downstream analyses were based on the clean data with high quality.
Assembly: Reference genome and gene model annotation files were downloaded from genome website directly. Index of the reference genome was built using Hisat2 v2.0.5 and paired-end clean reads were aligned to the reference genome using Hisat2 v2.0.5. We selected Hisat2 as the mapping tool for that Hisat2 can generate a database of splice junctions based on the gene model annotation file and thus a better mapping result than other non-splice mapping tools.
Supplementary files format and content: featureCounts v1.5.0-p3 was used to count the reads numbers mapped to each gene. And then FPKM of each gene was calculated based on the length of the gene and reads count mapped to this gene. FPKM, expected number of Fragments Per Kilobase of transcript sequence per Millions base pairs sequenced, considers the effect of sequencing depth and gene length for the reads count at the same time, and is currently the most commonly used method for estimating gene expression levels.

Submission date

Jun 05, 2024

Last update date

Jun 11, 2024

Contact name

xueting gu

E-mail(s)

0622730@zju.edu.cn

Phone

13761068574

Organization name

Zhejiang university