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Sample GSM5415587

Query DataSets for GSM5415587

Status

Public on Jul 05, 2021

Title

T9D-1

Sample type

SRA

Source name

leaf

Organism

Carya illinoinensis

Characteristics

tissue: leaf
treatment: drought treated for 9 days

Extracted molecule

total RNA

Extraction protocol

The leaves were flash frozen with liquid nitrogen, and the total RNA of leaves was extracted using Trizol reagent kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol.
After total RNA was extracted, eukaryotic mRNA was enriched by Oligo(dT) beads, while prokaryotic mRNA was enriched by removing rRNA by Ribo-ZeroTM Magnetic Kit (Epicentre, Madison, WI, USA). Then the enriched mRNA was fragmented into short fragments using fragmentation buffer and reverse transcripted into cDNA with random primers. Second-strand cDNA were synthesized by DNA polymerase I, RNase H, dNTP and buffer. Then the cDNA fragments were purified with QiaQuick PCR extraction kit (Qiagen, Venlo, The Netherlands), end repaired, A base added, and ligated to Illumina sequencing adapters. The ligation products were size selected by agarose gel electrophoresis, PCR amplified, and sequenced using Illumina Novaseq6000 by Gene Denovo Biotechnology Co. (Guangzhou, China).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Data processing

Raw reads obtained from the sequencing machines were further filtered by fastp (version 0.18.0).
Short reads alignment tool Bowtie2 (version 2.2.8) was used for mapping reads to ribosome RNA (rRNA) database. The rRNA mapped reads then will be removed. The remaining clean reads were further used in assembly and gene abundance calculation.
An index of the reference genome was built, and paired-end clean reads were mapped to the reference genome using HISAT2. 2.4 with “-rna-strandness RF” and other parameters set as a default.
The mapped reads of each sample were assembled by using StringTie v1.3.1 in a reference-based approach. For each transcription region, a FPKM (fragment per kilobase of transcript per million mapped reads) value was calculated to quantify its expression abundance and variations, using RSEM software.
GO enrichment analysis provides all GO terms that significantly enriched in DEGs comparing to the genome background, and filter the DEGs that correspond to biological functions. Firstly all DEGs were mapped to GO terms in the Gene Ontology database (http://www.geneontology.org/), gene numbers were calculated for every term, significantly enriched GO terms in DEGs comparing to the genome background were defined by hypergeometric test. KEGG is the major public pathway-related database. Pathway enrichment analysis identified significantly enriched metabolic pathways or signal transduction pathways in DEGs comparing with the whole genome background. The calculating formula is the same as that in GO analysis.
Genome_build: Carya illinoinensis (GigaDB), release date 2019/02/13.
Supplementary_files_format_and_content: pecan_drought_fpkm.txt: Matrix table with FPKM values for every gene and every sample.

Submission date

Jul 02, 2021

Last update date

Jul 05, 2021

Contact name

pinghua Fan

E-mail(s)

fph@njfu.edu.cn

Phone

15295537732

Organization name

Nanjing Forestry University