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Sample GSM4663897

Query DataSets for GSM4663897

Status

Public on Sep 13, 2020

Title

Panax notoginseng for MCAO in rats_3A

Sample type

SRA

Source name

Brain tissue

Organism

Rattus norvegicus

Characteristics

tissue: brain
treatment: Panax notoginseng for MCAO

Extracted molecule

total RNA

Extraction protocol

Total RNA was extracted using Trizol reagent kit (Invitrogen, Carlsbad, CA,USA) according to the manufacturer’s protocol. RNA quality was assessed on an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) and checked using RNase free agarose gel electrophoresis. 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, poly(A) added, and ligated to Illumina sequencing adapters. The ligation products were size selected by agarose gel electrophoresis, PCR amplified, and sequenced using Illumina HiSeq2500 by Gene Denovo Biotechnology Co. (Guangzhou, China)
RNA libraries were prepared for sequencing using standard Illumina protocols

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

HiSeq X Ten

Data processing

Reads obtained from the sequencing machines includes raw reads containing adapters or low quality bases which will affect the following assembly and analysis. Thus, to get high quality clean reads, reads were further filtered by fastp (version 0.18.0). The parameters were as follows:1) removing reads containing adapters;2) removing reads containing more than 10% of unknown nucleotides (N);3) removing low quality reads containing more than 50% of low quality (Q-value≤20) bases.
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 StringTie software.

Submission date

Jul 09, 2020

Last update date

Sep 13, 2020

Contact name

huang qing

E-mail(s)

20171002018@stu.ymcn.edu.cn

Organization name

Youjiang Medical University for Nationalities

Street address

No.18, Zhongshan 2nd Road, Youjiang District