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Sample GSM4153722

Query DataSets for GSM4153722

Status

Public on Jan 01, 2022

Title

stress_1

Sample type

SRA

Source name

S. cerevisiae_stress_10h

Organism

Saccharomyces cerevisiae

Characteristics

strain: CY
treatment: stress condition (36°C, 10% glucose) for 10hrs

Treatment protocol

Cells were harvested by centrifuge and followed by washing in sterile water.

Growth protocol

Strains were incubated in 20 mL YPD medium (2 % peptone, 1 % yeast extract, 2% or 10% glucose) at an initial OD600 of 0.5 and grown at 30°C or 36°C, and 200 rpm for 10 h.

Extracted molecule

total RNA

Extraction protocol

RNA was extracted using the TRIzol® method following the manufacturer’s protocol. Bulk cells or filamentous fungus are grounded to a powder using liquid nitrogen, and the powder was transferred into the 2ml tube contains 1.5ml Trizol reagent. The mix was shaked for 3min, and kept for 5min at room temperature, then was Centrifuged at 10,000×g for 5min at 4°C. The supernatant was added 200μL of chloroform/isoamyl alcohol (24:1) with 1ml lysis reagents. After centrifuged at 10,000×g for 10min at 4°C, the supernatant was transferred into another new tube with equal volume of isopropanol and put in the refrigerator at -20°C for 1h. After centrifuged at 13600×g for 20min at 4°C, the supernatant was precipitated by 1 mL of 75% ethanol and let dry for 3-5min. The RNA pellet was dissolved with 30-100μL of DEPC water or RNase-free water. The concentration of the extracted RNA samples was determined using a Nanodrop system (NanoDrop, Madison, USA), and the integrity of the RNA was examined by the RNA integrity number (RIN) using an Agilent 2100 bioanalyzer (Agilent, Santa Clara, USA).
Oligo(dT)-attached magnetic beads were used to purified mRNA. Purified mRNA was fragmented into small pieces with fragment buffer at appropriate temperature. Then First-strand cDNA was generated using random hexamer-primed reverse transcription, followed by a second-strand cDNA synthesis. afterwards, A-Tailing Mix and RNA Index Adapters were added by incubating to end repair. The cDNA fragments obtained from previous step were amplified by PCR, and products were purified by Ampure XP Beads, then dissolved in EB solution. The product was validated on the Agilent Technologies 2100 bioanalyzer for quality control. The double stranded PCR products from previous step were heated denatured and circularized by the splint oligo sequence to get the final library. The single strand circle DNA (ssCir DNA) was formatted as the final library. The final library was amplified with phi29 to make DNA nanoball (DNB) which had more than 300 copies of one molecular, DNBs were loaded into the patterned nanoarray and single end 50 bases reads were generated on BGIseq500 platform (BGI-Shenzhen, China).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

BGISEQ-500

Description

stress_1 FPKM

Data processing

Illumina Casava1.7 software used for basecalling.
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence using software SOAPnuke (v1.4.0)(parameter: -l 5 -q 0.5 -n 0.1)
Reads were then mapped to Saccharomyces_cerevisiae_S288C whole genome using software HISAT2 (v2.1.0) (parameter: --dta --phred64 unstranded --new-summary -x index -1 read_r1 -2 read_r2（PE）)
Clean reads were aligned using software Bowtie2 (v2.2.5) (parameter: -q --phred64 --sensitive --dpad 0 --gbar 99999999 --mp 1,1 --np 1 --score-min L,0,-0.1 -p 16 -k 200)
Genes were anotated with PHI-base using DIAMOND (v0.8.31) (parameter: --evalue 1e-5 --outfmt 6 --max-target-seqs 1 --more-sensitive) (screening factor: query coverage >= 50%、identity >= 40%). Gene expressions were calculated using RSEM (v1.2.8).
Genome_build: GCF_000146045.2_R64 (Saccharomyces_cerevisiae_S288C)
Supplementary_files_format_and_content: xls files include FPKM values for each Sample

Submission date

Nov 07, 2019

Last update date

Jan 01, 2022

Contact name

Lei Qin

E-mail(s)

qinlei@bit.edu.cn

Organization name

Beijing Institute of Technology

Street address

ZhongGuanCunNan Street 5