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Sample GSM3814034

Query DataSets for GSM3814034

Status

Public on May 13, 2024

Title

54_C3

Sample type

SRA

Source name

DMSO 100μg/L replicates3

Organism

Drosophila melanogaster

Characteristics

cell line: S2
drug concentration: 100 microgram/L
treatment: DMSO
library type: fr-firststrand (dUTP)

Treatment protocol

S2 cells were treated with different concentration of TMPyP4 (25μmol/L, 50μmol/L, 100μmol/L, respecticely), and DMSO was severed as control. After 48 hours, the S2 cells were sampled for sequencing.

Growth protocol

Drosophila line 2 cells (S2) were maintained at 28°C in Hyclone TNM-FH insect medium containing 10% Gibico serum and antibiotics (0.5 U/ml penicillin and 0.5 μg/ml streptomycin).

Extracted molecule

polyA RNA

Extraction protocol

We extracted the total RNA using the Trizol method. We added 1 ml of Trizol (invitrogen) to the sample (4-7 million cells) stored at -80 ° C, and carefully piped the sample with a pipette, which was the homogenate of the tissue cells. Then, 200 μl of chloroform was added to the homogenate, and the sample was shaken for 15 seconds using a vortex shaker until the phase separation disappeared. After standing at room temperature for 2-3 minutes, the sample was centrifuged at 12,000 g for 15 minutes at 4 °C. The colorless supernatant was transferred to another new centrifuge tube, mixed well with an equal volume of isopropanol, held at -20 ° C for 10 minutes, and then centrifuged at 12,000 g for 10 minutes at 4 ° C to precipitate RNA. The supernatant was carefully removed, the precipitate was gently washed 2-3 times with 1 ml of fresh 75% ethanol, dried at room temperature for 2 to 3 minutes and dissolved in 50 μl of RNase-free water.
Stranded PolyA+ RNA libraries were prepared at Majorbio (Shanghai, China) with in-house kits. A total of 12 qualified cDNA libraries were constructed and were sequenced on the Illumina HiSeq XTen System (Illumina Inc., San Diego, CA).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

HiSeq X Ten

Description

mixed species RNA preparation prepared with zebrafish RNA

Data processing

RNA-Seq: High-throughput sequencing raw image data files were converted to reads using fqtools_plus analysis.
RNA-Seq: We used FastqC (0.11.8) to evaluate the raw reads quality. Sequencing report tool was MultiQC (0.9).
RNA-Seq: Reads were mapped to the corresponding reference genomes (flybase dm6) by Hisat2 (2.0.5) with parameters “-x --rna-strandness RF” and “--known-splicesite-infile” followed by gene annotation in GTF format
RNA-Seq: The output of Hisat2 were converted to BAM format, sorted and reads with MAPQ <30 were discarded by Samtools (1.7)
RNAseq: HTSeq (0.9.1) was used for counting reads with parameters “-t exon -i gene_id -r name -s reverse”.
Genome_build: Dm6
Supplementary_files_format_and_content: Tab-delimited text files include raw or normalized read counts of all the genes for each RNAseq sample.

Submission date

May 23, 2019

Last update date

May 13, 2024

Contact name

Zhen-Xia Chen

E-mail(s)

zhen-xia.chen@mail.hzau.edu.cn

Organization name

Huazhong Agricultural University

Department

College of Life Science and Technology