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Sample GSM2219179

Query DataSets for GSM2219179

Status

Public on Sep 10, 2016

Title

RL(+)_rep2

Sample type

SRA

Source name

RL(+)

Organism

Cyanidioschyzon merolae

Characteristics

strain: 10D
genotype: wild type

Treatment protocol

For the blue light (BL) experiments, exponentially growing cells were sub-cultured to OD750=0.6 (3×10^7 cells/ml) in 50 mL of 2×MA and then placed into complete darkness for 36 hours by covering the tubes with aluminum foil. Then, the cells were exposed to BL (15 µmole m-2s-1) for 30 minutes. BL intensity was measured with a UVX Digital Radiometer at 365 nm. For red light (RL) experiments, dark kept cells were exposed to RL for 30 minutes. RL intensity was measured by Thorlabs PM100 console system at 630 nm. To assess the effect of reactive oxygen species, cells grown under complete darkness were treated with 600 µM hydrogen peroxide for 45 minutes.

Growth protocol

C. merolae 10D cells were grown in 2× Allen’s medium (2×MA) at pH 2.3. Fifty-mL flasks containing C. merolae cultures were shaken under continuous light (100 µmole m-2s-1) at 42 °C.

Extracted molecule

total RNA

Extraction protocol

Total RNA was extracted from OD750=0.6, 5 ml cell culture by using TRIzol reagent according to the manufacturer’s instructions.
After quality and quantity measurements by using 2100 BioAnalyzer, the total RNA was treated with RNase-free DNase I at a concentration of 1 U/µg to remove residual genomic DNA. Next, mRNAs were purified from 1 µg of total RNA using oligo (dT) magnetic beads and fragmented using fragmentation buffer with the TruSeq mRNA Sample Preparation Kit according to manufacturer's instructions. The cleaved short RNA fragments were used for the first-strand cDNA synthesis using random hexamer-primer, and then the second strand was synthesized by using DNA polymerase I and RNase H. The double strand cDNAs were purified with AMPure XP beads and eluted with resuspension buffer followed by 3’end adenine nucleotide addition. Finally, sequencing adaptors were ligated to the fragments and cDNA fragments were enriched by PCR amplification. Enriched cDNA libraries were used for cluster generation and sequencing. Paired-end sequencing was performed for the two cDNA libraries (two biological replicates) for each condition using the Illumina MiSeq sequencing platform. All sequence data are PE 2x75 bp. Image processing, base calling, and quality calue calculation were performed by the Illumina data processing pipeline (v1.5). High quality reads were saved in FASTQ format.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina MiSeq

Data processing

Illumina data processing pipeline (v1.5) was used for base calling.
FastQC (v0.11.5) was used to evaluate the initial quality of the raw reads.
The following steps were performed to obtain high quality clean reads using Trimmomatic (v0.32) tool: (1) removing the reads with adaptor contamination; (2) filtering the low-quality reads with ambiguous sequences ‘N’; (3) removing the low-quality bases (quality score < Q30).
All downstream analyses were based on clean, high quality data. The TopHat-cufflink-cuffmerge-cuffcompare-cuffdiff pipeline was used to discover novel transcripts and to identify differentially expressed genes. TopHat (v2.0.5) was used to align reads to the C. merolae 10D reference genome. Following TopHat alignment, Qualimap software (v2.1) was used to evaluate the coverage of the alignment data and a Pearson’s correlation analysis was conducted to obtain the transcript-level R2 value between replicates
We used the q-value < 0.01 (FDR) and the absolute value of |Fold change| ≥ 2 as the threshold to judge the significant differences of gene expression.
Genome_build: ASM9120v1
Supplementary_files_format_and_content: Microsoft Office Open XML Spreadsheet (RNA-seq abundance measurements in FPKM values across samples)

Submission date

Jun 28, 2016

Last update date

May 15, 2019

Contact name

mehmet tardu

E-mail(s)

mtardu@umich.edu

Organization name

University of Michigan

Department

Chemistry