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Sample GSM2522407

Query DataSets for GSM2522407

Status

Public on Nov 13, 2017

Title

EMS_rep1

Sample type

SRA

Source name

EMS

Organism

Chlorella vulgaris

Characteristics

strain: SAG 211-12
genotype: EMS mutant (EMS-25)

Treatment protocol

250 µl culture of C. vulgaris with the cell count of 4×10e8 cells/ml was spread on solid Bold’s basal media and then exposed to UV irradiation (254 nm) with an intensity of 2.9 ×10-2 W/cm2 for 0.5 to 10 minutes at a distance of 15 cm. UVX Digital Radiometer (UVP, USA) was used to measure the UV-light intensity. The UV-irradiated plates were kept in dark for 24 h to prevent light-induced repair. The plates were then maintained under normal light for 2 weeks. Single colonies appearing on plates were selected and transferred individually into 2 ml Bold’s basal medium. After 10 days of culturing, the cell densities were determined by measuring OD at 680 nm.

Growth protocol

Chlorella vulgaris (SAG 211-12) was obtained from the collection of algal cultures at the University of Göttingen, Germany. The inoculum was grown in Bold’s Basal medium consisting of (per liter): 0.25 g NaNO3, 0.075 g MgSO4.7H2O, 0.075 g K2HPO4, 0.175 g KH2PO4, 0.025 g NaCl, 0.025 g CaCl2.2H2O, 8.82 mg ZnSO4.7H2O, 0.44 mg MnCl2.4H2O, 0.71 mg MoO3, 1.57 mg CuSO4.5H2O, 0.49 mg CoCl2.6H2O, 11.42 mg H3BO3, 50 mg EDTA, 31 mg KOH, 4.98 mg feSO4.7H2O. Cultures were maintained at 25◦C under continuous illumination by four fluorescent lamps.

Extracted molecule

total RNA

Extraction protocol

Total RNA was extracted from 14-days old stationary phase reached (OD680=5) 5 ml cell culture using TRIzol reagent (Invitrogen, USA) according to the manufacturer’s instructions. Briefly, after quality and quantity measurement using 2100 BioAnalyzer (Agilent, USA), it was treated with RNase-free DNase I (Thermo Scientific, USA) at a concentration of 1 U/µg to remove residual genomic DNA. Then, RNA-seq library preparation was performed using TruSeq mRNA Sample Preparation Kit (Illumina, USA) according to the manufacturer’s instructions.
mRNAs were purified from the 1 µg of total RNA using oligo (dT) magnetic beads and fragmented using fragmentation buffer. Afterthat, the cleaved short RNA fragments were used for first-strand cDNA synthesis using first strand synthesis mix, and the second strand was synthesized using second strand marking master mix. The double strand cDNAs were purified with AMPure XP beads (Beckman Coulter, USA) 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. 75x2 paired-end sequencing of three cDNA libraries (WT, UV-715 and EMS-25) with two biological replicates were performed using the Illumina MiSeq sequencing platform (Illumina, USA). 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. De novo assembly of the high quality reads was performed using Trinity, which is composed of three independent software modules: Inchworm, Chrysalis and Butterfly. By Inchworm, RNA-seq data was assembled into unique sequences through establishing a k-mer graph (K = 25). By Chrysalis, the contigs generated in the previous step were clustered, and a de Bruijn graph was constructed for each cluster. Subsequently, by Butterfly, these Bruijn graphs were processed in parallel and the paths were traced according to reads and pairs of reads within graphs to obtain full-length transcripts for alternative splicing isoforms and distinguish transcripts of paralogous genes. Finally, the distributed situation of length of result sequences was counted and analyzed using RSEM method. A Pearson’s correlation analysis was performed to obtain the transcript-level R2 between biological replicates.
For count-based differential expression testing between WT, UV-715 and EMS-25 cells, edgeR was used. We used min_rowSum_counts of 10, stringent q-value < 0.01 (false discovery rate, FDR) and the absolute value of | log2 fold change| ≥ 1 as the threshold to judge the significant differences in gene expression between WT and mutant cells.
Genome_build: De novo assembly
Supplementary_files_format_and_content: matrix text file (transcript count values of rsem output across samples)

Submission date

Mar 06, 2017

Last update date

May 15, 2019

Contact name

mehmet tardu

E-mail(s)

[email protected]

Organization name

University of Michigan

Department

Chemistry