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Sample GSM720643

Query DataSets for GSM720643

Status

Public on Dec 30, 2011

Title

UV_RNA-Seq

Sample type

SRA

Source name

Deinococcus gobiensis I-0

Organism

Deinococcus gobiensis

Characteristics

strain: I-0
treatment: UV

Treatment protocol

Cells were harvested at the late-log phase (2 x 108 CFUs mL-1) and then washed twice with equal volumes of potassium phosphate buffer (100 mM, pH 7.0). UV light (254 nm, 200 uW cm-2) was used to irradiate a 20 mL suspension for 5 min in a 9 cm plate with stirring. As a control, an additional non-irradiated suspension was incubated for the same duration. After treatment, cells were harvested by centrifugation at 8,000 rpm for 3 min.

Growth protocol

Cells of Deinococcus gobiensis I-0 (= DSM 21396) were grown in TGY broth (1.0 % peptone, 0.5 % yeast extract, 0.1 % glucose) at 30oC and were harvested at the late-log phase (2 x 108 CFUs mL-1)

Extracted molecule

total RNA

Extraction protocol

Bacterial cells were collected and ground into a fine powder in liquid nitrogen. Total RNA was isolated using Trizol reagent (Invitrogen), subsequently purified using RNeasy MinElute Cleanup Kit (Qiagen) and eluted in RNase-free water. Bacterial ribosomal RNAs were removed via a mixed treatment using the MICROB Express kit (Ambion) and the mRNA-ONLY Prokaryotic mRNA isolation Kit (Epeicentre® Biotech.) according to the manufacturer's instructions. Heating at 94oC fragmented the mRNA. First strand cDNA was synthesized with random hexamer primers, and second strand cDNA was synthesized with DNA polymerase. Double strand cDNA was end-repaired, a single adenosine was added, and the Illumina adapters were ligated. Gel-electrophoresis was used to select DNA fragments between 200-250 bp. Libraries were amplified by PCR using Phusion polymerase.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina Genome Analyzer IIx

Description

Suspension of cells were exposed to UV (254 nm, 200 Î¼W cm-2) in potassium phosphate buffer

Data processing

High-throughput cDNA sequencing was performed using the Firecrest, Bustard and GERALD programs. The low quality bases (Q<5) at the ends of the reads were trimmed. The reads that were longer than 20 bps were kept and aligned to the Deinococcus gobiensis genome using Burrows-Wheeler Aligner (BWA) . The reads that were mapped into the rRNA regions were not included in further analysis. A transcript coverage map was calculated based on the alignment of whole transcript reads. For each of the genes, the 5'-end of the translation regions were defined as positions supported by at least 5 reads summarized in both of the samples. To identify the non-coding RNA, the continuous regions (30 bp) with an average sequencing depth of 15 times/bp in the intergenic regions were extracted and compared against GenBank using blastx and blastn. Those regions that lacked similarities to known protein coding genes or were similar to known ncRNAs were classified as possible non-coding RNAs. To compare the different samples, the fragments per kb of CDS per million mapped reads (FPKM) value were used to normalize the data and represent the overall gene expression. The differently expressed genes between the two samples were selected according to their significance in Chi-square tests (p = 0.05, with Bonferroni correction) and at least 2-fold differences.

Submission date

May 05, 2011

Last update date

May 15, 2019

Contact name

Menglong Yuan

E-mail(s)

[email protected]

Organization name

Biotechnology Research Institute, Chinese Academy of Agricultural Sciences,

Street address

Zhongguancunnan 12th