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Sample GSM3564340

Query DataSets for GSM3564340

Status

Public on Jan 17, 2019

Title

Ep_2

Sample type

SRA

Source name

Ep_2

Organism

Sulfolobus islandicus REY15A

Characteristics

genotype/variation: wild type with pSeSD plasmid
growth phase: log phase
od600: 0.2

Growth protocol

Strains were cultured in ACVy medium at 75°C to log phase (OD600=0.2)

Extracted molecule

total RNA

Extraction protocol

Total RNA was isolated using Trizol reagent (Invitrogen, Carlsbad, CA, USA), NEBNext Ultra™ RNA Library Prep Kit for Illumina (NEB, USA) was used with 3 ug of total RNA for the construction of sequencing libraries.
RNA libraries were prepared for sequencing using standard Illumina protocols. rRNA is removed using a specialized kit that leaves the mRNA. Fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer(5X). First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase(RNaseH-). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. In the reaction buffer, dNTPs with dTTP were replaced by dUTP. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3’ ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 150~200 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter,Beverly, USA). Then 3 μl USER Enzyme (NEB，USA) was used with size-selected, adaptor-ligated cDNA at 37°C for 15 min followed by 5 min at 95°C before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index(X) Primer. At last, products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system. The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumia) according to the manufacturer’s instructions. After cluster generation, the library preparations were sequenced on an Illumina Hiseq platform and paired-end reads were generated.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

HiSeq X Ten

Description

Ep_2

Data processing

Illumina Casava HiSeqX HD 3.4.0.38 software used for basecalling.
Raw data (raw reads) of fastq format were firstly processed through in-house perl scripts. In this step, clean data (clean reads) were obtained by removing reads containing adapter, reads containing ploy-N and low quality reads from raw data. At the same time, Q20, Q30 and GC content sequence of the cleandata were calculated. All the downstream analyses were based on the clean data with high quality.
Reference genome and gene model annotation files were downloaded from genome website directly. Both building index of reference genome and aligning clean reads to reference genome were used Bowtie2-2.2.3. (Langmead, B. and S.L. Salzberg, 2012)
HTSeq v0.6.1 was used to count the reads numbers mapped to each gene. And then FPKM of each gene was calculated based on the length of the gene and reads count mapped to this gene. FPKM, expected number of Fragments Per Kilobase of transcript sequence per Millions base pairs sequenced, considers the effect of sequencing depth and gene length for the reads count at the same time, and is currently the most commonly used method for estimating gene expression levels (Trapnell, Cole, et al., 2010).
Differential expression analysis of two groups (two biological replicates per condition) was performed using the DESeq R package (1.18.0). DESeq provide statistical routines for determining differential expression in digital gene expression data using a model based on the negative binomial distribution. The resulting P-values were adjusted using the Benjamini and Hochberg’s approach for controlling the false discovery rate . Genes with an adjusted P-value <0.05 found by DESeq were assigned as differentially expressed.
Genome_build: S. islandicus REY15A (accession no. NC_017276)
Supplementary_files_format_and_content: fpkm, Differential expression analysis of two groups

Submission date

Jan 16, 2019

Last update date

Jan 17, 2019

Contact name

Qing Ye

Phone

027-87281267

Organization name

Huazhong Agricultural University

Department

state key laboratory of agricultural microbiology