GEO Accession viewer

NCBI > GEO > Accession Display

Not logged in | Login

GEO help: Mouse over screen elements for information.

Sample GSM7474423

Query DataSets for GSM7474423

Status

Public on Oct 20, 2023

Title

delta-Sisdbp, biol rep 2

Sample type

SRA

Source name

microbial cells

Organism

Saccharolobus islandicus

Characteristics

tissue: microbial cells
cell line: REY15A
cell type: microbial cells
genotype: Sisdbp knockout
treatment: no treatment

Treatment protocol

None

Growth protocol

S. islandicus strains were grown in MTSUV medium (Basic mineral medium+Typtone+Sucrose+Uracil+Vatamin) at 75 oC.

Extracted molecule

total RNA

Extraction protocol

Cells were pelleted at 6,000 g for 10 min and washed in PBS, then frozen with liquid nitrogen and transported with dry ice. Total RNA was extracted using the Trizol reagent (Ambion, Austin, TX, USA) and assessed by Aagilent 2100 bioanalyzer.
mRNA was purified from total RNA using probes to remove rRNA. Fragmentation was carried out using divalent cations under elevated temperature in First Strand Synthesis Reaction Buffer(5X). First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase, then use RNaseH to degrade the RNA. And in the DNA polymerase I system, use dUTP to replace the dNTP of dTTP as the raw material to synthesize the second strand of cDNA. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3’ ends of DNA fragments, Adaptor with hairpin loop structure were ligated to prepare for hybridization. Then USER Enzyme was used to degrade the second strand of cDNA containing U, In order to select cDNA fragments of preferentially 370~420 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Description

The Sisdbp deletion strain, biol rep 2

Data processing

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 N base and low quality reads from raw data.
At the same time, Q20, Q30 and GC content the clean data were calculated. All the downstream analyses were based on the clean data with high quality.
Reference genome S. islandicus REY15A (Guo, et al., 2011) and its 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.
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.
Differential expression analysis of two conditions/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
Assembly: REY15A
Supplementary files format and content: tab-delimited text files
Supplementary files format and content: tab-delimited text files include FPKM values for each Sample

Submission date

Jun 12, 2023

Last update date

Oct 20, 2023

Contact name

Qihong Huang

E-mail(s)

[email protected]

Phone

15063382551

Organization name

Shandong University