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Sample GSM4998729

Query DataSets for GSM4998729

Status

Public on Mar 17, 2021

Title

Bifidobacterium longum W13 transcriptom sequence 3

Sample type

SRA

Source name

Bifidobacterium longum W13 total RNA

Organism

Bifidobacterium longum

Characteristics

strain: W13

Extracted molecule

total RNA

Extraction protocol

Total RNA was extracted from the tissue using TRIzol® Reagent according the manufacturer’s instructions (Invitrogen) and genomic DNA was removed using DNase I (TaKara). Then RNA quality was determined using 2100 Bioanalyser (Agilent) and quantified using the ND-2000 (NanoDrop Technologies). High-quality RNA sample (OD260/280=1.8~2.2, OD260/230≥2.0, RIN≥6.5, 28S:18S≥1.0,>10μg) is used to construct sequencing library.
RNA-seq strand-specific libraries were prepared following TruSeq RNA sample preparation Kit from Illumina (San Diego, CA), using 5μg of total RNA. Shortly, rRNA removal by RiboZero rRNA removal kit (Epicenter), fragmented using fragmentation buffer. cDNA synthesis, end repair, A-base addition and ligation of the Illumina-indexed adaptors were performed according to Illumina’s protocol. Libraries were then size selected for cDNA target fragments of 200-300 bp on 2% Low Range Ultra Agarose followed by PCR amplified using Phusion DNA polymerase (NEB) for 15 PCR cycles. After quantified by TBS380, Paired-end libraries were sequenced by Illumina NovaSeq 6000 sequencing (150bp*2.).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Description

LR20I10FP3_Clean_Data

Data processing

The raw paired end reads were trimmed and quality controlled by Trimmomatic with parameters (SLIDINGWINDOW:4:15 MINLEN:75) (version 0.36 http://www.usadellab.org/cms/uploads/supplementary/Trimmomatic)
Then clean reads were separately aligned to reference genome with orientation mode using Rockhopper (http://cs.wellesley.edu/~btjaden/Rockhopper/) software. Rockhopper was a comprehensive and user-friendly system for computational analysis of bacterial RNA-seq data. As input, Rockhopper takes RNA sequencing reads generated by high-throughput sequencing technology. This software was used to calculate gene expression levels with default parameters.
To identify DEGs (differential expression genes) between the two different samples, the expression level for each transcript was calculated using the fragments per kilobase of read per million mapped reads (RPKM) method. edgeR (https://bioconductor.org/packages/release/bioc/html/edgeR.html) was used for differential expression analysis. The DEGs between two samples were selected using the following criteria: i) the logarithmic of fold change was greater than 2 and the false discovery rate (FDR) should be less than 0.05.
To understand the functions of the differential expressed genes, GO functional enrichment and KEGG pathway analysis were carried out by Goatools (https://github.com/tanghaibao/Goatools) and KOBAS (http://kobas.cbi.pku.edu.cn/home.do) respectively. DEGs were significantly enriched in GO terms and metabolic pathways when their Bonferroni-corrected P-value was less than 0.05.
Genome_build: Bifidobacterium_W13; https://www.ncbi.nlm.nih.gov/assembly/GCF_004936435.1
Supplementary_files_format_and_content: tab-delimited text files include gene count for each Sample

Submission date

Jan 03, 2021

Last update date

Mar 17, 2021

Contact name

Tang jiao

E-mail(s)

xutingtingga@sina.com

Phone

15536096530

Organization name

Tianjin University Of Science and Technology