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Sample GSM2438795

Query DataSets for GSM2438795

Status

Public on Mar 21, 2018

Title

NCC2_B2

Sample type

SRA

Source name

NC colon

Organism

Rattus norvegicus

Characteristics

strain: Wistar
mouse model: control (NC)
tissue: colon

Extracted molecule

polyA RNA

Extraction protocol

Total RNA was extracted from 30 mg of ground tissue by using hot phenol method. The RNA was further purified with two phenol-chloroform treatments and then treated with RQ1DNase (Promega) to remove DNA. The quality and quantity of the purified RNA we redetermined by measuring the absorbance at 260 nm/280 nm (A260/A280) using Smartspec Plus (BioRad).
For each sample, 10 μg of total RNA was used for RNA-seq library preparation. Polyadenylated mRNAs were purified and concentrated with oligo(dT)-conjugated magnetic beads (Invitrogen) before used for directional RNA-seq library preparation. The purified mRNAs were then iron fragmented at 95°C followed by end repair and 5' adaptor ligation. Then reverse transcription was performed with RT primer harboring 3' adaptor sequence and randomized hexamer.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NextSeq 500

Description

Data processing

Illumina Casava1.7 software used for basecalling.
Raw reads were first discarded if containing more than 2-N bases, then reads were processed by clipping adaptor, removing low quality bases, and discarding too short reads (less than 16nt). FASTX-Toolkit (Version 0.0.13) was used to get the clean reads.
After that, clean reads were aligned to the Rattus norvegicus (Rnor 5.0) by tophat2 (Kim et al., 2013). Aligned reads with more than one genome location were discarded due to their ambiguous location.
Uniquely localized reads were used to calculate reads number and RPKM value (RPKM represents reads per kilobase and per million) for each gene according to reads and genes genome location. Other statistical results, such as gene coverage and depth, reads distribution around start codon and stop codon, were also obtained.
After getting the Expression level of all genes in all samples, differentially expressed genes were analyzed by using edgeR(Robinson et al., 2010), one of R packages.
Genome_build: Rnor 5.0
Supplementary_files_format_and_content: tabular data of txt , the RPKM of expressed genes

Submission date

Dec 22, 2016

Last update date

May 15, 2019

Contact name

Dong Chen

Organization name

ABLife, Inc.

Department

Center for Genome Analysis

Street address

388 GaoXin 2nd Road, East Lake Hi-Tech Development Zone