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Sample GSM2335744

Query DataSets for GSM2335744

Status

Public on Oct 05, 2018

Title

J.R2.LB.input

Sample type

SRA

Source name

pig adipose tissue

Organism

Sus scrofa

Characteristics

tissue: adipose tissue
breed: Jinhua

Growth protocol

Two males of the Landrace (a leaner breed) and Two males of the Jinhua (a fatty breed) at 180-day-old were used in this study.

Extracted molecule

total RNA

Extraction protocol

total RNA was extracted from tissue by TRIzol. mRNA was further purified using Dynabeads mRNA DIRECT Kit. For IP samples, mRNA was sonicated and subjected to IP using m6A antibody (Synaptic Systems, 202003).
Libraries were constructed using TruSeq Stranded mRNA Library Prep Kit (Illumina).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 4000

Data processing

All samples were sequenced by Illumina Hiseq 4000 with single-end 50-bp read length. The deep sequencing data were mapped to pig reference genome (Sscrofa10.2/susScr3).
Data analysis for each experiment: (1) for m6A-seq, reads were aligned to the reference genome using Tophat v2.0.14 with parameter -g 1 --library-type=fr-firststrand. RefSeq Gene structure annotations were downloaded from UCSC Table Browser. The longest isoform was used if the gene had multiple isoforms. Aligned reads were extended to 150 bp (average fragments size) and converted from genome-based coordinates to isoform-based coordinates, in order to eliminate the interference from introns in peak calling. The peak calling method was modified from published work (Dominissini et al., 2012). To call m6A peaks, the longest isoform of each gene was scanned using a 100 bp sliding window with 10 bp step. To reduce bias from potential inaccurate gene structure annotation and the arbitrary usage of the longest isoform, windows with read counts less than 1/20 of the top window in both m6A-IP and input sample were excluded. For each gene, the read counts in each window were normalized by the median count of all windows of that gene. A Fisher exact test was used to identify the differential windows between IP and input samples. The window was called as positive if the FDR < 0.01 and log2(Enrichment Score) >= 1. Overlapping positive windows were merged. The following four numbers were calculated to obtain the enrichment score of each peak (or window): reads count of the IP samples in the current peak/window (a), median read counts of the IP sample in all 100 bp windows on the current mRNA (b), reads count of the input sample in the current peak/window (c), and median read counts of the input sample in all 100 bp windows on the current mRNA (d). The enrichment score of each window was calculated as (a×d)/(b×c). (2) for mRNA-seq (the input samples of m6A-seq), reads were mapped with Tophat and Cufflink (v2.2.1) was used to calculate the FPKM of each gene to represent their mRNA expression level.
Genome_build: pig reference genome (Sscrofa10.2/susScr3)
Supplementary_files_format_and_content: excel file containing processed FPKM from input samples and m6A peak list from IP samples

Submission date

Oct 05, 2016

Last update date

May 15, 2019

Contact name

zhike lu

E-mail(s)

zhikelu@gmail.com

Organization name

University of Chicago

Department

department of chemistry