GEO Accession viewer

NCBI > GEO > Accession Display

Not logged in | Login

GEO help: Mouse over screen elements for information.

Sample GSM3566997

Query DataSets for GSM3566997

Status

Public on Jul 03, 2019

Title

Rat_liver_MazF

Sample type

SRA

Source name

Rat liver tissue

Organism

Rattus norvegicus

Characteristics

tissue: liver
treatment: MazF

Growth protocol

Human HEK293T cell line was cultured with DMEM (Corning) supplemented with 10% (v/v) FBS (Gibco) at 37℃ in the presence of 5% CO2, and tested to be negative for mycoplasma contamination.

Extracted molecule

polyA RNA

Extraction protocol

Total RNA from HEK293T cells was extracted using Trizol reagent (Thermo Fisher Scientific) and the mRNA was purified using Dynabeads mRNA Purification Kit (Thermo Fisher Scientific, Cat. No. 61006). The mRNA of human tissues (brain, liver and kidney), mouse tissues (brain, liver, heart, testis and kidney) and rat tissues (brain, liver and kidney) were purchased from Takara Bio.
mRNA samples were heated to denature and digested into fragments by MazF (Takara) at 37℃ for 30 min. The fragmented mRNA was purified and end-repaired using T4 polynucleotide kinase at 37℃ for 30 min. The sample was purified for small RNA library construction.
Parallel samples demethylated by FTO in advance were as the negative controls. The FTO treatment was in 20 ul of reaction mixture containing 100-200 ng mRNA, 2.5 ug FTO demethylase, 283 μM of (NH4)2Fe(SO4)2·6H2O, 300 μM of α-KG, 2 mM of L-ascorbic acid, 20 U RNase inhibitor and 50 mM of Tris·HCl buffer (pH 7.5). After incubating at room temperature for 3h, the reaction was stopped by adding 40 mM EDTA.
Small RNA library construction protocol

Library strategy

OTHER

Library source

transcriptomic

Library selection

other

Instrument model

HiSeq X Ten

Description

Rat_liver_Aimseq_m6A.txt

Data processing

Library strategy: Aim-seq
The raw reads were filtered adaptors by cutadapt with at least 15 bp remaining length of paired-end reads. The clean reads were mapped to the reference genome (hg19) using Hisat2.
Reads with ACA/TGT motif internal or at the end were used to identify transcriptome-wide candidate m6A sites.
The RNAfold program of ViennaRNA package was used to predict intramolecular secondary structure of each read which supported the candidate sites, and sites with the high pairing probability were discarded.
The FTO demethylation treatment was negative control. Only the sites with at least 10% methylation ratio decrease in FTO treatment were retained as m6A sites.
Genome_build: hg19,mm10,rn6
Supplementary_files_format_and_content: text format with the information of chromosome number, site loction, strand, #reads with internal ACA in MazF, #reads sum in MazF,#reads with internal ACA in FTO, #reads sum in FTO, in one or more replicates

Submission date

Jan 17, 2019

Last update date

Jul 03, 2019

Contact name

Zhang Zhang

E-mail(s)

lylazhang@foxmail.com, zhangzhang@mail.sysu.edu.cn

Organization name

Sun Yat-sen University

Street address

Xingangxi Roda 135#