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Sample GSM5606965

Query DataSets for GSM5606965

Status

Public on Oct 01, 2023

Title

Liver-cKO-3, IP

Sample type

SRA

Source name

Liver

Organism

Mus musculus

Characteristics

strain: C57BL/6
age: 8 weeks
genotype: Mettl3 cKO
Sex: male
tissue: liver

Extracted molecule

total RNA

Extraction protocol

Livers were removed, flash frozen on dry ice, and RNA was harvested using Trizol reagent.
MeRIP-Seq was performed by Cloudseq Biotech Inc. (Shanghai, China) according to the published procedure (Meyer et al., 2012) with slight modifications. Briefly, fragmented RNA was incubated with anti-m6A polyclonal antibody (Synaptic Systems, 202003) in IPP buffer for 2 hours at 4°C. The mixture was then immunoprecipitated by incubation with protein-A beads (Thermo Fisher) at 4°C for an additional 2 hours. Then, bound RNA was eluted from the beads with N6-methyladenosine (BERRY & ASSOCIATES, PR3732) in IPP buffer and then extracted with Trizol reagent (Thermo Fisher) by following the manufacturer’s instruction. Purified RNA was used for RNA-seq library generation with NEBNext® Ultra™ RNA Library Prep Kit (NEB). Both the input sample without immunoprecipitation and the m6A IP samples were subjected to 150 bp paired-end sequencing on Illumina HiSeq sequencer.

Library strategy

RIP-Seq

Library source

transcriptomic

Library selection

other

Instrument model

Illumina HiSeq 4000

Data processing

Briefly, Paired-end reads were harvested from Illumina HiSeq 4000 sequencer, and were quality controlled by Q30. After 3’ adaptor-trimming and low quality reads removing by cutadapt software (v1.9.3). First, clean reads of all libraries were aligned to the reference genome (UCSC MM10) by Hisat2 software (v2.0.4). Methylated sites on RNAs (peaks) were identified by MACS software. Differentially methylated sites were identified by diffReps. These peaks identified by both softwares overlapping with exons of mRNA were figured out and choosed by home-made scripts. GO and Pathway enrichment analysis were performed by the differentially methylated protein coding genes.
Paired-end reads were harvested from Illumina HiSeq 4000 sequencer, and were quality controlled by Q30. After 3’ adaptor-trimming and low quality reads removing by cutadapt software (v1.9.3), the high quality clean reads were aligned to the reference genome (UCSC MM10) with hisat2 software (v2.0.4). Then, guided by the Ensembl gtf gene annotation file, cuffdiff software (part of cufflinks) was used to get the gene level FPKM as the expression profiles of mRNA, and fold change and p-value were calculated based on FPKM, differentially expressed mRNA were identified. GO and Pathway enrichment analysis were performed based on the differentially expressed mRNAs.
Genome_build: mm10
Supplementary_files_format_and_content: For MeRIP-seq, xlsx files contain each peak of associated genes; for RNA-seq ,xlsx files include FPKM values for each Sample.

Submission date

Oct 01, 2021

Last update date

Oct 01, 2023

Contact name

Xiang Zhang

Organization name

the Fourth Military Medical University

Street address

Changle west road #169

City

Xi'an