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Sample GSM7433506

Query DataSets for GSM7433506

Status

Public on Oct 31, 2023

Title

IP Sham_1

Sample type

SRA

Source name

heart ventricular tissue

Organism

Mus musculus

Characteristics

tissue: heart ventricular tissue
genotype: Sham
treatment: Sham

Treatment protocol

Silencing and inhibiting NAT10

Extracted molecule

total RNA

Extraction protocol

For acRIP-seq: Briefly, the concentration of total RNA was measured by Qubit RNA HS assay kit (Invitrogen, Q32852). Total RNA was fragmented into 100-200 nt RNA fragments using ZnCl2. Subsequently, the purified RNA was incubated with anti-ac4c polyclonal antibody for 3 h at 4℃ and then with protein A/G magnetic beads (Thermo Fisher Scientific, 88802) at 4 ℃ for an additional 2 h to obtain immunoprecipitated RNA fragments. The ac4c-enriched RNA was eluted from the beads in RLT buffer supplied in RNeasy Mini Kit (QIAGEN, 74106). The RNA-seq library was prepared by NEBNext® Ultra™ II RNA Library Prep Kit for Illumina® (NEB, E7775). For Ribo-seq:tissue were pulverized manually under liquid nitrogen and treated by ice-cold PBS containing 100 μg/mL cycloheximide. Ribosome profiling was performed using Epi™ Ribosome Profiling Kit (Epibiotek, R1814). Subsquently, RPFs (ribosome-protected RNA fragments) was extracted using RNA clean&ConcentratorTM-5 kit (ZYMO, R1016). EpiTM RiboRNA Depletion Kit (Epibiotek, R1805) was used for rRNA depletion. Sequencing libraries were constructed using QIAseq miRNA Library kit (QIAGEN, 1103679).
RNA libraries were prepared for sequencing using standard Illumina protocols
acRIP-seq; Ribo-seq; RNA-seq

Library strategy

OTHER

Library source

transcriptomic

Library selection

other

Instrument model

Illumina NextSeq 500

Data processing

Cutadapt (v2.5) was used to trim adapters and filter for sequences.
For acRIP-seq: The 150 bp paired-end reads were mapped to the reference genome by using hisat2 (v2.1.0).
For acRIP-seq: The m6A peaks were identified using exomePeak R package (v2.13.2) under parameters: “PEAK_CUTOFF_PVALUE=0.05,PEAK_CUTOFF_FDR=NA,FRAGMENT_LENGTH=200”. Identified m6A peaks which p value <0.05 were chosen for the de novo motif analysis using homer (v4.10.4) under parameters:”-len 6 -rna”.
For Ribo-seq: The reads were aligned to rRNA and tRNA sequences so as to remove rRNA and tRNA reads using bowtie2 software, remaining reads were used to align to reference genome and transcriptome (Ensembl Version 91) using hisat23 and bowtie2 software separately.
For Ribo-seq:ORF identification was performed using Price5 software. Read counts were calculated using featureCounts6 software. Raw counts were further normalized as RPKM values using fpkm function in DESeq27 package.
Assembly: mm10 and hg38
Supplementary files format and content: mRNA-seq_9 mouse_All.count.txt
Supplementary files format and content: Ribo-seq_9 mouse_All.count.txt
Supplementary files format and content: 12hsa_acRIP-seq_input.count.txt
Supplementary files format and content: 12mmu_acRIP-seq_input.count.txt

Submission date

May 30, 2023

Last update date

Oct 31, 2023

Contact name

Chuanxi Yang

E-mail(s)

2205515@tongji.edu.cn

Organization name

School of Medicine, Southeast University

Department

Department of Cardiology