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Status |
Public on Jul 24, 2020 |
Title |
Heart_NC_2 weeks_IP |
Sample type |
SRA |
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Source name |
NC overexpressed heart
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Organism |
Mus musculus |
Characteristics |
strain background: C57BL/6 genotype/variation: NC-overexpressed tissue: heart antibody: Anti-N6-methyladenosine (m6A) antibody (Abcam, ab208577)
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Treatment protocol |
CHAPIR mimic (30 mg/kg) was administered using implanted osmotic minipumps (Alzet model 1002; Alza Corp.). A scrambled mimic served as a negative control (NC) and it was administered using the same procedure of mimic treatment. Pumps were dorsally implanted in the mice for 14 days.
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Extracted molecule |
total RNA |
Extraction protocol |
Total RNAs were extracted from CHAPIR mimic and negative control (NC) mice hearts using Trizol reagent (Invitrogen). RNA were quantified using a NanoDrop ND-1000 instrument. mRNAs were isolated from total RNA sample and they were chemically fragmented to 100-nucleotides-long fragments. MeRIP was performed to enrich m6A methylated mRNAs using Anti-N6-methyladenosine (m6A) antibody (Abcam, ab208577). We used KAPA Stranded mRNA-seq Kit (Illumina) for RNA-seq library preparation of both m6A enriched RNAs and input mRNAs. The libraries were subjected to denaturation to obtain single-stranded DNA molecules and captured on Illumina flow cells. Then, they were amplified in situ as sequencing clusters and sequenced for 150 cycles on Illumina HiSeq 4000 system as per the manufacturer’s instructions. The image analysis and base calling were carried out using Solexa pipeline v1.8 (Off-Line Base Caller software, v1.8). The sequencing quality was examined by FastQC software and trimmed reads (pass Illumina quality filter, trimmed adaptor bases by cutadapt) were aligned to genome sequences from Ensembl using HISAT2 software (v2.1.0). The aligned reads were used for peak calling by exomePeak and a statistically significant MeRIP enriched regions (peaks) were identified for each transcript and compared by exomePeak. The MeRIP enriched regions (peaks) were annotated by the overlapped gene using the newest version of Ensembl database. Then the statistical analysis of m6A peak in each transcript region was done.
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Library strategy |
OTHER |
Library source |
transcriptomic |
Library selection |
other |
Instrument model |
Illumina HiSeq 4000 |
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Data processing |
Illumina HiSeq raw sequencing read data that passed the Illumina chastity flter were used for the following analysis. The IP and Input trimmed reads (with 5’, 3’-adaptor bases removed) were aligned to genome reference sequences using HISAT2 software (v2.1.0). Aligned reads were used for peak calling of the MeRIP regions, statistically signifcant MeRIP enriched regions (peaks) were identifed for each sample at a p value threshold of 0.05. The MeRIP enriched regions (peaks) were annotated by the overlapped gene using the newest Ensembl database. The alignment statistical analysis was applied to retain the valid sequences for subsequent signifcant differentially methylated regions identifed. Hierarchical clustering, scatter plots and volcano plots were performed for the methylated enriched regions analysis in R or Python environment for statistical computing and graphics. Genome_build: Ensembl genes (GRCm38) Supplementary_files_format_and_content: The fold enrichment within the peak in the IP sample compared with the input sample
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Submission date |
Jul 19, 2020 |
Last update date |
Jul 25, 2020 |
Contact name |
Kun Wang |
E-mail(s) |
wangk696@qdu.edu.cn
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Organization name |
Qingdao University
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Street address |
308 Ningxia Road
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City |
Qingdao |
State/province |
Shandong |
ZIP/Postal code |
266021 |
Country |
China |
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Platform ID |
GPL21103 |
Series (1) |
GSE154699 |
Analysis of m6A methylome in CHAPIR overexpressed heart [MeRIP] |
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Relations |
BioSample |
SAMN15579816 |
SRA |
SRX8770585 |
Supplementary file |
Size |
Download |
File type/resource |
GSM4677601_NC_peaks_allRNA.xlsx |
2.5 Mb |
(ftp)(http) |
XLSX |
SRA Run Selector |
Raw data are available in SRA |
Processed data provided as supplementary file |
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