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| Status |
Public on Aug 19, 2021 |
| Title |
GR-Input |
| Sample type |
SRA |
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| Source name |
Mouse insulinoma cells
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| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
cell type: Mouse insulinoma cells
treatment: transfected GR expression vector
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| Treatment protocol |
Before transfection, the cell state was observed, and a proper amount of cells were inoculated into 10cm dishes, and the cell density was controlled at 50%~70%. After 16-24 hours, Lipofectamine 2000(Invitrogen) was used for transfection, and 10ug plasmid and 12 ul Lipofectamine 2000 were added to each 10cm dish. 24 hours after transfection, RNAs were extracted by trizol reagent.
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| Growth protocol |
The Min6 cells were cultured in DMEM medium containing 25mM D- glucose, 15％ FBS, 10mM HEPES, 1mM sodium pyruvate, 100 U/mL penicillin, 100 ug/mL streptomycin and 50μmol/L beta- mercaptoethanol, and incubated in a cell incubator at 37℃ with 95% air and 5% CO2.
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA in cells was extracted by Trizol, m6A on mRNA was specifically identified by m6A antibody, and methylated RNA fragments were enriched by RNA immunoprecipitation method
The mRNA m6A was sequenced by MeRIP-seq at Novogene (Beijing, China). Briefly, a total of 300 µg RNAs were extracted from the Min6 cells. The integrity and concentration of extracted RNAs were detected using an Agilent 2100 bioanalyzer (Agilent) and simpliNano spectrophotometer (GE Healthcare), respectively. Fragmented mRNA (~100 nt) was incubated for 2 hr at 4℃ with anti-m6A polyclonal antibody (Synaptic Systems) in the immunoprecipitation experiment. Then, immunoprecipitated mRNAs or Input was used for library construction with NEBNext ultra RNA library prepare kit for Illumina (New England Biolabs). The library preparations were sequenced on an Illumina Novaseq or Hiseq platform with a paired-end read length of 150 bp according to the standard protocols.
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| Library strategy |
RIP-Seq |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
total RNA
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| Data processing |
Quality control: The library preparations were sequenced on an Illumina Novaseq or Hiseq platform with a paired-end read length of 150 bp according to the standard protocols. The sequencing was carried out with Min6 cells independent biological replicates.
Reads mapping to the reference genome: Reference genome and gene model annotation files were downloaded from genome website directly. Index of the reference genome was built using BWA v0.7.12 and clean reads were aligned to the reference genome using BWA mem v 0.7.12.
Peak calling: After mapping reads to the reference genome, exomePeak R package (version 2.16.0) was used for the m6A peak identification in each anti-m6A immunoprecipitation group with the corresponding input samples serving as a control, and q-value threshold of enrichment of 0.05 was used for all data sets. The m6A-enriched motifs of each group were identified by HOMER (version 4.9.1).
Peak annotation: In the peak calling result, each peak was corresponding on gene, in which the peak was located in its exon. These genes were supposed as peak related genes, and then Gene Ontology (GO) enrichment analysis was performed to identify the function enrichment results. GO enrichment analysis was implemented by the GOseq R package, in which gene length bias was corrected. GO terms with corrected P-value less than 0.05 were considered significantly enriched by peak related genes. KEGG is a database resource for understanding high-level functions and utilities of the biological system, such as the cell, the organism and the ecosystem, from molecular-level information, especially large-scale molecular datasets generated by genome sequencing and other high-through put experimental technologies (http://www.genome.jp/kegg/). We used KOBAS software (version 3.0) to test the statistical enrichment of peak related genes in KEGG pathways.Besides, the distribution of peak on different function regions, such as 5’ UTR, CDS, 3’UTR was performed.
Different peak analysis: Differential peak calling was performed using exomePeak R package (version 2.16.0) with parameters of P-value less than 0.05 and fold_change more than 1. Using the same method, genes associated with different peaks were identified and also do GO and KEGG enrichment analysis.
Genome_build: mm10
Supplementary_files_format_and_content: differ peak text files and BigWig files(can be observed by IGV or uploaded to browsers such as UCSC for observation )
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| Submission date |
Aug 17, 2021 |
| Last update date |
Aug 21, 2021 |
| Contact name |
shao yi xue |
| E-mail(s) |
shaoyixue@outlook.com
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| Phone |
15911663318
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| Organization name |
Nanjing Medical University
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| Street address |
LongMian street
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| City |
Nanjing |
| ZIP/Postal code |
211112 |
| Country |
China |
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| Platform ID |
GPL24247 |
| Series (2) |
| GSE182268 |
m6A-seq of Min6 cells were transfected with the GR cDNA expression vector or control vector |
| GSE182269 |
RNA-seq and m6A-seq of Min6 cells were transfected with the GR cDNA expression vector or control vector |
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| Relations |
| BioSample |
SAMN20838789 |
| SRA |
SRX11801288 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5525953_GR_input.bw |
56.6 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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