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| Status |
Public on Nov 06, 2024 |
| Title |
BMDM-RIP-Rbm25 |
| Sample type |
SRA |
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| Source name |
BMDM
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| Organism |
Mus musculus |
| Characteristics |
cell line: Mouse primary bone marrow-derived macrophages
genotype: C57BL/6J
treatment: RIP lysate anti-Rbm25
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| Extracted molecule |
total RNA |
| Extraction protocol |
RIP-seq was performed using Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore), according to the product instructions. Briefly, 1x10^7 macrophages were harvested and re-suspended in RIP Lysis Buffer. Rbm25-binding RNA was immunoprecipitated by antibody against Rbm25 (Bethyl) and then purified for use.
First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase（RNase H-）. Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3’ ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 150~200 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then 3 µl USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37°C for 15 min followed by 5 min at 95 °C before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system.
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| Library strategy |
RIP-Seq |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
Because the interrupted fragment of RIP-seq is usually small, some sequenced reads contain junction sequences. After pruning, the joint sequence was removed. If the length of the remaining reads after pruning was long enough, it could still be used for subsequent analysis. The original reads were pruned to produce clean reads for subsequent analysis.
BWA (Burrows Wheeler Aligner) was used to aligned clean reads to reference genomes mm10. MACS2 software (threshold: qvalue=0.05) was used for peak calling analysis, and the number, width and distribution of peaks were statistically analyzed, and peak related genes were screened out.
The enrichment factor can also be called signal Value, which represents the digital display of the peak signal in the process of calling peak. The larger the value, the more reads enriched to the peak. The concentration ratio of peaks is used as an index to calculate the number of peaks. HOMER software was used for identification of the motifs on RNAs in protein-binding peaks.
Assembly: mm10
Supplementary files format and content: bw file that includes RNA information
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| Submission date |
Aug 04, 2023 |
| Last update date |
Nov 06, 2024 |
| Contact name |
Xingguang Liu |
| E-mail(s) |
[email protected]
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| Organization name |
Naval medical university
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| Department |
National Key Laboratory of Immunity & Inflammation
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| Street address |
Xiangyin road 800
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| City |
Shanghai |
| ZIP/Postal code |
200433 |
| Country |
China |
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| Platform ID |
GPL24247 |
| Series (2) |
| GSE240159 |
Identification of Rbm25-binding RNAs in bone-marrow-derived macrophages by RIP-seq |
| GSE240160 |
Next Generation Sequencing Facilitates Quantitative Analysis of wild type and Rbm25-deficient Bone Marrow Derived macrophages |
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| Relations |
| BioSample |
SAMN36844467 |
| SRA |
SRX21256488 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7683556_RIP.bw |
9.7 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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