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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Jun 07, 2023 |
| Title |
BMMCs, Input, 2 |
| Sample type |
SRA |
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| Source name |
BMMCs
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| Organism |
Mus musculus |
| Characteristics |
cell type: BMMCs
genotype: wild type
treatment: Input
antibody: anti-m6A antibody (SynapticSystem)
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Total RNA was isolated with TRIzol. For mRNA purification, two rounds of poly(A) selection were perfomed using Dynabeads mRNA purification kit (Ambion) according to the manufacturer’s instructions, which was followed by fragmentation using RNA fragmentation reagents (Invitrogen). To verify the size of fragmented mRNAs, the mRNA samples were analyzed with the Agilent RNA 6000 pico kit on an Agilent 2100 Bioanalyzer System. The fragmented mRNA samples were incubated with anti-m6A antibody (Synaptic Systems) and cross-linked twice with 150 mJ/cm2 UV light (254 nm) in a Stratalinker. After cross-linking, the solution was further incubated with protein A beads. After stringent washing, RNA 3’ ends were dephosphorylated with T4 PNK and subsequentlys the 3’ adaptor was ligated. The samples were radioactively end-labled and were subjected to NuPAGE gel electrophoresis and tranferred onto Nitrocellulose Membrane. The RNA was isolated from the membrane by treatment with proteinase K. After phonol/chloroform extraction and precipitation, the RNA was reversed transcribed and cDNA was purified with Dynabeads MyOne Silane (Invitrogen). The 5’ adaptor was ligated. First-strand cDNA was synthesized with 6 cycles and size-selected using ProNex Size-Selective Purification System (Promega). Libraries were PCR amplified for 8–13 cycles and sequencing was performed on an Illumina sequencer.
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| Library strategy |
RIP-Seq |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
NextSeq 1000 |
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| Data processing |
Reads were de-multiplexed based on the experimental barcode and adapter sequences were removed from the read ends (Flexbar v3.5.0). UMIs were trimmed and added to the read names. Individual samples were mapped to the respective genome (assembly version GRCm38.p6 for all mouse samples) and its annotation (GENCODE release M25 for all mouse samples) using STAR (v2.7.6a). Reads were de-duplicated if they had identical UMIs. Significant crosslink sites at single-nucleotide resolution were called using PureCLIP (v.1.3.1) with default parameters, and individual crosslink sites within a distance of 8 bp of each other were merged into wider binding regions (peaks). For assigning a host gene to each PureCLIP peak, transcript annotations were taken from GENCODE release M32, mm39.
Assembly: GRCm39
Supplementary files format and content: Bigwig files were generated usingbed GraphToBigWig.
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| Submission date |
Mar 30, 2023 |
| Last update date |
Jun 07, 2023 |
| Contact name |
Cristina Leoni |
| E-mail(s) |
cristina.leoni@irb.usi.ch
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| Organization name |
Institute for Research in Biomedicine
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| Department |
Molecular Immunology
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| Lab |
120
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| Street address |
Via F. Chiesa 5
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| City |
Bellinzona |
| State/province |
Ticino |
| ZIP/Postal code |
6500 |
| Country |
Switzerland |
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| Platform ID |
GPL32159 |
| Series (2) |
| GSE228612 |
The mRNA methyltransferase Mettl3 modulates cytokine mRNA stability and limits functional responses in mast cells [m6A-CLIP-Seq] |
| GSE228615 |
The mRNA methyltransferase Mettl3 modulates cytokine mRNA stability and limits functional responses in mast cells |
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| Relations |
| BioSample |
SAMN34001071 |
| SRA |
SRX19832960 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7133963_Input2.minus.bw |
1.5 Mb |
(ftp)(http) |
BW |
| GSM7133963_Input2.plus.bw |
1.9 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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