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| Status |
Public on Jun 04, 2020 |
| Title |
Analysis of High Molecular Weight RNA-induced silencing complex (HMW-RISC) in CD8+ T cells identifies miR-7 as a modulator of T cell activation |
| Organism |
Mus musculus |
| Experiment type |
Non-coding RNA profiling by high throughput sequencing
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| Summary |
To identify the miRNAs expected to be potent in suppressing targets, we followed HMW RISC formation upon activation of CD8+ T cells. We show that while most miRNAs distribute between HMW and LMW RISC in activated T cells, several miRNAs were dominant in one complex over the other. Among these, miR-7 is enriched in HMW RISC and inhibition of miR-7 upon T cell activation leads to increased production of IL-2 and expression of IL-2-regulated proteins including the α-subunit of the IL-2 receptor, CD25; transferrin receptor, CD71; and amino acid transporter, CD98, which are direct miR-7 targets. Our data support a model where recruitment of miR-7 to HMW RISC restrains IL-2 signalling and the metabolic processes regulated by IL-2 and thus modulates T cell activation.
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| Overall design |
RNA was isolated from Ago2-IP samples, as well as the IP input and unbound samples. The samples were resuspended in QIAzol lysis reagent and RNA was extracted using the miRNeasy kit (Qiagen) and eluted in 100µl dH2O. The RNA was then ethanol precipitated by adding 2.5 x volume 100% ethanol, 30µl sodium acetate and 1µl GlycoBlue Coprecipitant (Thermo Scientific) to the samples which were then incubated overnight at -20oC. The following day, the samples were centrifuged at full speed for 30 min, then washed twice with 70% ethanol. The pellets were air-dried on ice then resuspended in 10-15µl dH2O and quantified with Qubit 3.0 fluorometer (Thermo Scientific) using the Qubit RNA HS Assay kit (Thermo Scientific). Small RNA libraries were prepared using the CleanTagTM Small RNA Library Preparation kit (Trilink) using manufacturer’s instructions and 21 cycles. The libraries were size selected to include 145-160 bps and the input, IP-unbound and IP-bound libraries were pooled at a 1:1:2 ratio. The samples were sequenced using NovaSeq 50bp paired end sequencing at Edinburgh Genomics.
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| Contributor(s) |
Toivakka M, Gordon K, Kumar S, Zamoyska R, Buck A |
| Citation(s) |
37879863 |
| |
| Submission date |
Jun 01, 2020 |
| Last update date |
Jan 16, 2024 |
| Contact name |
Amy Buck |
| E-mail(s) |
amybucklab@gmail.com, a.buck@ed.ac.uk, sujai.kumar@ed.ac.uk
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| Organization name |
The University of Edinburgh
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| Department |
IIIR
|
| Lab |
Buck Lab
|
| Street address |
Ashworth Labs, Charlotte Auerbach Road
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| City |
Edinburgh |
| ZIP/Postal code |
EH9 3FL |
| Country |
United Kingdom |
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| Platforms (1) |
| GPL24247 |
Illumina NovaSeq 6000 (Mus musculus) |
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| Samples (13)
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| GSM4585159 |
OT1 CD8+ T cells activated with N4 Peptide rep1-HMW IP |
| GSM4585160 |
OT1 CD8+ T cells activated with N4 Peptide rep2-HMW IP |
| GSM4585161 |
OT1 CD8+ T cells activated with N4 Peptide rep3-HMW IP |
| GSM4585162 |
OT1 CD8+ T cells activated with N4 Peptide rep1-LMW IP |
| GSM4585163 |
OT1 CD8+ T cells activated with N4 Peptide rep2-LMW IP |
| GSM4585164 |
OT1 CD8+ T cells activated with N4 Peptide rep3-LMW IP |
| GSM4585165 |
OT1 CD8+ T cells activated with N4 Peptide rep1-total IP |
| GSM4585166 |
OT1 CD8+ T cells activated with N4 Peptide rep2-total IP |
| GSM4585167 |
OT1 CD8+ T cells activated with N4 Peptide rep3-total IP |
| GSM4585168 |
OT1 CD8+ T cells activated with N4 Peptide rep4-total IP |
| GSM4585169 |
Naïve OT1 CD8+ T cells rep1-total IP |
| GSM4585170 |
Naïve OT1 CD8+ T cells rep2-total IP |
| GSM4585171 |
Naïve OT1 CD8+ T cells rep3-total IP |
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| Relations |
| BioProject |
PRJNA636360 |
| SRA |
SRP265486 |
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