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| Status |
Public on May 01, 2024 |
| Title |
SG RNA3 |
| Sample type |
SRA |
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| Source name |
macrophage
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| Organism |
Mus musculus |
| Characteristics |
cell type: macrophage
rna fraction: SG-associated RNA
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| Treatment protocol |
Previous studies already showed that stressed RNA granule pellet could be a reasonable proxy for full SG preparation,[1] thus we used SG core enriched fraction to validate the enriched transcripts.After House dust mite treatment for 30min, one 15 cm-dish cells were washed twice with ice-cold PBS quickly (within 10s) and then harvested in 1.5 ml ice-cold SG lysis buffer (50mM Tris pH 7.6, 50mM NaCl, 5mM MgCl2,0.1% NP-40, 1mM b-mercaptoethanol, 1% EDTA-free protease inhibitor cocktail, and 0.4 U/mL RNase inhibitor) on ice for 20 min. After that, cell suspensions were given 30 strokes in a Dounce homogenizer. 1/10 of cell suspensions were separated as total cell fractions. The other cell suspensions was centrifuged at 2,000 g for 5 min at 4 . The supernatant contains cytosolic fraction, and the pellet contains nuclear fraction. Then, 1 ml of the supernatant was centrifuged at 18,000 g for 20 min at 4 to isolate the insoluble pellet fraction. After discarding the supernatant, the pellet was re-suspended in 1 ml lysis buffer and centrifuged at 18,000 g for another 20 min at 4 . Then, the supernatant was discarded, whereas the pellet was re-suspended in 300 ml SG lysis buffer and centrifuged at 850 g for 2 min at 4 . The supernatant represented SG core enriched fractions. The RNAs from total cell fractions, and SG core-enriched fractions were extracted using QIAzol (79306, Qiagen).
reference: 1. QKI shuttles internal m7G-modified transcripts into stress granules and modulates mRNA metabolism. Zhao et al., 2023, Cell 186, 1 C19.
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| Growth protocol |
DMEM, 10% fetal bocine serum, 1×penicilin/streptomycin. Cells were cultured at 37℃ with 5% CO2.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using Trizol reagent
Total RNA was extracted followed by library preparation according to Illumina standard instruction (VAHTS Universal V6 RNA-seq Library Prep Kit for Illumina® ). Agilent 4200 bioanalyzer was employed to evaluate the concentration and size distribution of cDNA library before sequencing with an Illumina novaseq6000. The protocol of high-throughput sequencing was fully according to the manufacturer's instructions (Illumina).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
The RNA is purified by phenol:chloroform extraction.
The raw reads were filtered by Seqtk before mapping to genome using Hisat2 (version:2.0.4). The fragments of genes were counted using stringtie(v1.3.3b) followed by TMM (trimmed mean of M values) normalization. Significant differential expressed genes (DEGs) were identified as those with a False Discovery Rate (FDR) value above the threshold (Q< 0.05)
Assembly: mm39
Supplementary files format and content: tab-delimited with raw counts
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| Submission date |
Apr 12, 2024 |
| Last update date |
May 01, 2024 |
| Contact name |
Ye Zhou |
| E-mail(s) |
yzhou1989@126.com
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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
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| City |
Shanghai |
| ZIP/Postal code |
200433 |
| Country |
China |
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| Platform ID |
GPL24247 |
| Series (1) |
| GSE263848 |
Stress granule assembly impairs macrophage efferocytosis [SG RNA-seq] |
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| Relations |
| BioSample |
SAMN40943561 |
| SRA |
SRX24234666 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
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