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50 lines
2.2 KiB
Markdown
50 lines
2.2 KiB
Markdown
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# Morning Push Checklist - 2025-05-06
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## Ybx1 Knockdown mRNA Stability Project Initiation
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### Project Setup
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- [x] Create project entry for Post-transcriptional regulation by Ybx1
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- [x] Create subproject for mRNA stability measurements
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- [x] Create protocol for Ybx1 knockdown mRNA stability assay
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- [x] Generate experiment YAML for initial Ybx1 knockdown study (EXP-0225)
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- [x] Create data directories for experiment results
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### Day 1 Tasks (May 6, 2025) - Jack Zhao
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- [ ] Thaw HEK293T cells and check viability
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- [ ] Prepare transfection master mixes
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- [ ] siRNA targeting Ybx1
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- [ ] Control siRNA
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- [ ] Set up reverse transfection in 24-well plates
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- [ ] Label all plates and wells
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- [ ] Prepare Lipofectamine RNAiMAX and Opti-MEM mixtures
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- [ ] Add siRNA to appropriate wells
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- [ ] Add cells to all wells
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- [ ] Place plates in 37°C incubator with 5% CO2
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- [ ] Update experiment status in repository
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### Upcoming Tasks
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- [ ] Day 3 (May 8): Verify Ybx1 knockdown by qPCR
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- [ ] Day 3 (May 8): Begin actinomycin D time course experiment
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- [ ] Collect t=0 samples before actinomycin D addition
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- [ ] Add actinomycin D (5 μg/ml) to remaining wells
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- [ ] Collect samples at 2h, 4h, and 8h timepoints
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- [ ] Day 4 (May 9): Complete RNA extractions
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- [ ] Day 4 (May 9): Synthesize cDNA and perform qPCR
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- [ ] Create issue for RNA collection on day 4 (issue for Jack Zhao) - **NOTE: Create manually in GitHub interface since authentication is required**
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### Materials to Order/Check
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- [ ] Confirm Actinomycin D stock availability
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- [ ] Ensure sufficient RNA extraction reagents are available
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- [ ] Check qPCR primer stocks for all target genes
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### Pull Request Items
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- [ ] Request PI review of experimental design
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- [ ] Share preliminary results after day 3 knockdown verification
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- [ ] Final data submission after completion of all qPCR analyses
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### Notes
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* This experiment is the first in a series to understand how Ybx1 affects mRNA stability
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* Target genes include those with known or suspected m5C/m6A modifications
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* Pay special attention to transfection efficiency - critical for meaningful results
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* Submit data to repository as soon as results are analyzed
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* **IMPORTANT: Manually create GitHub issues for each major step as GitHub CLI authentication is pending**
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