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Morning Push Checklist - 2025-05-06

Ybx1 Knockdown mRNA Stability Project Initiation

Project Setup

  • Create project entry for Post-transcriptional regulation by Ybx1
  • Create subproject for mRNA stability measurements
  • Create protocol for Ybx1 knockdown mRNA stability assay
  • Generate experiment YAML for initial Ybx1 knockdown study (EXP-0225)
  • Create data directories for experiment results

Day 1 Tasks (May 6, 2025) - Jack Zhao

  • Thaw HEK293T cells and check viability
  • Prepare transfection master mixes
    • siRNA targeting Ybx1
    • Control siRNA
  • Set up reverse transfection in 24-well plates
    • Label all plates and wells
    • Prepare Lipofectamine RNAiMAX and Opti-MEM mixtures
    • Add siRNA to appropriate wells
    • Add cells to all wells
  • Place plates in 37°C incubator with 5% CO2
  • Update experiment status in repository

Upcoming Tasks

  • Day 3 (May 8): Verify Ybx1 knockdown by qPCR
  • Day 3 (May 8): Begin actinomycin D time course experiment
    • Collect t=0 samples before actinomycin D addition
    • Add actinomycin D (5 μg/ml) to remaining wells
    • Collect samples at 2h, 4h, and 8h timepoints
  • Day 4 (May 9): Complete RNA extractions
  • Day 4 (May 9): Synthesize cDNA and perform qPCR
  • Create issue for RNA collection on day 4 (issue for Jack Zhao) - NOTE: Create manually in GitHub interface since authentication is required

Materials to Order/Check

  • Confirm Actinomycin D stock availability
  • Ensure sufficient RNA extraction reagents are available
  • Check qPCR primer stocks for all target genes

Pull Request Items

  • Request PI review of experimental design
  • Share preliminary results after day 3 knockdown verification
  • Final data submission after completion of all qPCR analyses

Notes

  • This experiment is the first in a series to understand how Ybx1 affects mRNA stability
  • Target genes include those with known or suspected m5C/m6A modifications
  • Pay special attention to transfection efficiency - critical for meaningful results
  • Submit data to repository as soon as results are analyzed
  • IMPORTANT: Manually create GitHub issues for each major step as GitHub CLI authentication is pending