PcG perturbation experiment planning

RNAi

• Cavalli lab (Gonzalez et al) do RNAi based visual screen of RNAi knockdown, look at Pc.
• distributions of Pc fluorescence across cells in Pc knockdown is pretty tight — the remaining variability could be background auto-fluorescence. This looks promising.
• recommend dsRNA targeting th and rho as transfection controls, these produce cell death and large nuclear size.
• flyRNAi protocol: http://www.flyrnai.org/DRSC-PRS.html (from Drosophila RNAi screening center at Harvard Medical School).
• see also http://www.genomernai.org/About,

Questions

• do I just look up tested-primer sequences (with T7 additions) for my targets and use these to amplify from my in-house genomic DNA?
• or can I order DNA template from the DRSC?

gene deletion

• Nice PSC Su(Z)2 deletion: Su(Z)2-1.b8
• could order corresponding fly line to make late-stage embryos as well: 24467. Deletion over eve-LacZ marked balancer SM6 (Cy[1]).
• should contact Perrimon lab.

To order

• more Sneider’s medium (for culture of deletion line and for RNAi protocols).
• Fly stock: 24467