Brain Tissue Homogenization for rAAV Genome Recovery

Half of a frozen brain hemisphere (0.3 g approx.) was homogenized with a 2 ml glass homogenizer (Sigma Aldrich; D8938) or a motorized plastic pestle (Fisher Scientific;12-141-361, 12-141-363) (for smaller tissues) or beads using BeadBug Homogenizers (1.5–3.0 mm zirconium or steel beads per manufacturer recommendations) (Homogenizers, Benchmark Scientific, D1032–15, D1032–30, D1033–28) and processed using Trizol as described in our prior work^{26 (link)} (also see Supplementary Note 17). From deep sequencing data analysis, we observed that the amount of tissue processed was sufficient for rAAV genome recovery.

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Kumar S.R., Miles T.F., Chen X., Brown D., Dobreva T., Huang Q., Ding X., Luo Y., Einarsson P.H., Greenbaum A., Jang M.J., Deverman B.E, & Gradinaru V. (2020). Multiplexed Cre-dependent selection yields systemic AAVs for targeting distinct brain cell types. Nature methods, 17(5), 541-550.

Publication 2020

D8938 BeadBug homogenizers Homogenizers

Brain hemisphere Frozen Genome Steel Tissue Trizol Zirconium

Corresponding Organization :

Other organizations : California Institute of Technology

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Variable analysis

independent variables

Homogenization method: Glass homogenizer, motorized plastic pestle, or BeadBug homogenizers with 1.5–3.0 mm zirconium or steel beads

dependent variables

Amount of rAAV genome recovered

control variables

Amount of tissue processed (0.3 g approx. of half a frozen brain hemisphere)

controls

No positive or negative controls were explicitly mentioned.

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