Library Information

The genome is arrayed as 250ng of dsRNA resuspended in 5ul of water on 53x 384 plates. The library is arrayed in either white Grenier Bio One Ltd plates (#781073), for luminescent assays, black Thermo Matrix plates (#4318), for GFP reporter assays or clear bottomed View Plates from Perkin Elmer (#60017460), for high content microscopy screens.

The dsRNA collection we use is from Prof. Michael Boutros' lab and is the redesigned, non-off target effect library, HD2.0 generated using the software next-rnai (developed by Thomas Horn). Low complexity regions and sequence motifs that induce off-target effects have been excluded. dsRNA probe sizes vary from 81 to 800bp covering ~14000 protein encoding genes and ~1000 non-coding genes (~98.8% coverage).

The dsRNA design covers every isoform of each gene and have been optimised for specificity and avoidance of low complexity regions The HD2 library has been arrayed with controls in mind and has been reformatted according to the generic library plate layout (see layout figure for a visulalisation of each plate).

Each plate contains the following controls: 4 wells with no dsRNA in the wells; A1,A2,B1,B2, 4 wells of DIAP1 dsRNA (variable between plates) and 16 empty wells containing no dsRNA (for screen-specific controls). The left-hand corner empty wells act as no dsRNA controls, the DIAP1 wells causes cell death and act as a control to see if RNAi is working.

The 16 empty wells are spiked with 4 known regulators of the process you are investigating in duplicate and 4 control dsRNAs (ie, lac-Z, GFP etc). You will need to supply your control dsRNAs, but we have a range of "neutral controls".

Luminometry