siRNA Circadian Assay
To identify additional clock genes and modifiers, we conducted a genome-wide screen of ~90,000 siRNAs (QIAGEN Inc) in a human cellular model of clock function. Knockdown of nearly a thousand genes reduced ..more
To identify additional clock genes and modifiers, we conducted a genome-wide screen of ~90,000 siRNAs (QIAGEN Inc) in a human cellular model of clock function. Knockdown of nearly a thousand genes reduced
rhythm amplitude; these are presumably due to toxic effects on cells. Hundreds of genes potently modified period length or increased amplitude.
Cell Culture Growth Conditions: U2OS cells were obtained from the American Type Culture Collection (ATCC), and were grown in regular DMEM supplemented with 10% FBS and antibiotics. Lentiviral
Bmal1-dLuc reporter was introduced into U2OS cells via lentivirus-mediated infection as described (Liu et al., PLoS Genetics, 2008). Stable cell lines were selected with blasticidin and clonal lines were sorted by FACS-based single cell sorting in 96-well plates. The clonal lines are genetically and morphologically indistinguishable from parental cells, and represent the average period length of the infected cell population.
Transfection: Human genome-wide siRNA set against 22,468 known or predicted genes was purchased from Qiagen Inc, and pre-spotted into 384-well plates with 2 siRNA/well, 2 well/gene in duplicates, and 294 plates in total. Subsequently, U2OS circadian reporter cells were reverse transfected with siRNAs in 384-well plates. Briefly, we trypsinized rapidly growing cells and re-suspended them in DMEM containing 20% FBS without antibiotics at 0.1X106 cells/ml. We next added 20 ul of transfection reagent mixture (3.3 ul/ml or 0.066 ul/well Lipofectamine 2000 in Opti-MEM) to each well containing pre-spotted siRNA constructs (1 pmol; 0.5 pmol for each siRNA; final concentration of 12.5 nM), incubated at room temperature for 20 min, and added 20 ul of cells (2000 cells/well) with our robotic system. Approximately 18 hr after transfection, we replaced this media with 60 ul pre-warmed fresh DMEM containing 10% FBS and antibiotics and allow the cells to grow for an additional two days.
Luminescence recording: Three days post-transfection, we replaced this media with 60 ul HEPES-buffered explant medium supplemented with luciferin (1 uM) (Promega) and B-27 supplements (Invitrogen), and the plates were sealed with an optically clear film (USA Scientific). We next loaded these plates in a 36oC incubator and recorded bioluminescence expression with a ViewLux (Perkin Elmer). We measured bioluminescence for 30 seconds every two hours for four days.
The data contain plate and well position and bioluminescence value from the primary screen. X-axis and Y-axis indicate time-course (from 0 to 78h) and plate/well position, respectively. Time 0 is set up as 8h after the change of luciferin-containing-medium /recording starts. Bioluminescence data was detrended by subtracting a best fit line (first order polynomial).
† RNAi Target.
Data Table (Concise)