VALUE = RMA signal
Arrays were scanned on Nimblegen's 2-μm, high-resolution MS 200 Microarray Scanner. Data was extracted from the images at Roche NimbleGen, according to the protocol described NimbleScan v2.5 Software User's Guide, Version 2.5, October 2008.
Raw ChIP-chip data were retrieved from Nimblegen and analyzed using MA2C (Song et al., Genome Biol, 2007). MA2C analysis was performed with the following settings: # MA2C Score Method (median), Band Width (300), p-value cut off (-6), and other parameters were set as default. WS180 was used to annotate gene names. MA2C-defined LIN-54 ChIP peaks are listed in the file 'GSE28852_MA2C_processed_LIN-54_ChIP_peaks.txt'.
Statistical analyses were performed using R, a system for statistical computation and graphics ( http://www.r-project.org). The rma method in the affy package from Bioconductor was used in R to summarize probe level data and to normalize the dataset to remove across array variation. Log transformed data were used in subsequent analysis and plotting. WormBase version WS190 was used. To determine differentially expressed genes between wild-type and mutants, moderated T Statistics in limma was used with p-value ≤0.01, fold change ≥1.5.
Array scanning was performed according to the manufacturer's instruction (Affymetrix)
cDNA probes of three replicates were hybridized to GeneChip C. elegans genome arrays (Affymetrix). Probe-preparation, hybridization, and scanning for DNA microarray were performed at the Genomics Core facility at University of Massachusetts Medical School.
Strains for embryo or germline were cultured at 25o or 20oC, respectively, using standard methods.
Fluorescence-labeled cDNA probes were prepared using the One-Cycle kit (Affymetrix) and the Enzo HighYield RNA Transcript Labeling Kit (Enzo) for embryo, and the 3' IVT Express Kit (Affymetrix) for germline
Embryos from young adults were harvested by the bleach-alkaline method, and filtered through 100 micron mesh. Germlines were dissected in 1X egg buffer to excise the germline including mitotic tip through meiotic late pachytene 24 hours after L4 stage.
200 µL of embryo pellet was suspended in 1 mL of Tri reagent (Molecular Research Center, Inc. TR118), flash-frozen, and dounced. Total RNA was purified with RNAeasy mini kits (Qiagen), treated with DNase, and integrity examined on agarose gel. Germline RNA was isolated as described in Chi and Reinke (2006), and linearly amplified once using MessageAmp II aRNA Amplification Kit (Ambion).
Samples were then hybridized on the Nimblegen C. elegans ChIP-chip 385K Whole-Genome Tiling - 3 Array Set (GPL7482, GPL7483, GPL7484) using standard Nimblegen protocols.
ChIP was performed as described (Mukhopadhyay et al., Nat. Protocol., 2008). Briefly, mixed stage wild-type worms were cultured in S-basal at 20oC. Lysates were cross-linked in 1% formaldehyde, sonicated, and immunoprecipitated with anti-LIN-54 antibody or pre-bleed antibody control. ChIP samples including the input were subjected to two rounds of linear amplification, using the genomePlex complete whole genome amplification kit (Sigma), and minimum difference between original precipitates and amplified precipitate confirmed by qPCR.
For each experiment, a control sample as well as an experimental sample were prepared with a concentration of ~250 ng/ul. At least 4 ug of each of these samples was then sent to Nimblegen where the samples were labeled with respectively Cy-3 and Cy-5 dyes using standard Nimblegen protocols.
Lysates were cross-linked in 1% formaldehyde, sonicated, and immunoprecipitated with pre-bleed antibody control.
mixed stage wild-type worms were cultured in S-basal at 20oC.
Lysates were cross-linked in 1% formaldehyde, sonicated, and immunoprecipitated with anti-LIN-54 antibody
Lysates were cross-linked in 1% formaldehyde, sonicated, and immunoprecipitated with anti-LIN-54 antibody.