Human breast cancer cell lines were grown in RPMI-1640, 10% fetal bovine serum, and 1% penicillin/streptomycin
Extracted molecule
total RNA
Extraction protocol
Total RNA from DMSO or drug –treated cells was extracted using a RNeasy Mini kit with QIAshredder spin column for homogenization and an on-column DNase digestion (79254, Qiagen) following the manufacturer’s instructions and re-suspended in 50 μL nuclease-free water (Ambion, AM9938). Total RNA was spiked-in with ERCC RNA Spike-In Mix (Ambion, 4456740) proportional to cell number (Loven et al., 2012), treated with DNA-freeTM DNase I (Ambion, AM1906), and analyzed on Agilent 2100 Bioanalyzer for integrity.
Label
biotin
Label protocol
For microarray analysis, 100 ng of total RNA containing ERCC RNA Spike-In Mix (see above) was used to prepare biotinylated aRNA (cRNA) according to the manufacturer’s protocol (30 IVT Express Kit, Affymetrix 901228). Briefly, total RNA undergoes T7 oligo(dT)-primed reverse transcription to synthesize first-strand cDNA containing a T7 promoter sequence. This cDNA is then converted into a double-stranded DNA template for transcription using DNA Polymerase and RNase H to simultaneously degrade the RNA and synthesize second strand cDNA. In vitro transcription synthesizes aRNA and incorporates a biotin-conjugated nucleotide. The aRNA is then purified to remove unincorporated NTPs, salts, enzymes, and inorganic phosphate. Fragmentation of the biotin-labeled aRNA prepares the sample for hybridization onto GeneChip 3’ expression arrays.
Hybridization protocol
Samples were prepared for hybridization using 10 µg of biotinylated aRNA in a 1X hybridization cocktail according the Affymetrix hybridization manual. Additional hybridization cocktail components were provided in the Affymetrix GeneChip Hybridization, Wash and Stain Kit. GeneChip arrays (Human PrimeView, Affymetrix 901837) were hybridized in a GeneChip Hybridization Oven at 45C for 16 hours at 60 RPM. Washing was done using a GeneChip Fluidics Station 450 according to the manufacturer’s instructions, using the buffers provided in the Affymetrix GeneChip Hybridization, Wash and Stain Kit.
Scan protocol
Images were extracted with Affymetrix GeneChip Command Console (AGCC), and analyzed using GeneChip Expression Console. A Primeview CDF that included probe information for the ERCC controls (GPL16043), provided by Affymetrix, was used to generate .CEL files.
Data processing
A CDF provided by Affymetrix, which contained the ERCC probes (PrimeView_withERCC_binary.cdf) was used instead of the standard PrimeView cdf. The data were analyzed with Bioconductor using the MAS5 normalization method. Then, these data were renormalized to the external spike-ins. See Loven, Orlando et al., Cell, 2012 for additional details.
