BE2C cells were plated at 500,000 cells/ mL and incubated in media containing 3uM JQ1 and 125nM THZ1, or with DMSO, for 1, 4, or 12 hrs. At the end of the experiment cells were collected by centrifugation and cell numbers were determined by manually counting cells using C-Chip disposable hemocytometers (Digital Bio, DHC-N01). 5,000,000 cells were isolated from each treatment condition.
Growth protocol
BE2C cells were grown in RPMI medium supplemented with 1% glutamine and 10% FBS. Cells were cultured at 37 degrees C in a humidified chamber in the presence of 5% CO2, unless otherwise noted.
Extracted molecule
total RNA
Extraction protocol
Total RNA from biological replicates (equivalent to 5 million cells per replicate) was subsequently isolated using TRIzol (Thermo Fisher, cat# 15596026) 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) 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 (3' 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 per batch (B1/B2) to the external spike-ins. See Loven, Orlando et al., Cell, 2012 for additional details.
