cell line: Loucy treatment: Cells were treated with DMSO (1/1000 dilution) for 4 hours
Treatment protocol
Jurkat/ Loucy/ MM.1S or Hela S3 cells were plated at the appropriate densities: 250,000-1,000,000 cells/ mL for Jurkat/Loucy/MM.1S and 500,000 to 2,000,000 cells per 60-mm plate for Hela S3 (per experimental protocols) and incubated in media containing CDK7-IN-1, CDK7-IN-1R, Flavopiridol, or Actinomycin D at the indicated concentrations or with DMSO for the specified duration of time. 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).
Growth protocol
Jurkat, Loucy, and MM.1S cells were grown in RPMI medium supplemented with 1% glutamine and 10% FBS and Hela S3 cells were grown in DMEM medium supplemented with 10% FBS and 1% penn/strep/glutamine. All 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 and sample preparation was performed as previously described (Loven et al. 2012). Briefly, biological duplicates (equivalent to 5 million cells per replicate for Jurkat/ Loucy/ MM.1S and 6,000,000 cells for Hela S3) were subsequently collected and homogenized in 1 ml of TRIzol Reagent (Life Technologies, 15596-026), purified using the mirVANA miRNA isolation kit (Ambion, AM1560) 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), 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
Batches of microarray experiments were normalized within themselves to ERCC spike-in probes as described in (Loven et al. 2012). Briefly, probes were combined into probesets using expresso with mas5 normalization. Probeset expression profiles were normalized using loess from the affy R package to equilibrate ERCC spike-in probes across microarrays in a given batch. Where possible, log2 values of biological replicates were averaged. Fold-changes were taken by subtracting average log2 DMSO signal from average log2 treatment signal. Expressed genes were those with log2(expression) > log2(100) in the corresponding DMSO sample.
