H1.2 (and control) knock-down cell lines were established from T47D-MTVL cells: Human breast cancer cell line T47D modified to contain one stably integrated copy of Luciferase reporter gene driven by the Mouse Mammary Tumor Virus promoter, named T47D-MTVL (Truss, M., J. Bartsch, A. Schelbert, R. J. Hache, and M. Beato. 1995). Initially, a cell line expressing the Dox-responsive KRAB repressor and RedFP (ptTR-KRAB-Red) was generated. Then, this cell line was infected with viruses for expression of the different H1 variants shRNAs (pLVTHM). The inducible knocked-down cell lines were sorted in a FACSvantageSE (Becton Dickinson) for RedFP-positive and GFP-positive fluorescence after 3 days of Dox treatment. Then cells were amplified in the absence of Dox until an experiment was performed. Plasmids for the lentivirus vector-mediated drug-inducible RNA interference system (pLVTHM, ptTR-KRAB-Red, pCMC-R8.91 and pMD.G) were provided by D. Trono (University of Geneva) (Wiznerowicz M & Trono D (2003) Conditional suppression of cellular genes: lentivirus vector-mediated drug-inducible RNA interference. J Virol 77: 8957-8961).
Treatment protocol
Along a 6-day treatment with Dox, cells were passaged at day 3. Serum-containing media was replaced with serum-free media at day 4 for growth arrest.
Growth protocol
These cell lines were grown in RPMI 1640 medium, supplemented with 10% FBS, 2mM L-glutamine, 100 U/ml penicillin, and 100µg/ml streptomycin. Doxicycline (Sigma) was added at 2.5 µg/ml when indicated.
Extracted molecule
total RNA
Extraction protocol
RNeasy mini kit (QUIAGEN, cat.no. 74104) Quality assesment of Total RNA samples were analyzed using the Agilent Bioanalyzer 2100 and the RNA 6000 LabChip Kit (Agilent) with the Eukaryote Total RNA Nano Assay.
Label
Cy5
Label protocol
Linear T7-oligodT mediated mRNA amplification (described by Van Gelder et al. 1990 and Eberwine et al. 1992) 3 microgram of the in vitro amplified aRNA samples were reverse transcribed with 4 microliter 5X first strand buffer, 2 microliter 0.1 M DTT, 0.4 microliter low dT-dNTP mix (25 mM dA, dC, dG, 10 mM dT), 1.5 microliter Superscript II (Invitrogen) and 2 microliter 25 mM Cy3-dUTP or Cy5-dUTP (Amersham). The reaction was incubated at 42ºC for 2 hr, chilled on ice and quick spun to bring down condensation. To stop the reaction 1 microliter 1 M NaOH/20 mM EDTA, was added followed by 80 microliter MilliQ-H20 and 10 microliter 3M NaOAc pH 5. Labeled samples were purified with Quiaquick PCR purification columns (Qiagen) and eluted twice with 50 microliter EB (10 mM Tris pH 8.5). Labeling efficiency was calculated by quantification with Nanodrop, obtaining between 50-80 pmol/microliter of Cy3 or Cy5 labeled probes. Labeled samples for the same array were combined, 1 microliter 1 microgram/microliter Human Cot DNA (Invitrogen) was added, and the labeled mix was desiccated by Speed-Vac centrifugation.
Channel 2
Source name
Universal human reference RNA Stratagene® (cat.no.740000)
pool of ten different tumor cell lines from different human tissues Stratagene® (cat.no.740000)
Biomaterial provider
Stratagene® (cat.no.740000)
Treatment protocol
none
Growth protocol
none
Extracted molecule
total RNA
Extraction protocol
none
Label
Cy3
Label protocol
Linear T7-oligodT mediated mRNA amplification (described by Van Gelder et al. 1990 and Eberwine et al. 1992) 3 microgram of the in vitro amplified aRNA samples were reverse transcribed with 4 microliter 5X first strand buffer, 2 microliter 0.1 M DTT, 0.4 microliter low dT-dNTP mix (25 mM dA, dC, dG, 10 mM dT), 1.5 microliter Superscript II (Invitrogen) and 2 microliter 25 mM Cy3-dUTP or Cy5-dUTP (Amersham). The reaction was incubated at 42ºC for 2 hr, chilled on ice and quick spun to bring down condensation. To stop the reaction 1 microliter 1 M NaOH/20 mM EDTA, was added followed by 80 microliter MilliQ-H20 and 10 microliter 3M NaOAc pH 5. Labeled samples were purified with Quiaquick PCR purification columns (Qiagen) and eluted twice with 50 microliter EB (10 mM Tris pH 8.5). Labeling efficiency was calculated by quantification with Nanodrop, obtaining between 50-80 pmol/microliter of Cy3 or Cy5 labeled probes. Labeled samples for the same array were combined, 1 microliter 1 microgram/microliter Human Cot DNA (Invitrogen) was added, and the labeled mix was desiccated by Speed-Vac centrifugation.
Hybridization protocol
Slides were pre-hybridized in prewarmed 5X SCC, 0.1% SDS and 0.1% BSA at 42ºC for 45 min, rinsed under milliQ water and spun dry using a centrifuge for 5 min at 1500 rpm. Labeled samples were redissolved in 42ºC prewarmed 12 microliter of Hybridization buffer A (50% formamide, 6X SSC, 0.5% SDS and 5X Denhardt’s applied to a glass coverslip, covered with the spotted glass slide. Arrays were incubated in a corning hybridization chamber for 18 hr at 42ºC in a humid environment (In Slide Out oven, Boeckel). Arrays were washed at room temperature using an orbital shaker for 10 min with a high stringency wash buffer (0.1X SSC, 0.1% SDS), twice for 10 min with a low stringency wash buffer (0.1X SSC), rinsed for 5 min in milliQ water and spun dry for 5 min at 1500 rpm using a centrifuge.
Scan protocol
Fluorescent images were obtained using the G2565BA Microarray Scanner System (Agilent) with 100% laser power and 100% PMT settings and 16-bit TIFF images, one for each channel, were quantified using GenePix Pro 6.0 microarray analysis software (Molecular Devices, www.moleculardevices.com).
Description
Mean foreground and background intensities were extracted from the red (Cy5) and green (Cy3) channels for every spot on the microarray. The background intensities are used to correct the foreground intensities for local variation on the array surface, resulting in corrected red and green intensities.
Data processing
Raw data were processed using LIMMA, a microarray statistical analysis package of Bioconductor (http://www.bioconductor.org, Dudoit et al. 2003) that runs in the R programming environment (Gentleman et al. 2004, Wettenhall et al. 2004). Gene intensities were locally background subtracted using Normexp, taking mean of channel intensities and the median of background intensities. Spots with intensities smaller than two times the local background in both dye filter channels (Cy3 or Cy5), as well as control spots, were excluded from normalization and are left blank. Expression ratios were calculated as R/G, where R and G are the red and green color intensities, respectively. Ratios were transformed to logarithm base 2. Locally weighted linear regression (Lowess) analysis was used as a normalization method. We applied a local Lowess to each print-tip group or subgrid to correct any systematic spatial variation on the array, between spotting needles or variability on slide surface, by adjusting the mean of the Log2Ratio values in each subgrid to zero. We adjust the variance across all subgrids with a scaling factor for normalization, applying a smoothing factor f=0.2 to each subgrid in order to homogenize the variance of the log2Ratio within each print-tip. Normalized Log2Ratios across all arrays were scaled so that every array has the same median intensity and same absolute standard deviation, in order to give the same weight to every gene on the array. After normalization, replicated spots of the same gene were average.
