SUPPLEMENTARY INFORMATION
Supplementary Materials and methods
Illumina Microarray Hybridization
RNA samples were prepared with TRIzol reagent from Invitrogen using standard
procedures Hybridization was conducted by the gene expression analysis service at
Génome Québec, (Montréal, Québec, Canada). Briefly, Agilent Bioanalyzer nano chip
was used to check the RNA quality and integrity. cRNA amplification and labeling with
biotin were performed using Illumina TotalPrep RNA amplification kit (Ambion, Inc.,
Austin, TX) with 250 ng of total RNA as input material. cRNA yields were quantified
with Agilent Bioanalyzer, and 1.5 μg of cRNAs were hybridized to the HumanHT-12 v4
Expression BeadChip (Illumina, Inc., San Diego). Each chip contains six arrays, and each
array contains >48,000 gene transcripts, of which 46,000 were derived from human genes
at the National Center for Biotechnology Information (NCBI) Reference Sequence
(RefSeq) and UniGene databases. All reagents and equipment used for hybridization
were purchased from Illumina. The cRNA was hybridized to arrays for 16 h at 58°C,
washed and stained with streptavidin-Cy3 according to the manufacturer's protocol. Then
the bead chips were centrifuged to dry and scanned on the Illumina BeadArray Reader
confocal scanner. The statistical analysis of microarray data was performed using the
FlexArray 1.1.3 software package (Génome Québec, Montréal, Québec, Canada). The
data were also analyzed through the use of Ingenuity Pathway Analysis Ingenuity
Systems (Redwood City, CA).
RT-PCR
Total RNA was isolated by Trizol (Invitrogen), according to the manufacturer’s
instructions. The Protoscript M-MuLV Taq RT-PCR Kit (New England Biolabs,
Ipswich, MA, USA) was used to synthesize cDNA. From 0.5 ug of total RNA, cDNA
was synthesized using 10 U of M-MuLV reverse transcriptase, 2.5 mmol/l of
deoxyribonucleotide triphosphate mixture, and 100 pmol of Oligo d(T)23VN. The primer
d(T)23VN, RNA, and deoxyribonucleotide triphosphate mixture were combined and
heated for 5 min at 70°C. Following this step, the reverse transcriptase, buffer and
RNase inhibitor were added and the mixture was heated at 42°C for 1 hour. The enzyme
was inactivated at 80°C for 5 min. For reverse transcription PCR analysis in an intronspanning manner, 1ul of cDNA was amplified using the aforementioned Protoscript Kit
and the C1000 Thermal Cycler (Bio-Rad). The cycling program run was 95°C for 2 min,
followed by 25 PCR cycles (95°C for 30s, 55-65°C for 30s, 72°C for 45s) and a final
extension for 5 min at 72°C. All RT-PCR reactions included a 327-bp GAPDH fragment
which was amplified using the GAPDH primers supplied in the ProtoScript® M-MuLV
Taq RT-PCR Kit (New England Biolabs, Ipswich, MA, USA). Primer sequences used for
PCR are in Supplementary Table S3.
Supplementary Figure legends
Fig. S1. Semi-quantitative RT-PCR analysis to validate the effect of BRK on the
transcription of some of the genes identified by microarray analysis. Semi-quantitative
RT-PCR was performed for the indicated differentially expressed genes with the cDNAs
from HEK 293 cells stably expressing GFP, GFP-BRK-WT and GFP-BRK-YF. The
reactions were conducted using the ProtoScript® M-MuLV Taq RT-PCR Kit as
described by the manufacturer. The primers for GAPDH were supplied by the
manufacturer and were used for internal control reactions.