Supplementary Methods
Preparation of hybridization probes
Total RNA was converted to double-stranded cDNA using SuperScript II
Double-Stranded cDNA synthesis kit (Invitrogen) and an oligo-dT primer containing the
T7 RNA polymerase promoter.
In vitro transcription (IVT) was used to produce
biotin-labeled cRNA from cDNA using MEGAscript T7 kit (Ambion). Briefly, 1 µg
double-stranded cDNA was incubated with 7.5 mM ATP and GTP, 5.6 mM UTP and
CTP, 1.9 mM bio-11-CTP and bio-16 UTP (Enzo) and 1x T7 enzyme mix in 1x
transcription buffer for 5 h at 37˚C.
Before hybridization, cRNA was fragmented to
an average size of 50 to 200 bp by incubation in 100 mM potassium acetate, 30 mM
magnesium acetate, and 40mM tris-acetate for 35 min at 94˚C. Fragmentation was
monitored by a Bioanalyzer (Agilent).
Hybridization to oligonucleotide arrays
Microarrays were hybridized with 437 ng cRNA in 5.7 μL per well in the presence
of 50 mM MES, 0.5 M NaCl, 10 mM EDTA, and 0.005% (v/v) Tween-20 and 5.25%
glycerol for 16 h at 45˚C. Prior to application to the array, samples were heated to 95°
C for 30 sec, followed by incubation at 45˚C for 5 min, and spun at 14,000g for 5 min.
After hybridization, arrays were washed in non-stringent (NS) buffer (6x SSPE, 0.01%
[v/v] Tween-20) for 15 min at 45˚C, followed by washing in stringent buffer (100 mM
MES, 0.1 M NaCl, 0.01% Tween-20) for 30 sec.
After washing, arrays were stained
with streptavidin-Cy3 conjugate (Amersham) for 25 min at room temperature, followed
by a 5-min wash in NS buffer, a 10-sec rinse in super stringent buffer (0.06x SSPE,
0.0001% Tween-20), and then spin-dry step using high-speed centrifuge.
After
hybridization, the glass slides were scanned with an Axon 4000B 5 um scanner from
Molecular Devices Corp (USA). The scanned images were initially captured with
Axon GenePix4.0 and captured images used to call signal intensities and to generate the
expression data. Scanned images of hybridization signals were analyzed using
NimbleGen Scan Software (NimbleGen Systems).
Procedure for 3’- and 5’-RACE
Non-truncated mRNA without cap structure was prepared from polyA+ RNA (250
ng) of NbCD3-overexpressing N. benthamiana leaves by treatment of calf intestine
phosphatase and tobacco acid pyrophosphatase.
Oligo RNA (GeneRacer RNA
Oligo) was ligated to the 5’-end of prepared RNA, and single-stranded cDNA was
synthesized from this mRNA using GeneRacer Oligo-dT primer. First, 3’-RACE was
carried out using the 26-base SuperSAGE tag primer and an adapter primer at the 3’-end
of cDNA (GeneRacer 3’primer).
Amplified fragments were cloned into
pCR4-TOPO (Invitrogen) and sequenced.
From the sequence of 3’-RACE
fragments, other primers were designed and used for 5’-RACE PCR together with an
adapter primer annealing to the 5’-end of cDNA (GeneRacer 5’primer).
Similarly,
5’-RACE PCR fragments were cloned into pCR4-TOPO and sequenced.
The
sequences of 3’-RACE and 5’-RACE PCR fragments were assembled, and full-length
cDNA sequences obtained.
Equipment and settings
Fig.2
Scanning of hybridization signals, scanned image analysis and normalization of signal
values were described in “Hybridization to oligonucleotide arrays” in Supplementary
Methods online and “Hybridization and analysis of oligonucleotide array” in
METHODS.
Average and difference of normalized signal values were also calculated
as described in “Hybridization and analysis of oligonucleotide array” in METHODS by
Microsoft Excel X for Macintosh.
A file of calculated data was analyzed by Clusters
(ver. 2.11) and subsequently analyzed by Tree View (ver. 1.60) for visualizing the
difference of hybridization signal values between samples.
Fig.3
Scanning of hybridization signals, scanned image analysis and normalization of signal
values were described in “Hybridization to oligonucleotide arrays” in Supplementary
Methods online and “Hybridization and analysis of oligonucleotide array” in
METHODS. Average and difference of normalized signal values were also calculated
as described in “Hybridization and analysis of oligonucleotide array” in METHODS by
Microsoft Excel X for Macintosh.
A file of calculated data was analyzed by Clusters
(ver. 2.11) and subsequently analyzed by Tree View (ver. 1.60) for visualizing the
difference of hybridization signal values between samples.
Fig.4
(a): Scanning of hybridization signals, scanned image analysis and normalization of
signal values were described in “Hybridization to oligonucleotide arrays” in
Supplementary Methods online and “Hybridization and analysis of oligonucleotide
array” in METHODS.
Average and difference of normalized signal values were also
calculated as described in “Hybridization and analysis of oligonucleotide array” in
METHODS by Microsoft Excel X for Macintosh.
A file of calculated data was
analyzed by Clusters (ver. 2.11) and subsequently analyzed by Tree View (ver. 1.60) for
visualizing the difference of hybridization signal values between samples.
(c): RACE PCR products were run on 1% TAE-Agraose gel containing ethidium
bromide. Photo of the gel was taken on UV-transilluminator (Funakoshi, Japan), and
scanned by ES-8500 scanner (EPSON, Japan).
Scanned image of photo was
converted to a TIFF format file by Adobe Photoshop 7.0.