SUPPORTING INFORMATION
Figure S1. RNA isolation and pilot experiment of cDNA amplification cycles.
(A) Electrophoresis results of total RNA. Lane 1 to 3 represents 0μM, 5μM and 25μM AlCl3
treated sample RNA, respectively. (B-D) cDNA amplification of 0μM, 5μM and 25μM AlCl3
treated sample at 15, 18, 21, 24, 27 and 30 cycles, respectively. In order to reflect initial mRNA
amount, cDNA amplified at 17 cycles was chosen for further use.
Figure S2. PCR-select cDNA subtraction.
(A) RsaI digestion of amplified cDNA. Lane 1: amplified cDNA from 5μM AlCl3-treated
sample; Lane 2: RsaI digestion of cDNA in Lane 1; Lane 3: amplified cDNA from 25μM AlCl3treated sample; Lane 4: RsaI digestion of DNA in Lane 3; Lane 5: amplified cDNA from CK
sample. Lane 6: RsaI digestion of DNA in Lane 5. (B) Adapter ligation efficiency test. Lane 1, 3,
5, 7, 9 and 11: PCR reaction using internal primer and adapter primer. Lane 2, 4, 6, 8, 10 and 12:
PCR reaction using internal primers. (C-F) Subtractive efficiency tests. PCR results of first
forward and reverse hybridization from 5μM AlCl3-treated sample (C) and 25μM AlCl3-treated
sample (E). PCR results of second forward and reverse hybridization from 5μM AlCl3-treated
sample (D) and 25μM AlCl3-treated sample (F). Line 1 and 4: unsubtractive control results from
forward and reverse libraries. Line 2 and 3: subtractive results from forward and reverse libraries.
Figure S3. Representative electrophoretogram of amplification of cDNA inserts from four
libraries.
Red arrow shown non single insert clone while yellow arrow shown no amplification clone. Both of
them were eliminated in following differential screening.
Figure S4. Test of labeled cDNAs probes.
(A) Electrophoretogram of labeled Tester and Driver cDNAs probes.18S rRNA was use as internal
reference. (B) Detection of label efficiency.
Figure S5. Differential screening of cDNA clones from 25µM AlCl3-treated rice bean
forward SSH library.
Duplicate dot-blots were prepared and the membranes were hybridized with labeled probes. (A)
The clones from forward SSH library hybridized with tester probes. (B) The clones from forward
SSH library hybridized with driver probes.
Figure S6. Differential screening of cDNA clones from 5µM AlCl3-treated rice bean reverse
SSH library.
Duplicate dot-blots were prepared and the membranes were hybridized with labeled probes. (A)
The clones from reverse SSH library hybridized with tester probes. (B) The clones from reverse
SSH library hybridized with driver probes.
Figure S7. Differential screening of cDNA clones from 25µM AlCl3-treated rice bean
reverse SSH library.
Duplicate dot-blots were prepared and the membranes were hybridized with labeled probes. (A)
The clones from reverse SSH library hybridized with tester probes. (B) The clones from reverse
SSH library hybridized with driver probes.