1
Appendix S1. Supporting Methods.
2
3’ RACE analysis of pri-miR165a
3
We determined the 3’ end of pri-miR165a by 3’ RACE. One microgram of total RNA
4
was treated with DNase I (Life technologies, Carlsbad, CA). The DNase-treated RNA
5
was used in the reverse transcription (RT) with the SuperScript® III First-Strand
6
Synthesis
7
(5’-GACTCGAGTCGACATCGATTTTTTTTTTTTTTTTT-3’) as the RT primer. The
8
cDNA samples were used in the PCR reaction with AmpliTaq Gold® PCR Master Mix
9
(Life technologies), and pre-165a-F (5’-GGATATTATAGATATATACATGTG-3’) and
10
ADP (5’-GACTCGAGTCGACATCGA-3’) were used as the PCR primers. The PCR
11
products were cloned into the pT7 Blue vector (Merck Millipore, Darmstadt, Germany),
12
and the nucleotide sequences were determined. The 3’ end of the pre-miR165a was
13
located 365 bases from the 5’ end, and the full-length pri-miR165a was 494 bases with
14
no introns (Figure S4a).
System
(Life
technologies),
and
we
used
the
ADT
oligomer
15
16
Cloning of MIR165Amu fragments having different lengths of the 3’ region
17
The primer sequences that were used to construct the MIR165Amu fragments having
18
different lengths of the 3’ region are listed in Table S4. The 3’-region-less MIR165Amu
19
fragments were amplified from the MIR165Amu fragment using the primer sets as
20
follows: for MIR165Amu(241), SalI-165A(-)3927-F and 165A(+)241-BamHI-R; for
21
MIR165Amu(365),
22
MIR165Amu(496),
23
MIR165Amu(628), SalI-165A(-)3927-F and 165A(+)628-SmaI-R. The fragments that
24
were digested by restriction enzymes were ligated into the multi-cloning site of pBIN40
25
binary vectors (Miyashima et al., 2011). All of the constructs were introduced into the
26
35S:miGFP-M line by the Agrobacterium-mediated transformation method as described
27
in the text.
SalI-165A(-)3927-F
SalI-165A(-)3927-F
and
and
165A(+)365-SmaI-R;
165A(+)496-SmaI-R;
for
and
for
28
29
30
References for Supporting information
31
Miyashima,
32
Non-cell-autonomous microRNA165 acts in a dose-dependent manner to regulate
33
multiple differentiation status in the Arabidopsis root. Development 138, 2303-2313.
S.,
Koi,
S.,
Hashimoto,
1
T.
and
Nakajima,
K.
(2011)
1
2
Xie,, Z.X., Allen, E., Fahlgren, N., Calamar, A., Givan, S.A. and Carrington, J.C.
3
(2005) Expression of Arabidopsis MIRNA genes. Plant Physiol. 138, 2145-2154
4
5
Yao, X.Z., Wang, H., Li, H., Yuan, Z.H., Li, F.P., Yang, L. and Huang H. (2009) Two
6
types of cis-acting elements control the abaxial epidermis-specific transcription of the
7
MIR165a and MIR166a genes. FEBS Lett. 583, 3711-3717
8
9
10
2