- Reaction progress can be monitored using the Kaiser test.
- The resin should be washed until the waste solution is colorless.
- We monitored the reaction progress with LC-MS.
- The yield is normally expected to be around 80% or higher. If
the yield is considerably lower than this value, we recommend
that the whole process should be repeated with new reagents;
especially the Cy3-NHS stock solution, the cleavage solution,
and HCTU. - In the preparation of the first DNA pool, we used a sufficient
amount of DNA template to maintain the diversity of the
library. - A NAP-5 column is equilibrated with three column-volumes of
RNase-free water. - The percentage of RNA bound to the resin is calculated as 100
(AbsTAbsF)/AbsT, where AbsT is the UVabsorbance of the
RNA solution before binding to the resin, and AbsF is that of
the flow-through fraction. - PCR was stopped during the positive acceleration phase to
avoid nonspecific amplification. The purity of PCR products
was assessed by 8 w/v % native polyacrylamide gel
electrophoresis. - M13-RV was used as a universal primer for sequencing.
- The RNA targeting arms are connected with a three-base-pair
stem segment through U mononucleotide bridges. - We normally single out an effective siRNA site as an RT-
aptamer recognition sequence. Such a site might exhibit high
levels of exposure for hybridization. - Cell images were taken with a vertical range of 4μm and
displayed as maximum intensity projection (MIP). Time-lapse
images were taken at 20–30 s intervals for up to 10 min. The
cell images were taken with a vertical range of 2μm, and each
image stack was then projected onto a single plane. - We added the BHQ1-Cy3 probe from a stock solution in
DMSO (1 mM) and Hoechst 33,342 from a stock solution in
H 2 O (1 mg/mL) to wells filled with media. - Overstaining (>5 min) results in an increase of background
fluorescence.
Acknowledgment
This work was supported in part by JSPS (26220206 to M.U. and
26440005 to S.S.), ZE Research Program, IAE (ZE27B-18 to S.
S.), and an iCeMS research acceleration grant. iCeMS is supported316 Shin-ichi Sato et al.