2. Add 7 μL RNase-free water to each sample to dissolve RNA.
Prepare a mix for 3
0 ligation system: 2.5 μL 50 μM 3
0 adapter,
2 μL 10Â T4 3
0 ligase buffer (no ATP), 1 μL 10 Â BSA, and
6 μL 50% DMSO. The total volume is 11.5 μL. Multiply the
volume for one reaction by 1.1-fold of sample numbers due to
pipetting variations.
3. Mix well. Incubate the reaction mixture at 95
C for 1 min to
denature the RNA and immediately place on ice for 2 min.
4. Add 1.5 μL Rnl2 K227Q to each sample. Tap tubes to mix well.
Centrifuge at 5000 g for 1 min at 4
C. Keep samples on ice in
4
C overnight.
5. Add 20 μL FA buffer to each sample. Run gel electrophoresis
and recover RNA as mentioned in Subheading 3.5. The image
is shown in Fig. 3c.
3.7 5
0 Ligation
1. Centrifuge at 21,130 g for 30 min at 4
C. Discard supernatant
and spin down again. Discard residual and air-dry pellet in a
clear area for 5 min.
2. Add 6.1 μL RNase-free water to each sample to dissolve RNA.
Prepare a mix for 5
0 ligation system: 1 μL 100 μM 5
0 adapter
(Table 2), 2 μL 10 Â RNA ligation buffer, 0.4 μL 10 mM ATP,
2 μL 10Â BSA, 6 μL 50% DMSO. The total volume is 11.4 μL.
Multiply the volume for one reaction by 1.1-fold of sample
numbers due to pipetting variations.
3. Mix well. Incubate the reaction mixture at 95
C for 1 min to
denature the RNA and immediately place on ice for 2 min.
4. Add 1 μL RNase Inhibitor, 1.5 μL Rnl1. Tap to mix well.
Incubate samples at 37
C for 3 h.
5. Add 20 μL FA buffer to each sample. Run gel electrophoresis
and recover RNA as described in Sect. 3.5 but add 1 μL 50 μM
SR-RT primer to each sample together with 350 μL NaCl
(DEPC treated). The image is shown in Fig. 3d.
3.8 Reverse
Transcription
1. Centrifuge at 21,130 g for 30 min at 4
C. Discard supernatant
and spin down again. Discard residual and air-dry pellet in a
clear area for 5 min.
2. Add 10.8 μL RNase-free water and 1.2 μL dNTP mix (10 mM
each) to each sample to dissolve RNA. Prepare a mix containing
2 μL 0.1 M DTT and 5Â first strand buffer 4 μL for each
sample. The total volume is 6 μL. Multiply the volume for
one reaction by 1.1-fold of sample numbers due to pipetting
variations.
3. Mix well. Incubate the reaction mixture at 90
C for 1 min and
immediately place samples on ice for 2 min.
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Di Sun et al.
Prepare a mix for 3
0 ligation system: 2.5 μL 50 μM 3
0 adapter,
2 μL 10Â T4 3
0 ligase buffer (no ATP), 1 μL 10 Â BSA, and
6 μL 50% DMSO. The total volume is 11.5 μL. Multiply the
volume for one reaction by 1.1-fold of sample numbers due to
pipetting variations.
3. Mix well. Incubate the reaction mixture at 95
C for 1 min to
denature the RNA and immediately place on ice for 2 min.
4. Add 1.5 μL Rnl2 K227Q to each sample. Tap tubes to mix well.
Centrifuge at 5000 g for 1 min at 4
C. Keep samples on ice in
4
C overnight.
5. Add 20 μL FA buffer to each sample. Run gel electrophoresis
and recover RNA as mentioned in Subheading 3.5. The image
is shown in Fig. 3c.
3.7 5
0 Ligation
1. Centrifuge at 21,130 g for 30 min at 4
C. Discard supernatant
and spin down again. Discard residual and air-dry pellet in a
clear area for 5 min.
2. Add 6.1 μL RNase-free water to each sample to dissolve RNA.
Prepare a mix for 5
0 ligation system: 1 μL 100 μM 5
0 adapter
(Table 2), 2 μL 10 Â RNA ligation buffer, 0.4 μL 10 mM ATP,
2 μL 10Â BSA, 6 μL 50% DMSO. The total volume is 11.4 μL.
Multiply the volume for one reaction by 1.1-fold of sample
numbers due to pipetting variations.
3. Mix well. Incubate the reaction mixture at 95
C for 1 min to
denature the RNA and immediately place on ice for 2 min.
4. Add 1 μL RNase Inhibitor, 1.5 μL Rnl1. Tap to mix well.
Incubate samples at 37
C for 3 h.
5. Add 20 μL FA buffer to each sample. Run gel electrophoresis
and recover RNA as described in Sect. 3.5 but add 1 μL 50 μM
SR-RT primer to each sample together with 350 μL NaCl
(DEPC treated). The image is shown in Fig. 3d.
3.8 Reverse
Transcription
1. Centrifuge at 21,130 g for 30 min at 4
C. Discard supernatant
and spin down again. Discard residual and air-dry pellet in a
clear area for 5 min.
2. Add 10.8 μL RNase-free water and 1.2 μL dNTP mix (10 mM
each) to each sample to dissolve RNA. Prepare a mix containing
2 μL 0.1 M DTT and 5Â first strand buffer 4 μL for each
sample. The total volume is 6 μL. Multiply the volume for
one reaction by 1.1-fold of sample numbers due to pipetting
variations.
3. Mix well. Incubate the reaction mixture at 90
C for 1 min and
immediately place samples on ice for 2 min.
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