3. Add the following (can be prepared as a master mix) (total
volume 30 μL):
(a) 1 μL of murine RNase inhibitor.
(b) 10 μL of 50% PEG 8000.
(c) 3 μL of 10Â T4 RNA ligase I buffer.
(d) 3 μL of 10 mM ATP.
(e) 3 μL of DMSO.
(f) 1 μL of T4 RNA ligase I, standard concentration version
(10,000 units/mL).
4. Incubate at 20
C overnight.
5. Add 70 μL of RNase-free water and purify with Zymo Clean
and Concentrator kit as follows in steps 6–13.
6. Add 200 μL of RNA Binding Buffer and mix.
7. Add 300 μL of 100% ethanol, mix well by pipetting, and load
sample onto Zymo spin IIC column.
8. Centrifuge for 30 s at >10,000 Â g and discard flow-through.
9. Apply 500 μL of Prep Buffer to the spin column, centrifuge for
30 s at >10,000 Â g, and discard flow-through.
10. Repeat step 9 twice (three washes in total), using 400 μL of
RNA Wash Buffer, and increase centrifugation to 2 min at the
third wash.
11. After discarding the flow-through from the third wash, centrifuge column for 1 min at maximum speed.
12. Transfer column to a clean 1.5 mL tube, pipet 50 μL of water
directly onto the membrane, and centrifuge for 1 min at
>10,000 Â g.
13. Concentrate in a vacuum microcentrifuge to 5 μL.
3.3 RNA
Fragmentation
and cDNA Synthesis
1. Combine 5 μL of sample from Subheading 3.2 with 1 μL of
oligo SSS397 (1μg/μl) and 4 μL of NEBNext First Strand
Synthesis Reaction Buffer.
2. Heat at 85
C for 6 min in thermocycler.
3. Set aside on ice.
4. Prepare a master mix containing the following amounts per
reaction:
(a) 3.5 μL nuclease-free water.
(b) 0.5 μL murine RNase inhibitor.
(c) 5 μL 0.1 mg/mL Actinomycin D.
(d) 1 μL Protoscript II.
5. Add 10 μL of master mix to each cooled RNA/oligo mix
on ice.
520
M. Carla Martini et al.
volume 30 μL):
(a) 1 μL of murine RNase inhibitor.
(b) 10 μL of 50% PEG 8000.
(c) 3 μL of 10Â T4 RNA ligase I buffer.
(d) 3 μL of 10 mM ATP.
(e) 3 μL of DMSO.
(f) 1 μL of T4 RNA ligase I, standard concentration version
(10,000 units/mL).
4. Incubate at 20
C overnight.
5. Add 70 μL of RNase-free water and purify with Zymo Clean
and Concentrator kit as follows in steps 6–13.
6. Add 200 μL of RNA Binding Buffer and mix.
7. Add 300 μL of 100% ethanol, mix well by pipetting, and load
sample onto Zymo spin IIC column.
8. Centrifuge for 30 s at >10,000 Â g and discard flow-through.
9. Apply 500 μL of Prep Buffer to the spin column, centrifuge for
30 s at >10,000 Â g, and discard flow-through.
10. Repeat step 9 twice (three washes in total), using 400 μL of
RNA Wash Buffer, and increase centrifugation to 2 min at the
third wash.
11. After discarding the flow-through from the third wash, centrifuge column for 1 min at maximum speed.
12. Transfer column to a clean 1.5 mL tube, pipet 50 μL of water
directly onto the membrane, and centrifuge for 1 min at
>10,000 Â g.
13. Concentrate in a vacuum microcentrifuge to 5 μL.
3.3 RNA
Fragmentation
and cDNA Synthesis
1. Combine 5 μL of sample from Subheading 3.2 with 1 μL of
oligo SSS397 (1μg/μl) and 4 μL of NEBNext First Strand
Synthesis Reaction Buffer.
2. Heat at 85
C for 6 min in thermocycler.
3. Set aside on ice.
4. Prepare a master mix containing the following amounts per
reaction:
(a) 3.5 μL nuclease-free water.
(b) 0.5 μL murine RNase inhibitor.
(c) 5 μL 0.1 mg/mL Actinomycin D.
(d) 1 μL Protoscript II.
5. Add 10 μL of master mix to each cooled RNA/oligo mix
on ice.
520
M. Carla Martini et al.