2.12 Distinguishing
TSSs from
Cleavage Sites
1. R programming package.
3 Methods
3.1 Conversion of 5
0
Triphosphates to 5
0
Monophosphates
For each sample, perform one reaction with RppH and one mock
reaction. Each of these will be carried through all subsequent steps
to create the “converted” and “nonconverted” libraries, respectively (see Fig. 1).
1. Combine:
(a) 0.25–1 μg of rRNA-depleted RNA.
(b) 2 μL of 10Â RppH buffer.
(c) 2 μL of RppH (or 2 μL of RNase-free water for mock
reaction).
(d) RNase-free water to a total reaction volume of 20 μL.
2. Incubate at 37 ˚C for 1 h.
3. Add 80 μL of RNase-free water to each reaction and purify with
Zymo Clean and Concentrator kit as follows in steps 4–11.
4. Add 200 μL of RNA Binding Buffer and mix.
5. Add 300 μL of 100% ethanol, mix well by pipetting and load
sample onto Zymo spin IIC column.
6. Centrifuge for 30 s at >10,000 Â g and discard flow-through.
7. Apply 700 μL of RNA Wash Buffer to the spin column, centrifuge for 30 s at >10,000 Â g, and discard flow-through.
8. Repeat step 7 twice (three washes in total), using 400 μL of
RNA Wash Buffer and increase centrifugation time to 2 min at
the third wash.
9. After discarding the flow-through from the third wash, centrifuge column for 1 min at maximum speed.
10. Transfer column to a clean 1.5 mL tube, pipet 50 μL of water
containing Murine RNase Inhibitor (1 μL inhibitor/mL
water) directly onto the membrane, and centrifuge for 1 min
at >10,000 Â g.
11. Add 0.5 μL of 100 mM Tris pH 7.5.
12. Concentrate in a vacuum centrifuge to 8 μL.
3.2 Adapter Ligation
1. Combine 8 μL sample from Subheading 3.1, step 12, with
1 μL oligo SSS392.
2. Denature RNA at 65 ˚C for 10 min, then snap-cool in an
ice-water bath.
RNA 5
0
End Mapping
519
TSSs from
Cleavage Sites
1. R programming package.
3 Methods
3.1 Conversion of 5
0
Triphosphates to 5
0
Monophosphates
For each sample, perform one reaction with RppH and one mock
reaction. Each of these will be carried through all subsequent steps
to create the “converted” and “nonconverted” libraries, respectively (see Fig. 1).
1. Combine:
(a) 0.25–1 μg of rRNA-depleted RNA.
(b) 2 μL of 10Â RppH buffer.
(c) 2 μL of RppH (or 2 μL of RNase-free water for mock
reaction).
(d) RNase-free water to a total reaction volume of 20 μL.
2. Incubate at 37 ˚C for 1 h.
3. Add 80 μL of RNase-free water to each reaction and purify with
Zymo Clean and Concentrator kit as follows in steps 4–11.
4. Add 200 μL of RNA Binding Buffer and mix.
5. Add 300 μL of 100% ethanol, mix well by pipetting and load
sample onto Zymo spin IIC column.
6. Centrifuge for 30 s at >10,000 Â g and discard flow-through.
7. Apply 700 μL of RNA Wash Buffer to the spin column, centrifuge for 30 s at >10,000 Â g, and discard flow-through.
8. Repeat step 7 twice (three washes in total), using 400 μL of
RNA Wash Buffer and increase centrifugation time to 2 min at
the third wash.
9. After discarding the flow-through from the third wash, centrifuge column for 1 min at maximum speed.
10. Transfer column to a clean 1.5 mL tube, pipet 50 μL of water
containing Murine RNase Inhibitor (1 μL inhibitor/mL
water) directly onto the membrane, and centrifuge for 1 min
at >10,000 Â g.
11. Add 0.5 μL of 100 mM Tris pH 7.5.
12. Concentrate in a vacuum centrifuge to 8 μL.
3.2 Adapter Ligation
1. Combine 8 μL sample from Subheading 3.1, step 12, with
1 μL oligo SSS392.
2. Denature RNA at 65 ˚C for 10 min, then snap-cool in an
ice-water bath.
RNA 5
0
End Mapping
519