8. Add 350 μL 0.4 M NaCl to each tube and tumble the tubes in
the Eppendorf Thermomixer overnight at 4
C.
9. Centrifuge at 12,000 g for 5 min at 4
C. Take supernatant out
to a new tube. Add 100 μL 0.4 M NaCl to each old tube and
tumble for additional 30 min at 4
C.
10. Centrifuge at 12,000 g for 5 min at 4
C. Take the supernatant
out to the new tube and add 1 mL ethanol, mix well, and
incubate at À80
C at least 1 h, or À20
C for an overnight.
11. Centrifuge at 21,130 g for 15 min at 4
C. Discard supernatant. Use 70% ethanol to wash the pellet. Centrifuge at
21,130 g for 5 min. Discard supernatant and spin down
again. Discard residual through pipetting and air-dry the pellet
in a clear area for 5 min.
12. Add water to a final concentration of 50 μM.
3.3 Marker Labeling
In our sRNA library preparation, we do size selection prior to
ligation. We typically make RNA oligonucleotides through
in vitro T7 transcription in our laboratory. However, one could
simply purchase commercially synthesized RNA oligonucleotides
of certain lengths as markers to spike the sRNAs of interest. The
lengths can be optimized according to the size of sRNAs to be
detected. Here we use 19 nt and 24 nt RNA oligonucleotides
(Table 1) as examples. In order to make them easily detectable,
we label the markers with
32 P-ATP and T4 PNK. The RNA markers
can also be ligated with adapters together with samples through
experiments until their clearance from the samples through Pme I
digestion in a final step. Thus, they can also be used to monitor the
ligation efficiency and degradation scenario (see Note 5).
3.3.1 CIP Treatment of
RNA Oligomers with 5
0
Mono- or Tri-phosphate
(See Note 6)
1. Prepare CIP reaction system for each marker on ice as following: 2 μL marker solution dissolved in RNase-free water
(10 μM), 2 μL ten-fold NEB Buffer 3, 2 μL CIP, 1 μL RNase
inhibitor (final concentration 1 U/μL), and RNase-free water
13 μL. Mix well and incubate at 37
C for 30 min.
2. Add 220 μL RNase-free water to each reaction mixture above.
3. Add 240 μL of phenol:chloroform:isoamyl alcohol (25:24:1,
pH 4.3) to each mixture. Vortex for 30 s to mix well. Centrifuge for 5 min at room temperature by maximum speed.
4. Carefully take 200 μL upper phase liquid to a new labeled tube,
respectively. Add 200 μL chloroform:isoamyl alcohol (24:1) to
each tube. Vortex for 30 s to mix well. Centrifuge for 5 min at
room temperature by maximum speed.
5. Carefully take 170 μL upper phase liquid to a new labeled tube,
respectively. Add 510 μL ethanol, 17 μL 3 M NaAc (pH 5.2),
Identification and Quantification of sRNAs
239
the Eppendorf Thermomixer overnight at 4
C.
9. Centrifuge at 12,000 g for 5 min at 4
C. Take supernatant out
to a new tube. Add 100 μL 0.4 M NaCl to each old tube and
tumble for additional 30 min at 4
C.
10. Centrifuge at 12,000 g for 5 min at 4
C. Take the supernatant
out to the new tube and add 1 mL ethanol, mix well, and
incubate at À80
C at least 1 h, or À20
C for an overnight.
11. Centrifuge at 21,130 g for 15 min at 4
C. Discard supernatant. Use 70% ethanol to wash the pellet. Centrifuge at
21,130 g for 5 min. Discard supernatant and spin down
again. Discard residual through pipetting and air-dry the pellet
in a clear area for 5 min.
12. Add water to a final concentration of 50 μM.
3.3 Marker Labeling
In our sRNA library preparation, we do size selection prior to
ligation. We typically make RNA oligonucleotides through
in vitro T7 transcription in our laboratory. However, one could
simply purchase commercially synthesized RNA oligonucleotides
of certain lengths as markers to spike the sRNAs of interest. The
lengths can be optimized according to the size of sRNAs to be
detected. Here we use 19 nt and 24 nt RNA oligonucleotides
(Table 1) as examples. In order to make them easily detectable,
we label the markers with
32 P-ATP and T4 PNK. The RNA markers
can also be ligated with adapters together with samples through
experiments until their clearance from the samples through Pme I
digestion in a final step. Thus, they can also be used to monitor the
ligation efficiency and degradation scenario (see Note 5).
3.3.1 CIP Treatment of
RNA Oligomers with 5
0
Mono- or Tri-phosphate
(See Note 6)
1. Prepare CIP reaction system for each marker on ice as following: 2 μL marker solution dissolved in RNase-free water
(10 μM), 2 μL ten-fold NEB Buffer 3, 2 μL CIP, 1 μL RNase
inhibitor (final concentration 1 U/μL), and RNase-free water
13 μL. Mix well and incubate at 37
C for 30 min.
2. Add 220 μL RNase-free water to each reaction mixture above.
3. Add 240 μL of phenol:chloroform:isoamyl alcohol (25:24:1,
pH 4.3) to each mixture. Vortex for 30 s to mix well. Centrifuge for 5 min at room temperature by maximum speed.
4. Carefully take 200 μL upper phase liquid to a new labeled tube,
respectively. Add 200 μL chloroform:isoamyl alcohol (24:1) to
each tube. Vortex for 30 s to mix well. Centrifuge for 5 min at
room temperature by maximum speed.
5. Carefully take 170 μL upper phase liquid to a new labeled tube,
respectively. Add 510 μL ethanol, 17 μL 3 M NaAc (pH 5.2),
Identification and Quantification of sRNAs
239