25. Incubate at 90
C for 1 min and return the tubes to ice. Once
chilled add 1 μL of T4 Rnl2(1-249)K227Q (1 μg/μL), mix
gently, and incubate on ice overnight (place the ice bucket in a
refrigerator).
26. Terminate 3
0 ligation by adding 20 μL of denaturing 2Â formamide gel loading solution and incubating at 90
C for 1 min.
27. Load samples on a 15% denaturing urea-PAGE gel, image by
autoradiography as described in step 21 (Fig. 4b, see Note 3),
and excise ligated footprints (Fig. 4) using the ligated RNA size
markers as reference.
28. Transfer the excised gel pieces in a gel breaker tube and extract
the RNA as follows: (1) Centrifuge at maximum speed for
1 min. (2) Add 350 μL of 0.3 M NaCl and shake at 60
C for
1 h. (3) Transfer the suspension into filter tubes and Centrifuge at 5000 Â g for 1 min. (4) Add ~10 μg of glycogen and
1200 μL of 100% ethanol, vortex, and incubate at À80
C for
>1 h. (5) Centrifuge at maximum speed for 15 min, discard the
supernatant, and add 0.5 mL of 75% ethanol. Without mixing,
centrifuge at >12,000 Â g for 7 min, thoroughly remove the
75% ethanol, and air-dry the pellet by leaving the tubes open
on the bench for 5 min. Do not let the pellet overdry.
29. Resuspend the RNA pellet in 9 μL of water.
30. For 5
0 adapter ligation to each 9 μL RNA add 9 μL mix
containing 6 μL 50% DMSO, 2 μL 10Â RNA ligase buffer
with ATP, and 1 μL of 100 μM RNA 5
0 adapter (Table 1).
31. Incubate at 90
C for 1 min and return the tubes to ice. Once
chilled, add 2 μL of T4 Rnl1 (1 μg/μL), mix gently, and
incubate at 37
C for 1 h.
32. Terminate 5
0 adapter ligation by adding 20 μL of denaturing
2Â formamide gel loading solution and incubating at 90
C for
1 min.
33. Load samples on a 12% denaturing urea-PAGE gel, image by
autoradiography overnight as described in step 21 (Fig. 4b, see
Note 3), and excise ligated footprints (Fig. 4) using the ligated
RNA size markers as reference.
34. Put the excised gel pieces in a gel breaker tube and extract the
RNA as follows: (1) Centrifuge at maximum speed for 1 min.
(2) Add 350 μL of 0.3 M NaCl and shake at 60
C for 1 h.
(3) Transfer the suspension into filter tubes and centrifuge at
5000 Â g for 1 min. (4) Add ~10 μg of glycogen and 1200 μL
of 100% ethanol, vortex, and incubate at À80
C for >1 h.
(5) Centrifuge at maximum speed for 15 min, discard the
supernatant, and add 0.5 mL of 75% ethanol. Without mixing,
centrifuge at >12,000 Â g for 7 min, thoroughly remove the
supernatant, and air-dry the pellet by leaving the tubes open on
the bench for 5 min. Do not let the pellet overdry.
298
Daniel Benhalevy and Markus Hafner
C for 1 min and return the tubes to ice. Once
chilled add 1 μL of T4 Rnl2(1-249)K227Q (1 μg/μL), mix
gently, and incubate on ice overnight (place the ice bucket in a
refrigerator).
26. Terminate 3
0 ligation by adding 20 μL of denaturing 2Â formamide gel loading solution and incubating at 90
C for 1 min.
27. Load samples on a 15% denaturing urea-PAGE gel, image by
autoradiography as described in step 21 (Fig. 4b, see Note 3),
and excise ligated footprints (Fig. 4) using the ligated RNA size
markers as reference.
28. Transfer the excised gel pieces in a gel breaker tube and extract
the RNA as follows: (1) Centrifuge at maximum speed for
1 min. (2) Add 350 μL of 0.3 M NaCl and shake at 60
C for
1 h. (3) Transfer the suspension into filter tubes and Centrifuge at 5000 Â g for 1 min. (4) Add ~10 μg of glycogen and
1200 μL of 100% ethanol, vortex, and incubate at À80
C for
>1 h. (5) Centrifuge at maximum speed for 15 min, discard the
supernatant, and add 0.5 mL of 75% ethanol. Without mixing,
centrifuge at >12,000 Â g for 7 min, thoroughly remove the
75% ethanol, and air-dry the pellet by leaving the tubes open
on the bench for 5 min. Do not let the pellet overdry.
29. Resuspend the RNA pellet in 9 μL of water.
30. For 5
0 adapter ligation to each 9 μL RNA add 9 μL mix
containing 6 μL 50% DMSO, 2 μL 10Â RNA ligase buffer
with ATP, and 1 μL of 100 μM RNA 5
0 adapter (Table 1).
31. Incubate at 90
C for 1 min and return the tubes to ice. Once
chilled, add 2 μL of T4 Rnl1 (1 μg/μL), mix gently, and
incubate at 37
C for 1 h.
32. Terminate 5
0 adapter ligation by adding 20 μL of denaturing
2Â formamide gel loading solution and incubating at 90
C for
1 min.
33. Load samples on a 12% denaturing urea-PAGE gel, image by
autoradiography overnight as described in step 21 (Fig. 4b, see
Note 3), and excise ligated footprints (Fig. 4) using the ligated
RNA size markers as reference.
34. Put the excised gel pieces in a gel breaker tube and extract the
RNA as follows: (1) Centrifuge at maximum speed for 1 min.
(2) Add 350 μL of 0.3 M NaCl and shake at 60
C for 1 h.
(3) Transfer the suspension into filter tubes and centrifuge at
5000 Â g for 1 min. (4) Add ~10 μg of glycogen and 1200 μL
of 100% ethanol, vortex, and incubate at À80
C for >1 h.
(5) Centrifuge at maximum speed for 15 min, discard the
supernatant, and add 0.5 mL of 75% ethanol. Without mixing,
centrifuge at >12,000 Â g for 7 min, thoroughly remove the
supernatant, and air-dry the pellet by leaving the tubes open on
the bench for 5 min. Do not let the pellet overdry.
298
Daniel Benhalevy and Markus Hafner