urea-PAGE gel autoradiography for alignment (see Notes 3
and 9), and properly discard the rest.
11. Wash the beads five times with 1 mL of PNK buffer
without DTT.
12. At this point the beads can be stored at À20
C. Note that the
beads are also radioactive and make sure to follow radiation
safety guidelines for storage.
13. Elute the RNA footprints by triple proteinase K digestions:
(a) Add 1.2 mg/mL proteinase K in 200 μL of proteinase K
buffer. Incubate at 50
C under vigorous shaking for 30 min.
(b) Add 0.75 mg/mL proteinase K in 150 μL proteinase K
buffer. Incubate at 50
C under vigorous shaking for 30 min.
(c) Add 0.75 mg/mL proteinase K in 150 μL proteinase K
buffer. Incubate at 50
C under vigorous shaking for 30 min.
14. Collect liquid from tube caps by briefly spinning on a tabletop
centrifuge and place in a magnetic rack. Transfer the supernatant, which contains the eluted RNA footprints, into a new
1.5 mL low-bind microcentrifuge tube.
Fig. 4 Illustration and demonstration of steps along the production of small RNA cDNA libraries (Subheading
3.6). (a) Scheme of the molecular procedures succeeding streptavidin affinity purification. (b) Autoradiographs
of the three denaturing gel steps along the procedure: Left, step 21—size selection of RNA footprints. Middle,
step 27—purification of 3
0 -ligated footprints and depletion of excess non-ligated 3
0 adapter. Right, step 33—
purification of 3
0 - and 5
0 -ligated footprints and depletion of excess non-ligated 5
0 adapter
Proximity-CLIP
295
and 9), and properly discard the rest.
11. Wash the beads five times with 1 mL of PNK buffer
without DTT.
12. At this point the beads can be stored at À20
C. Note that the
beads are also radioactive and make sure to follow radiation
safety guidelines for storage.
13. Elute the RNA footprints by triple proteinase K digestions:
(a) Add 1.2 mg/mL proteinase K in 200 μL of proteinase K
buffer. Incubate at 50
C under vigorous shaking for 30 min.
(b) Add 0.75 mg/mL proteinase K in 150 μL proteinase K
buffer. Incubate at 50
C under vigorous shaking for 30 min.
(c) Add 0.75 mg/mL proteinase K in 150 μL proteinase K
buffer. Incubate at 50
C under vigorous shaking for 30 min.
14. Collect liquid from tube caps by briefly spinning on a tabletop
centrifuge and place in a magnetic rack. Transfer the supernatant, which contains the eluted RNA footprints, into a new
1.5 mL low-bind microcentrifuge tube.
Fig. 4 Illustration and demonstration of steps along the production of small RNA cDNA libraries (Subheading
3.6). (a) Scheme of the molecular procedures succeeding streptavidin affinity purification. (b) Autoradiographs
of the three denaturing gel steps along the procedure: Left, step 21—size selection of RNA footprints. Middle,
step 27—purification of 3
0 -ligated footprints and depletion of excess non-ligated 3
0 adapter. Right, step 33—
purification of 3
0 - and 5
0 -ligated footprints and depletion of excess non-ligated 5
0 adapter
Proximity-CLIP
295