35. Resuspend the RNA pellet in 4.6 μL of water.
36. For reverse transcription denature the RNA at 90
C for 1 min,
then reduce the temperature to 50
C, and add 10.4 μL master
mix comprised of 1.5 μL of 100 mM DTT, 3 μL of 5Â firststrand buffer (Thermo), 4.2 μL of 2 mM dNTPs, 1 μL of
100 μM RT primer (Table 1), and 0.7 μL Superscript III.
37. Incubate at 50
C for 1 h.
38. Dilute the cDNA by adding 85 μL water, reaching a volume of
100 μL.
39. Use 6 μL of the diluted cDNA for calibration PCR by adding
0.6 μL of 100 μM multiplexed reverse PCR primer (Table 1)
and a mix comprised of 38.6 μL of water, 6 μL of 10Â buffer
without Mg
2+ , 1.8 μL of 50 mM MgCl 2 , 6 μL of 2 mM
dNTPs, 0.6 μL 100 μM forward PCR primer (Table 1), and
0.42 Taq polymerase.
40. Split the 60 μL reaction mix into 6 tubes and load on a PCR
19-cycle program. Remove one of the tubes after 9, 11, 13, 15,
17, and 19 cycles, and load the PCR products on a 2.5%
agarose gel for electrophoresis.
41. Image the gel to select the optimal number of PCR cycles,
where amplification of the library is favored relative to that of
linker-linker products derived from directly ligated 3
0 and 5
0
adapters.
42. Use the same reagents to set up an identical PCR with reaction
volume of 300 μL. Split the reaction to three 100 μL tubes and
run with the previously determined optimal number of cycles.
43. Clean and concentrate the PCR product using a standard
column-based purification kit and elute in 70 μL of water.
44. Load 30 μL of the purified PCR product for a Pippin Prep size
selection to deplete ligated 3
0 –5
0 adapters (126 bp long) and
enrich insert-containing library products. For footprints of
lengths 20–40 nt, the expected library size is 146–166 bp.
45. Use TapeStation with a D1000 ScreenTape to measure the
library final concentration and average length.
3.7 Transformation
of Intact RNA into
RNA-seq Libraries
1. Obtain the À80
C-stored 20% of beads for RNA-seq of intact
bound RNAs (Subheading 3.4, step 7).
2. Elute the bound RNA by triple proteinase K digestions:
(1) Add 1.2 mg/mL proteinase K in 200 μL of proteinase K
buffer. Incubate at 50
C under vigorous shaking for 30 min.
(2) Add 0.75 mg/mL proteinase K in 150 μL proteinase K
buffer. Incubate at 50
C under vigorous shaking for 30 min.
(3) Add 0.75 mg/mL proteinase K in 150 μL proteinase K
buffer. Incubate at 50
C under vigorous shaking for 30 min.
Proximity-CLIP
299
36. For reverse transcription denature the RNA at 90
C for 1 min,
then reduce the temperature to 50
C, and add 10.4 μL master
mix comprised of 1.5 μL of 100 mM DTT, 3 μL of 5Â firststrand buffer (Thermo), 4.2 μL of 2 mM dNTPs, 1 μL of
100 μM RT primer (Table 1), and 0.7 μL Superscript III.
37. Incubate at 50
C for 1 h.
38. Dilute the cDNA by adding 85 μL water, reaching a volume of
100 μL.
39. Use 6 μL of the diluted cDNA for calibration PCR by adding
0.6 μL of 100 μM multiplexed reverse PCR primer (Table 1)
and a mix comprised of 38.6 μL of water, 6 μL of 10Â buffer
without Mg
2+ , 1.8 μL of 50 mM MgCl 2 , 6 μL of 2 mM
dNTPs, 0.6 μL 100 μM forward PCR primer (Table 1), and
0.42 Taq polymerase.
40. Split the 60 μL reaction mix into 6 tubes and load on a PCR
19-cycle program. Remove one of the tubes after 9, 11, 13, 15,
17, and 19 cycles, and load the PCR products on a 2.5%
agarose gel for electrophoresis.
41. Image the gel to select the optimal number of PCR cycles,
where amplification of the library is favored relative to that of
linker-linker products derived from directly ligated 3
0 and 5
0
adapters.
42. Use the same reagents to set up an identical PCR with reaction
volume of 300 μL. Split the reaction to three 100 μL tubes and
run with the previously determined optimal number of cycles.
43. Clean and concentrate the PCR product using a standard
column-based purification kit and elute in 70 μL of water.
44. Load 30 μL of the purified PCR product for a Pippin Prep size
selection to deplete ligated 3
0 –5
0 adapters (126 bp long) and
enrich insert-containing library products. For footprints of
lengths 20–40 nt, the expected library size is 146–166 bp.
45. Use TapeStation with a D1000 ScreenTape to measure the
library final concentration and average length.
3.7 Transformation
of Intact RNA into
RNA-seq Libraries
1. Obtain the À80
C-stored 20% of beads for RNA-seq of intact
bound RNAs (Subheading 3.4, step 7).
2. Elute the bound RNA by triple proteinase K digestions:
(1) Add 1.2 mg/mL proteinase K in 200 μL of proteinase K
buffer. Incubate at 50
C under vigorous shaking for 30 min.
(2) Add 0.75 mg/mL proteinase K in 150 μL proteinase K
buffer. Incubate at 50
C under vigorous shaking for 30 min.
(3) Add 0.75 mg/mL proteinase K in 150 μL proteinase K
buffer. Incubate at 50
C under vigorous shaking for 30 min.
Proximity-CLIP
299