3.5 On-Beads Protein
Trypsinization
1. Transfer the beads (30% aliquot kept on ice) to the magnetic
rack to discard the remaining supernatant and resuspend the
beads in 30 μL freshly prepared 25 mM NH 4 HCO 3 and
20 mM DTT. Shake for 30 min at 25
C, then for 20 min at
37
C, and finally for 10 min at 56
C.
2. Add 6 μL of 200 mM iodoacetamide in 25 mM NH 4 HCO 3
and shake for 1 h at 25
C.
3. Collect liquid from tube caps by briefly spinning on a tabletop
centrifuge and transfer the tubes to a magnetic rack. Discard
the supernatant and wash the beads three times with 200 μL of
1 mM DTT in 25 mM NH 4 HCO 3 , to quench any remaining
iodoacetamide and to ensure full depletion of NP40.
4. Dissolve 20 μg of trypsin in 1 mL of 25 mM NH 4 HCO 3 .
5. Add 98 μL of 25 mM NH 4 HCO 3 and 2 μL of the trypsin
dilution (40 ng) to the beads. Shake overnight at 37
C; if
possible cover vortex with a heated lid to minimize condensation on tube lid.
6. Collect liquid from tube caps by briefly spinning on a tabletop
centrifuge and transfer the tubes to a magnetic rack. Collect the
liquid, which contains the proteolyzed peptides to clean tubes.
Peptides are ready for final cleanup and processing for mass
spectrometric analysis.
3.6 Preparation
of Small RNA cDNA
Libraries from RNP
Footprints for NGS
(General Scheme
in Fig. 4a)
1. Transfer the beads (50% aliquot kept on ice) to the magnetic
rack, discard the remaining supernatant, and resuspend the
beads in 100 μL RNase T1 buffer.
2. Add RNase T1 to a final concentration of 1 U/μL and incubate
at 22
C for 15 min. Then immediately cool the reaction on ice.
3. Wash beads twice with RNase T1 buffer and once with dephosphorylation buffer.
4. Resuspend the beads in 60 μL of dephosphorylation buffer
supplemented with 30 units of calf intestine phosphatase
(CIP). Incubate at 37
C for 10 min with shaking.
5. Wash beads twice with 1 mL of dephosphorylation buffer.
6. Wash beads twice with PNK buffer without DTT.
7. Resuspend beads in 60 μL of
32 P 5
0 labeling reaction mix: PNK
buffer (with DTT) supplemented with 60 units of PNK and
0.5 μCi γ32
P-ATP.
8. Incubate at 37
C for 30 min with shaking.
9. Add nonradioactive ATP to a final concentration of 100 μM
and incubate at 37
C for additional 5 min.
10. Spin down and place tubes in a magnetic rack. Keep 50 μL of
the supernatant, add into it 50 μL denaturing 2Â formamide
gel-loading solution to be used later to mark gel edges during
294
Daniel Benhalevy and Markus Hafner
Trypsinization
1. Transfer the beads (30% aliquot kept on ice) to the magnetic
rack to discard the remaining supernatant and resuspend the
beads in 30 μL freshly prepared 25 mM NH 4 HCO 3 and
20 mM DTT. Shake for 30 min at 25
C, then for 20 min at
37
C, and finally for 10 min at 56
C.
2. Add 6 μL of 200 mM iodoacetamide in 25 mM NH 4 HCO 3
and shake for 1 h at 25
C.
3. Collect liquid from tube caps by briefly spinning on a tabletop
centrifuge and transfer the tubes to a magnetic rack. Discard
the supernatant and wash the beads three times with 200 μL of
1 mM DTT in 25 mM NH 4 HCO 3 , to quench any remaining
iodoacetamide and to ensure full depletion of NP40.
4. Dissolve 20 μg of trypsin in 1 mL of 25 mM NH 4 HCO 3 .
5. Add 98 μL of 25 mM NH 4 HCO 3 and 2 μL of the trypsin
dilution (40 ng) to the beads. Shake overnight at 37
C; if
possible cover vortex with a heated lid to minimize condensation on tube lid.
6. Collect liquid from tube caps by briefly spinning on a tabletop
centrifuge and transfer the tubes to a magnetic rack. Collect the
liquid, which contains the proteolyzed peptides to clean tubes.
Peptides are ready for final cleanup and processing for mass
spectrometric analysis.
3.6 Preparation
of Small RNA cDNA
Libraries from RNP
Footprints for NGS
(General Scheme
in Fig. 4a)
1. Transfer the beads (50% aliquot kept on ice) to the magnetic
rack, discard the remaining supernatant, and resuspend the
beads in 100 μL RNase T1 buffer.
2. Add RNase T1 to a final concentration of 1 U/μL and incubate
at 22
C for 15 min. Then immediately cool the reaction on ice.
3. Wash beads twice with RNase T1 buffer and once with dephosphorylation buffer.
4. Resuspend the beads in 60 μL of dephosphorylation buffer
supplemented with 30 units of calf intestine phosphatase
(CIP). Incubate at 37
C for 10 min with shaking.
5. Wash beads twice with 1 mL of dephosphorylation buffer.
6. Wash beads twice with PNK buffer without DTT.
7. Resuspend beads in 60 μL of
32 P 5
0 labeling reaction mix: PNK
buffer (with DTT) supplemented with 60 units of PNK and
0.5 μCi γ32
P-ATP.
8. Incubate at 37
C for 30 min with shaking.
9. Add nonradioactive ATP to a final concentration of 100 μM
and incubate at 37
C for additional 5 min.
10. Spin down and place tubes in a magnetic rack. Keep 50 μL of
the supernatant, add into it 50 μL denaturing 2Â formamide
gel-loading solution to be used later to mark gel edges during
294
Daniel Benhalevy and Markus Hafner