Purified E. coli ribosome
(E. coli B strain) was purchased from New England
Biolabs (MA, USA) and diluted with dilution buffer (50 mM HEPES, 50
mM KCl, pH 7.5, 10 mM MgAc2) to a concentration of 1 mg/mL.
Purified recombinant Cas9 from S. pyogenes fused
with a Halo-tag was generated in house, as described by Deng et al.25 (link) DSBSO was synthesized similarly as described
by Burke et al.24 (link) For enrichment similar
as described by Kaake et al.15 (link) (BARAC Method),
Dibenzocyclooctyne-PEG4-biotin conjugate (#760749-5MG, Sigma-Aldrich)
was used without further purification. Biotin was pulled using Pierce
High Capacity Streptavidin Resin (# 20359, Thermo). DBCO beads were
synthesized in house: NHS-activated Sepharose fast flow (#17-0906-01,
GE Healthcare) was incubated to varying concentrations of dibenzocyclooctyne-amine
(DBCO-amine, #761540, Sigma-Aldrich). The prepared beads were stored
as 50% slurry in a 1:1 ethanol:water mixture. AF488-Azide (#CLK-1275-1,
Jena Bioscience) was used to test success of bead–DBCO coupling.
Trypsin gold was purchased from Promega (Mannheim, Germany) and lysyl
endopeptidase (LysC) was from Wako (Neuss, Germany). Benzonase—pharmaceutical
production purity—was from Merck (Darmstadt, Germany).
(E. coli B strain) was purchased from New England
Biolabs (MA, USA) and diluted with dilution buffer (50 mM HEPES, 50
mM KCl, pH 7.5, 10 mM MgAc2) to a concentration of 1 mg/mL.
Purified recombinant Cas9 from S. pyogenes fused
with a Halo-tag was generated in house, as described by Deng et al.25 (link) DSBSO was synthesized similarly as described
by Burke et al.24 (link) For enrichment similar
as described by Kaake et al.15 (link) (BARAC Method),
Dibenzocyclooctyne-PEG4-biotin conjugate (#760749-5MG, Sigma-Aldrich)
was used without further purification. Biotin was pulled using Pierce
High Capacity Streptavidin Resin (# 20359, Thermo). DBCO beads were
synthesized in house: NHS-activated Sepharose fast flow (#17-0906-01,
GE Healthcare) was incubated to varying concentrations of dibenzocyclooctyne-amine
(DBCO-amine, #761540, Sigma-Aldrich). The prepared beads were stored
as 50% slurry in a 1:1 ethanol:water mixture. AF488-Azide (#CLK-1275-1,
Jena Bioscience) was used to test success of bead–DBCO coupling.
Trypsin gold was purchased from Promega (Mannheim, Germany) and lysyl
endopeptidase (LysC) was from Wako (Neuss, Germany). Benzonase—pharmaceutical
production purity—was from Merck (Darmstadt, Germany).