Materials and Reagents

1. Purifying nucleic acids from proteins
   
   1. Purified protein and co-purified nucleic acids in purification buffer (e.g. TtAgo with siDNA guides)
      Note: Please refer to the protocol “Expression and Purification of the Thermus thermophilus Argonaute Protein” (Swarts et al., 2014b).
      
   2. Proteinase K solution (20 mg/ml) (Life Technologies, Ambion^®, catalog number: AM2548 )
      
   3. CaCl₂ solution (50 μM)
      
   4. Roti phenol/chloroform/isoamyl alcohol (pH 7.5-8.0) (Carl Roth, catalog number: A156 )
      
   5. 99% ethanol
      
   6. 70% ethanol (pre-cooled to -20 °C)
      
   7. Linear acrylamide (5 mg/ml) (Life Technologies, Ambion^®, catalog number: AM9520 )
      Note: Alternatively, a house-made Linear Acrylamide can be used (http://www.uvm.edu/~tpdelane/lab/protocols/LinearPolyAcryl.htm).
      
   8. RNase free MQ water
      
   9. Additional materials and reagents required for analysis of purified nucleic acids (optional)
      
      1. T4 polynucleotide kinase (PNK) (New England Biolabs, catalog number M0201 )
         
      2. T4 polynucleotide kinase reaction buffer (New England Biolabs, catalog number M0201)
   
   
2. Enriching and preparing ssDNA molecules for sequencing
   
   1. Purified single stranded DNA nucleotides in MQ water (e.g. see Procedure part A "purifying nucleic acids from proteins")
      
   2. RNase A (DNase and protease-free) (10 mg/ml) (Thermo Fisher Scientific, catalog number: EN0531 )
      
   3. Terminal Deoxynucleotidyl transferase (TdT) (recombinant) (Life Technologies, Invitrogen^TM, catalog number: 10533-073 )
      
   4. Terminal Deoxynucleotidyl transferase (TdT) buffer (Life Technologies, Invitrogen^TM, catalog number: 16314-015 )
      
   5. dATP (100 mM) (Thermo Fisher Scientific, catalog number: R0141 )
      
   6. QIAquick nucleotide removal kit (QIAGEN, catalog number: 28304 )
      
   7. Primer A (5’-GAGAGAGGATCCCGAATTGTGCAGCTGTCAATCAACC-3’) (5 μM)
      
   8. T4 RNA ligase 1 (New England BioLabs, catalog number: M0204 )
      
   9. T4 RNA ligase reaction buffer (10x, supplied with T4 RNA ligase 1)
      
   10. ATP (10 mM) (supplied with T4 RNA ligase 1)
       
   11. PEG8000 (supplied with T4 RNA ligase 1)
       
   12. Primer B (5’-GAGAGAGGATCCTTTTTTTTTTTTTTTTTTTTTTTTTTVN-3’) (5 μM)
       
   13. Pfu DNA polymerase (or equivalent)
       
   14. 10x Pfu DNA polymerase buffer (or equivalent, supplied with MgCl₂ or MgSO₄)
       
   15. dNTPs (5 μM)
       
   16. 2% agarose gel
       
   17. Generuler low range DNA ladder (Thermo Fisher Scientific)
       
   18. GeneJET gel extraction kit (Thermo Fisher Scientific, Fermentas, catalog number: K0691 )
       Note: Additional materials and reagents required only if ssDNA is not 5’-end phosphorylated: T4 polynucleotide kinase (PNK, New England Biolabs, catalog number: M0201) and T4 polynucleotide kinase reaction buffer (New England Biolabs, catalog number: M0201).

Equipment

1. Purifying nucleic acids from proteins
   
   1. Heat block
      
   2. PCR machine
      
   3. Cooled table top Eppendorf centrifuge
      
   4. Freezer (-20 °C)
   
   
2. Enriching and preparing ssDNA molecules for sequencing
   
   1. Heat block
      
   2. PCR machine
      
   3. Table top centrifuge
      
   4. Agarose gel electrophoresis equipment

Procedure