Terminal transferase (TdT) was ordered from New England Biolabs (#M0315L). Ddel restriction enzyme was ordered from New England Biolabs (#R0175L). Cy3-labeled ddUTP (5-propargylamino-ddUTP-Cy3) was ordered from Jena Bioscience (#NU-1619-CY3) and unlabeled ddTTP (2’,3’-dideoxythymidine-5’-triphosphate) was ordered from TriLink Biotechnologies (#N-4004). ATP (adenosine 5’-triphosphate) was ordered from Thermo Fisher Scientific (#R0441) and glucose was purchased from Sigma-Aldrich (#G8270). The strands for the switch screen were all purchased HPLC-purified from Integrated DNA Technologies (IDT); all purchased sequences are presented in Supplementary Tables 1 and 3 . The switching domains that were validated via plate-reader were purchased from the Stanford Protein and Nucleic Acid facility and the sequences are presented in Supplementary Table 2 . Streptavidin beads for the proof-of-concept TdT and Ddel experiments were purchased from Thermo Fisher Scientific (#88816). All other reagents were purchased from Sigma-Aldrich unless otherwise noted.
Terminal transferase tdt
Manufactured by New England Biolabs
Terminal transferase (TdT) is an enzyme that catalyzes the addition of deoxynucleotides to the 3' hydroxyl ends of DNA molecules in a template-independent manner. It is commonly used in molecular biology applications that require the incorporation of nucleotides into DNA strands.
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Lab products found in correlation
Cobalt chloride, by New England Biolabs (1 mentions)
Fastap thermosensitive alkaline phosphatase, by Thermo Fisher Scientific (1 mentions)
Buffer 4, by New England Biolabs (1 mentions)
Deoxyribonucleotides, by Roche (1 mentions)
T4 polynucleotide kinase pnk, by New England Biolabs (1 mentions)
Phix174 hinfi digest dna ladder, by Promega (1 mentions)
Dna oligonucleotide, by Integrated DNA Technologies (1 mentions)
Radiolabeled compounds, by PerkinElmer (1 mentions)
G 25 spin columns, by Harvard Apparatus (1 mentions)
Rnase if, by New England Biolabs (1 mentions)
Protoscript 2 reverse transcriptase, by New England Biolabs (1 mentions)
4 protocols using terminal transferase tdt
1
Terminal Transferase-Based Molecular Switching
2
Phosphatase Treatment and Ribo-Tailing of DNA
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The ribo-tailing reaction requires the presence of an intact 3′-hydroxyl group, which is lost during DNA strand breaks after the formation of abasic sites during DNA degradation40 (link). Phosphate groups at the 3′ end of the DNA were removed by treatment with phosphatase. The 13-μl reaction contained 1.3 μl of 10 × buffer 4 (New England Biolabs), 1 μl of 1 U μl−1 FastAP thermosensitive alkaline phosphatase (Thermo Scientific) and 200 ng gDNA in EB buffer. The reaction was adjusted with water to reach the reaction volume. The reaction was incubated at 37 °C for 1 h and then heat inactivated at 75 °C for 10 min. We found that there was a significant increase in library yield compared with the same samples without the phosphatase treatment (data not shown).
gDNA was heat denatured into single-stranded DNA at 95 °C for 5 min and then quickly chilled on ice before the ribo-tailing reaction with terminal transferase (TdT; New England Biolabs). The 7-μl TdT reaction consisted of 2.5 μl water, 0.7 μl of 10 × buffer 4, 2 μl of 25 mM cobalt chloride (New England Biolabs), 0.8 μl of 100 mM GTP (Takara), 20 U of TdT and denatured DNA. The 20-μl reaction was incubated at 37 °C for 30 min and then TdT was heat inactivated at 70 °C for 10 min.
gDNA was heat denatured into single-stranded DNA at 95 °C for 5 min and then quickly chilled on ice before the ribo-tailing reaction with terminal transferase (TdT; New England Biolabs). The 7-μl TdT reaction consisted of 2.5 μl water, 0.7 μl of 10 × buffer 4, 2 μl of 25 mM cobalt chloride (New England Biolabs), 0.8 μl of 100 mM GTP (Takara), 20 U of TdT and denatured DNA. The 20-μl reaction was incubated at 37 °C for 30 min and then TdT was heat inactivated at 70 °C for 10 min.
3
Enzymatic Manipulation of Nucleic Acids
4
SMRT-Cappable-seq: Detailed RNA Sequencing
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A volume of 20 μl of enriched RNA (SMRT-Cappable-seq) and 10 μl of non-enriched RNA (control)) were used in a 40 μl first strand cDNA synthesis reaction with 400 units of ProtoScript II Reverse Transcriptase (New England Biolabs) and 50 μM RT primer (Supplementary Table 1 ). Reactions were incubated at 42 °C for 1 h, then 50 units Rnase If (New England Biolabs) was added and incubated at 37 °C for another 30 min. The reactions were purified using AMPure beads and eluted in 20 μl Low TE.
The polyG was added to the 3′end of cDNA for second-strand synthesis using Terminal transferase (TdT, New England Biolabs). The purified cDNA/RNA duplex samples were incubated with 10 units TdT and 2 mM dGTP at 37 °C for 30 min. For the SMRT-Cappable-seq RNA, the reaction was purified using 30 μl hydrophilic streptavidin magnetic beads (New England Biolabs) as mentioned above but without Biotin buffer elution. The washed beads with the bound cDNA/RNA duplex were resuspended in 30 μl Low TE. For control RNA, the reaction products were purified using AMPure beads and eluted in 30 μl Low TE.
The polyG was added to the 3′end of cDNA for second-strand synthesis using Terminal transferase (TdT, New England Biolabs). The purified cDNA/RNA duplex samples were incubated with 10 units TdT and 2 mM dGTP at 37 °C for 30 min. For the SMRT-Cappable-seq RNA, the reaction was purified using 30 μl hydrophilic streptavidin magnetic beads (New England Biolabs) as mentioned above but without Biotin buffer elution. The washed beads with the bound cDNA/RNA duplex were resuspended in 30 μl Low TE. For control RNA, the reaction products were purified using AMPure beads and eluted in 30 μl Low TE.
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