Cobalt chloride

Since single-stranded DNA is more efficiently tailed with ribonucleoside 5’-triphosphates than is double-stranded DNA [24] , 10 µl genomic DNA (between 14–220 ng in total) were heat denatured at 95°C for 10 minutes and then quickly chilled on ice before the ribo-tailing reaction with terminal transferase (TdT). The reaction consisted of 1x buffer 4 (New England Biolabs), 2.5 mM cobalt chloride (New England Biolabs), 4 mM GTP (Takara), 10 U TdT (New England Biolabs) and 10 µl denatured genomic DNA in 20 µl reaction volume. Reactions were incubated at 37°C for 30 minutes and then TdT was heat-inactivated at 70°C for 10 minutes.

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.