For DNA end repair, we incubated 0.5–2 μg sonicated genomic DNA in 1× NEB end repair reaction buffer by adding 5 μl of NEB End Repair Enzyme mix (New England Biolabs, MA, USA) in a volume of 100 μl. The reaction mixture was incubated at 20°C for 30 min. The DNA was purified using QIAquick PCR purification kit (Qiagen) and eluted with 50 μl of water. To achieve dA-tailing, we incubated the end-repaired DNA in a 50 μl volume in 1× NEBNext dA-Tailing Reaction buffer (New England Biolabs) by adding 3 μl of NEB Klenow exo- and incubating at 37°C for 30 min. The DNA was purified using MinElute PCR Purification kit (Qiagen) and eluted twice in 10 μl of Qiagen elution buffer. The final volume was approximately 20 μl.
Subsequently, we performed linker ligation in a volume of 40 μl by incubating the dA-tailed DNA in 1× NEB T4 DNA ligase reaction buffer with 6 μl of a 20 μM stock solution of Illumina Tru-seq linkers in the presence of 1 μl of NEB T4 DNA ligase (400 U/μl). The reaction was carried out at 16°C overnight. The ligated DNA was purified using MinElute PCR Purification kit (Qiagen) and eluted in 10 μl of Qiagen elution buffer (twice), then combined into one tube. We saved 2 μl of linker-ligated DNA as control DNA (input).
Subsequently, we performed linker ligation in a volume of 40 μl by incubating the dA-tailed DNA in 1× NEB T4 DNA ligase reaction buffer with 6 μl of a 20 μM stock solution of Illumina Tru-seq linkers in the presence of 1 μl of NEB T4 DNA ligase (400 U/μl). The reaction was carried out at 16°C overnight. The ligated DNA was purified using MinElute PCR Purification kit (Qiagen) and eluted in 10 μl of Qiagen elution buffer (twice), then combined into one tube. We saved 2 μl of linker-ligated DNA as control DNA (input).