TGIRT-ssDNaseq of cfDNA
Purified cfDNA was dephosphorylated with FastAP (1 unit; Thermo Fisher) in 13.2 ml of a solution containing 20 mM Tris-HCl pH 7.5, 5 mM MgCl2, and 7.5 mM DTT for 20 min at 37 ĄC and denatured by heating at 95 ĄC for 5 min. The DNA was then used as substrate for TGIRT template-switching DNA synthesis from an initial templateĐprimer substrate consisting of a 34-nt RNA oligonucleotide (R2 RNA), which contains an Illumina Read 2 (R2) primer-binding site and a 3Ő-blocking group (C3 Spacer, 3SpC3; IDT), annealed to a complementary 35-nt DNA primer (R2R DNA), which leaves an equimolar mixture of A, C, G, or T single-nucleotide 3Ő overhangs. Reactions were done with 5-50 ng DNA substrate, 100 nM annealed template-primer substrate, and TGIRT-III enzyme (400 units, 1 mM; InGex, LLC; St. Louis) in 20 ml of reaction medium containing 420 mM NaCl, 5 mM MgCl2, 20 mM Tris-HCl, pH 7.5, 5 mM DTT and 1 mM dNTPs (an equimolar mix of 1 mM dATP, dCTP, dGTP, and dTTP). Prior to the reactions, the template-primer substrate was pre-incubated with TGIRT-III enzyme for 30 min at room temperature in 6 ml of reaction mixture containing 1.4 M NaCl, 5.8 mM MgCl2, 23.3 mM Tris HCl pH 7.5 and then mixed with the 13.2-ul dephosphorylation reaction mixture containing DNA substrate prior to initiating template-switching and DNA synthesis by adding dNTPs. Reactions were incubated at 60 ĄC for 20 min and terminated by adding 5 M NaOH to a final concentration of 0.25 M, incubating at 95Ą C for 3 min, and neutralizing with 5 M HCl. A 5ŐApp/3Ő-CpSp blocked UMI-containing R1R-adapter with a 13-nt UMI at its 5Ő end was ligated to the 3Ő end of the cDNA by using Thermostable 5Ő AppDNA/RNA Ligase as specified by manufacturerŐs protocol (New England Biolabs). The ligated product was amplified by PCR using a KAPA Library Amplification Kit (KAPA) with 500 nM of each Illumina multiplex and barcode primers, with the 5Ő primer adding a P5 capture site and the 3Ő primer adding a sequencing barcode and P7 capture site (Supplemental Table 1). PCR was done with initial denaturation at 98 ĄC for 30 sec followed by 10-12 cycles of 98ĄC for 45 sec, 60ĄC for 10 sec, and 65 ĄC extension for 10 sec. The PCR products were size-selected with 1.3X Agencourt AMPure XP beads (Beckman Coulter) to remove adapter dimers.