alias: PBD1
title: TGIRT-ssDNA bisulfite sequencing of cfDNA
design: Plasma cfDNA (20 ng) was concentrated to 20 ml with an Oligo Clean and Concentrator kit (Zymo). The concentrated DNA was treated with EZ DNA Methylation-Lightning kit (Zymo) following the manufacturerŐs protocol and eluted in 10 ml of elution buffer supplied by the kit. 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 and extra 5-bp fixed sequence (CATCG) 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 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.
identifiers.namespace: SUB2671096
identifiers.id: PBD1