TGIRT-ssDNaseq of K12 genome
E. coli K12 (MG1655) genomic DNA was obtained from the American Type Culture Collection (ATCC). 50 ng K12 gDNA was fragmented by NEB fragmentase for 30 minutes following manufacturerŐs protocol. After sequential size-selection with 0.6X and 1X Agencourt AMPure XP beads (Beckman Coulter) to remove larger and smaller DNA fragments, respectively, the remaining DNA fragments used for library preparation had an average length of ~250 bp. Fragmented E. coli genomic DNA (8-50 ng) 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 40 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. After clean up by using a Nucleospin Gel and PCR cleanup kit (Clontech), 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 cleaned up by Nucleospin Gel and PCR cleanup kit (Clontech) and 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 15 sec, and 72 ĄC extension for 30 sec, with a final extension of 72 ĄC for 5 min. The PCR products were size-selected with 1X Agencourt AMPure XP beads (Beckman Coulter) to remove adapter dimers.