title: GSM7431035: Bisulfite converted 5'-3' strand gDNA mCpG; Homo sapiens; OTHER
library_construction_protocol: Reaction mixtures for linear PCR contained 10 µM dGTP and 200 µM d(A/T/C)TP (each), 100 nM 109 bp forward primer, template DNA (3 fM 803 bp C or 5mC template generated by PCR, 62.5 ng HeLa gDNA native or CpG methylated (New England Biolabs)) and 300 nM RIV A8 KTq variant in 1x KTq reaction buffer (50 mM Tris HCl (pH 9.2), 16 mM (NH4)2SO4, 2.5 mM MgCl2, 0.1% (v/v) Tween 20) in 25 µL end volume. PCR was performed with an initial denaturation at 95°C for 3 min followed by amplification over 20 cycles with denaturation at 95°C for 10 s, annealing at 60°C for 30 s and elongation at 72°C for 10 min. Reaction mixtures were purified by using the NucleoSpin® Gel and PCR Clean-up XS kit (Macherey-Nagel) in combination with the NTC binding buffer according to the manufacturer's instructions for ssDNA PCR product purification. DNA was eluted with 18 µL Elution Buffer NE (5 mM Tris HCl (pH 8.5)). To use the linear PCR product as template for NGS library preparation, the ssDNA was exponentially amplified by a high-fidelity DNA polymerase to increase the 109 bp product concentration. Reaction mixtures contained 200 µM dNTPs (each), 100 nM 109 bp forward and reverse primer, 67% (v/v) of the PCR elution and 0.02 U/µL Q5® Hot Start High-Fidelity DNA Polymerase in 1x Q5® Reaction Buffer (New England Biolabs). PCR was performed in 25 µL reaction mixtures with an initial denaturation at 98°C for 30 s followed by amplification over 10 cycles with denaturation at 98°C for 5 s, annealing at 62°C for 10 s and elongation at 72°C for 5 s. Reaction mixtures were purified by using the NucleoSpin® Gel and PCR Clean-up XS kit (Macherey-Nagel) and a 1:2 dilution of the NTI buffer according to the manufacturer's instructions to optimise PCR product purification and yield. DNA was eluted with 18 µL Elution Buffer NE. Note, all reactions were performed in the Thermo Scientific™ Low Profile Tubes (Thermo Scientific) and DNA was eluted in Eppendorf DNA LoBind® Tubes (Eppendorf). 16.25 µL of purified linear PCR product dsDNA was applied as template for a 2-cycle UMI PCR. Reaction mixtures for UMI introduction contained 200 µM dNTPs (each), 200 nM forward and reverse UMI primer, 0.02 U/µL Q5® Hot Start High-Fidelity DNA Polymerase and 1x Q5® Reaction Buffer (New England Biolabs) in 25 µL end volume. PCR was performed with an initial denaturation at 98°C for 30 s followed by primer elongation over 2 cycles with denaturation at 98°C for 10 s, annealing at 48°C for 30 s and elongation at 72°C for 30 s. Final elongation was performed for 2 min at 72°C. 183 bp UMI PCR product was purified by preparative agarose gel electrophoresis using the NucleoSpin® Gel and PCR Clean-up XS kit (Macherey-Nagel) according to the manufacturer's instructions. Purified UMI PCR product was eluted with 18 µL Elution Buffer NE. DNA concentration was determined by qPCR according to the method described for NGS library preparation using template DNA generated by PCR. DNA repair using the PreCR® Repair Mix (New England Biolabs) and Amplicon PCR were performed according to the method described for NGS library preparation using template DNA generated by PCR. Amplicon PCR was performed with an initial denaturation at 98°C for 30 s followed by amplification over 30 cycles with denaturation at 98°C for 10 s, annealing at 70°C for 30 s and elongation at 72°C for 30 s. Final elongation was performed for 2 min at 72°C. 263 bp Amplicon PCR product was purified by preparative agarose gel electrophoresis using the NucleoSpin® Gel and PCR Clean-up XS kit (Macherey-Nagel) according to the manufacturer's instructions. To improve purification, non-diluted NTI binding buffer was used first for solubilising agarose gel slices and subsequently the solved mixture was diluted in total to 1:6.5 with Milli-Q water prior to DNA binding on the purification column. PCR product was eluted with Elution Buffer NE. Treatment of extracted DNA with PreCR® Repair Mix (New England Biolabs) and QIAEX II system (Qiagen) purification were performed according to the method described for NGS library preparation using template DNA generated by PCR. Repaired product DNA was eluted with 22 µL Milli-Q water. DNA library concentration determination, quality control and sequencing was performed as described for NGS library preparation using template DNA generated by PCR. Note, all reactions were performed in the Thermo Scientific™ Low Profile Tubes (Thermo Scientific) and DNA was eluted in Eppendorf DNA LoBind® Tubes (Eppendorf).