Single-stranded DNA oligonucleotides used for generation of double-stranded substrates with CpH or methylated CpG sites embedded in a 10 nucleotide random context were obtained from IDT. The second strand synthesis was conducted by a primer extension reaction using one universal primer. The obtained mix of double-stranded DNA oligonucleotides was methylated by murine DNMT3A or DNMT3B catalytic domain for 60 min at 37 °C in the presence of 0.8 mM S-adenosyl-L-methionine (Sigma) in reaction buffer (20 mM HEPES pH 7.5, 1 mM EDTA, 50 mM KCl, 0.05 mg/mL bovine serum albumin). Reactions were stopped by shock freezing in liquid nitrogen, then treated with proteinase K for 2 h. Afterward, the DNA was digested with the BsaI-HFv2 enzyme and a hairpin was ligated using T4 DNA ligase (NEB). The DNA was bisulfite-converted using EZ DNA Methylation-Lightning kit (ZYMO RESEARCH) according to the manufacturer protocol, purified and eluted with 10 µL ddH2O.
Libraries for Illumina Next-Generation Sequencing (NGS) were produced with the two-step PCR approach. In the first PCR, 2 µL of bisulfite-converted DNA were amplified with the HotStartTaq DNA Polymerase (QIAGEN) and primers containing internal barcodes using following conditions: 15 min at 95 °C, 10 cycles of 30 s at 94 °C, 30 s at 50 °C, 1 min and 30 s at 72 °C, and final 5 min at 72 °C; using a mixture containing 1x PCR Buffer, 1x Q-Solution, 0.2 mM dNTPs, 0.05 U/µL HotStartTaq DNA Polymerase, 0.4 µM forward and 0.4 µM reverse primers in a total volume of 20 µL. In the second PCR, 1 µL of obtained products were amplified by Phusion Polymerase (Thermo) with another set of primers to introduce adapters and indices needed for NGS (30 s at 98 °C, 10 cycles—10 s at 98 °C, 40 s at 72 °C, and 5 min at 72 °C). PCRII was carried out in 1x Phusion HF Buffer, 0.2 mM dNTPs, 0.02 U/µL Phusion HF DNA Polymerase, 0.4 µM forward and 0.4 µM reverse primers in a total volume of 20 µL. Obtained libraries were pooled in equimolar amounts and purified using NucleoSpin® Gel and PCR Clean-up kit (Macherey-Nagel), followed by a second purification step of gel extraction and size exclusion with AMPure XP magnetic beads (Beckman Coulter). Sequencing was performed at the Max Planck Genome Centre Cologne.
Bioinformatics analysis of obtained NGS data was conducted with the tools available on the Usegalaxy.eu server52 (link) and with home written programs. Briefly, fastq files were analyzed by FastQC, 3′ ends of the reads with a quality lower than 20 were trimmed and reads containing both full-length sense and antisense strands were selected. Next, using the information of both strands of the bisulfite-converted substrate the original DNA sequence and methylation state of both CpG sites was reconstituted. CpH and CpN data were split into CpG, CpA, CpT, and CpC, as appropriate. In each case average methylation levels of each NNCGNN and NNNCGNNN site were determined. Pearson correlation factors were calculated using Excel. Each experiment was performed in two independent repeats. For downstream analysis, DNMT3A data of repeat 1 and the combined data of DNMT3B were used for further analysis, based on their comparable methylation levels. Sequence logos were calculated using Weblogo 3 (http://weblogo.threeplusone.com/ ).
Libraries for Illumina Next-Generation Sequencing (NGS) were produced with the two-step PCR approach. In the first PCR, 2 µL of bisulfite-converted DNA were amplified with the HotStartTaq DNA Polymerase (QIAGEN) and primers containing internal barcodes using following conditions: 15 min at 95 °C, 10 cycles of 30 s at 94 °C, 30 s at 50 °C, 1 min and 30 s at 72 °C, and final 5 min at 72 °C; using a mixture containing 1x PCR Buffer, 1x Q-Solution, 0.2 mM dNTPs, 0.05 U/µL HotStartTaq DNA Polymerase, 0.4 µM forward and 0.4 µM reverse primers in a total volume of 20 µL. In the second PCR, 1 µL of obtained products were amplified by Phusion Polymerase (Thermo) with another set of primers to introduce adapters and indices needed for NGS (30 s at 98 °C, 10 cycles—10 s at 98 °C, 40 s at 72 °C, and 5 min at 72 °C). PCRII was carried out in 1x Phusion HF Buffer, 0.2 mM dNTPs, 0.02 U/µL Phusion HF DNA Polymerase, 0.4 µM forward and 0.4 µM reverse primers in a total volume of 20 µL. Obtained libraries were pooled in equimolar amounts and purified using NucleoSpin® Gel and PCR Clean-up kit (Macherey-Nagel), followed by a second purification step of gel extraction and size exclusion with AMPure XP magnetic beads (Beckman Coulter). Sequencing was performed at the Max Planck Genome Centre Cologne.
Bioinformatics analysis of obtained NGS data was conducted with the tools available on the Usegalaxy.eu server52 (link) and with home written programs. Briefly, fastq files were analyzed by FastQC, 3′ ends of the reads with a quality lower than 20 were trimmed and reads containing both full-length sense and antisense strands were selected. Next, using the information of both strands of the bisulfite-converted substrate the original DNA sequence and methylation state of both CpG sites was reconstituted. CpH and CpN data were split into CpG, CpA, CpT, and CpC, as appropriate. In each case average methylation levels of each NNCGNN and NNNCGNNN site were determined. Pearson correlation factors were calculated using Excel. Each experiment was performed in two independent repeats. For downstream analysis, DNMT3A data of repeat 1 and the combined data of DNMT3B were used for further analysis, based on their comparable methylation levels. Sequence logos were calculated using Weblogo 3 (
Free full text: Click here
