Bisulfite Conversion and Sequencing of hCMV-MIE DNA

Genomic DNA was extracted from recombinant CHO pools and treated with bisulphite as described previously12 (link). Bisulphite-converted hCMV-MIE DNA was amplified by PCR using primers 5′-GATATTGATTATTGATTAGTTATTAATAGTAATTAA-3′ and 5′-CAAATAAAAAAATCCCATAAAATCATATACTAA-3′ for amplicon 1 and primers 5′-TTAGTATATGATTTTATGGGATTTTTTTATTTG-3′ and 5′-TTCTAATACTAAACTCCTCTCCCAA-3′ for amplicon 2. Both amplicons were sequenced with the MiSeq system (Illumina, Inc., San Diego, USA). The concentration of amplified DNA was measured with the Qubit system (Life Technologies GmbH, Darmstadt, Germany). The library was generated according to the TruSeq Nano DNA Library Prep Guide (Part # 15041110 Rev. C, Illumina, Inc., San Diego, USA) using 100 ng of each amplified product. Library quality was checked by employing the Agilent 2100 bioanalyzer (Agilent Technologies, Waldbronn, Germany) and the High Sensitivity DNA Assay Kit (Agilent Technologies, Waldbronn, Germany). After verification of quality, 56 amplicons from different cell pools were pooled in equal shares and mixed with 10% PhiX before being run. Reads were mapped employing the Bismark software32 (link) which uses Bowtie233 (link) for alignment.