Truseq nano dna library prep guide

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.

Library preparation was performed according to the TruSeq Nano DNA Library Prep Guide (Illumina Inc., San Diego, CA, USA). In brief, 100 ng of 3 genomic DNA samples from each VN/HY/2019-ASFV1, VN/QP/2019-ASFV1, and VN/HY/2022-ASFV2 sample was sheared using an LE220 Focused-ultrasonicator (Covaris Inc., Woburn, MA, USA) with a duty factor of 15% and peak incident power of 450 W at 200 cycles per burst for 50 s. Sheared DNA fragments were then end-repaired, size-selected to obtain DNA fragments of approximately 350 bp, and adenylated according to the manufacturer’s instructions. After ligating index adapters to the ends of the DNA fragments, DNA libraries were enriched using eight cycles of PCR, according to the manufacturer’s instructions. After size selection and PCR amplification, the quality of the library and band size were assessed using D1000 Screen Tapes on a Tapestation 2200 (Agilent Technologies, Santa Clara, CA, USA). Finally, libraries were quantified using the PicoGreen dsDNA quantitation assay (Thermo Fisher Scientific, Waltham, MA, USA) and determined using a Victor3 plate reader (PerkinElmer, Waltham, MA, USA).