Spriworks fragment library system 1 kit

DNA extraction was performed after proteinase K and RNase A treatment, using a phenol/chloroform standard protocol. Glycoblue^TM (Life Technologies) was added at the precipitation step to avoid subsequent pellet loss. DNA was then resuspended in 130 µL Tris pH 8.0. DNA was fragmented on an S2 Focused-ultrasonicator (COVARIS). The size of the fragments was assessed on a Bioanalyzer (Agilent Technologies) using a High Sensitivity DNA Kit. The mean size of the fragments was 150 bp for the MeDIP experiment and 250 bp for the hMeDIP experiment. Non-methylated TruSeq DNA adapters (synthesized by Sigma), with different indexes (for sample multiplexing before sequencing), were ligated using a SPRIworks Fragment Library System I kit (Beckman) on an SPRI-TE instrument, according to the Illumina Truseq DNA sample prep kit protocol.

Purified DNA from plasmids showing different restriction patterns were sent for sequencing to Eurofins Genomics (GATC Biotech, Constance, Germany). Individual libraries were constructed using the SPRIworks Fragment Library System I Kit (Beckman Coulter, Brea, CA, USA) and pair-end sequenced (2 × 150 bp runs) in a HiSeq sequencer (Illumina, San Diego, CA, USA). Quality-filtered reads, trimmed or non-trimmed depending on the plasmid, were de novo assembled in contigs using SPAdes v3.6.2 software [89 (link)], with a k value of 127 and employing the only-assembler settings. The plasmidSPAdes algorithm [90 (link)], which uses coverage as a parameter to remove chromosomal contigs, was also used for assembly. When plasmids were not merged into a single molecule after assembly, primers were designed based on the end of the contigs, used in PCR reactions, and the sequences of the amplicons employed to join the segments. The Vector NTI program (Invitrogen, Carlsbad, CA, USA) was used to align the sequences of contigs and PCR amplicons. The same program was used for open reading frame (ORF) prediction. Deduced protein sequences larger than 50 amino acids were compared to those in the NCBI’s non-redundant protein database and manually analyzed using BLASTp.