Dna preparation kit

To perform a genome-wide DNA methylation analysis, libraries were prepared from 200 to 500 ng of genomic DNA digested sequentially with 60 units of TaqI and 30 units of MspI (New England Biolabs, Ipswich, MA, USA). The resulting size-selected TaqI-MspI fragments (40–120 bp and 120–350 bp) were filled in, and 3′-terminal-A extended, extracted with DNA Clean & Concentrator™ kit (Zymo Research, Irvine, CA, USA). Ligation of selected fragments to pre-annealed adapters containing 5′-methyl-cytosine instead of cytosine was performed by using the Illumina DNA preparation kit in accordance with the protocol of the manufacturer (Illumina Inc., San Diego, CA, USA). Purified, adaptor-ligated fragments were then bisulphite-treated by using the EZ DNA Methylation-Direct™ Kit (Zymo Research). Preparative-scale PCR was performed with the resulting fragments followed by purification of PCR products with DNA Clean & Concentrator™ (Zymo Research). Final size selection of the purified PCR products was performed by using 4% NuSieve 3:1 agarose gel. SYBR-green-stained gel slices of adapter-ligated fragments (130–210 bp or 210–460 bp in size) were excised, and library material was recovered by using the Zymoclean™ Gel DNA Recovery Kit (Zymo Research). Sequencing was performed on an Illumina HiSeq genome analyzer.

The concentration of extracted DNA samples was determined using 1X dsDNA High Sensitivity assay (Invitrogen, Waltham MA, USA) in a Qubit fluorometer (ThermoFisher Scientific, Waltham MA, USA). Sequencing libraries were prepared using the Illumina DNA preparation kit (Illumina, San Diego CA, USA). Library preparation protocol was adopted from the CDC PulseNet Nextera DNA Flex Standard operating protocol [26 (link)] and sequenced using the Illumina Miseq platform at the African Center of Excellence for Genomics of Infectious Diseases (ACEGID), Redeemer’s University, Nigeria.

Total RNAs from samples of three time points (PP, 2d PF, 4d PF) were extracted with an miRNeasy Mini kit (QIAGEN). Small RNA sequencing library are constructed by following the standard protocols of Illumina TruSeq Small RNA Sample Prep kit in the University of Utah Sequencing Core. Size from 145 to 160 bp small RNA with adaptors (118 bp) were isolated and sequenced on an Illumina Genome Analyzer IIx to generate 50-nt single end reads. Sequence reads from three time points (PP, 2d PF, 4d PF) are deposited in the SRA database under project accession number SRP021051. Additional pilot sequencing of the 4d PF time point, which had been previously prepared in a similar fashion and sequenced on an Illumina Genome Analyzer at University of British Columbia Sequencing center serves as replicate for comparison, though with considerably lower sequencing coverage.
The genomic sequencing library of strain FGSC 8820 was constructed by using Nextera Illumina DNA preparation kit with dual indexing primers and sequenced in the Genomic Core at the Institute of Integrative Genome Biology, University of California, Riverside, on an Illumina HiSeq2000 Genome Analyzer. The sequence coverage was approximately 80X, and the reads are deposited in the SRA database under project accession SRP021049.