Ion pgm sequencing kit

One hundred percent methylated DNA was commercially sourced from NEB (CpG Methylated Jurkat Genomic DNA, Cat# N4002S). Zero percent methylated DNA was created by performing whole-genome amplification of commercially bought human genomic DNA (Roche Human Genomic DNA Cat# 11691112001), using the Qiagen REPLI-g whole-genome amplification kit, according to the manufacturer’s instruction. Graduated methylated controls (that is, 25%, 50%, 75%) were made by determining the amount of amplifiable DNA in the 100% and 0% methylated DNA samples using qPCR and pooling 0% and 100% methylated DNA in the proportions needed to produce the final methylated control.
Sequencing was performed on either a MiSeq or Ion Torrent sequencer. MiSeq runs used the MiSeq Reagent Kit v2 (300 cycles; PN MS-102-2002). PGM runs used either the OT2 200 bp and 200 bp Ion PGM Sequencing Kit (Life Technologies) with a 314 chip or the OT2 400 bp kit and the 400 bp Ion PGM Sequencing Kit with a 314v2 chip. MiSeq sequencing utilized custom sequencing adaptors, as described in the Fluidigm Access Array manual.

Sequencing of the two RRLs was obtained using 200 ng of DNA that was purified by agarose gel electrophoresis as described above, enzymatically sheared, end-repaired, and adapter-ligated using the Ion Xpress Plus Fragment Library Kit (Life Technologies). Obtained DNA material was size-selected using the e-gel system (Invitrogen, Carlsbad, CA, USA) and bands corresponding to 100 bp of inserts were collected and quantified by qPCR using a StepOnePlus Real-Time PCR System (Life Technologies). Selected fragments were clonally amplified, purified, and sequenced using the Ion One Touch 100 Template Kit and the Ion PGM Sequencing Kit with two Ion 318 chips (Life Technologies), for the two RRLs.