GSM3231026: DC558_TB18 [SeqCap Epi]; Homo sapiens; OTHER
DNA from DCs was extracted using the PureGene DNA extraction kit (Gentra Systems). DNA from macrophages was extracted using the DNeasy Blood and Tissue Kit (Qiagen). Libraries were generated with KAPA Library Preparation Kit for Illumina Platforms (KAPA Biosystems), as per the manufacturer's instructions. Briefly, genomic DNA was fragmented to 100-300 bp with an S2 sonicator (Covaris). Fragments were then end-repaired, A-tailed, and ligated with methylated sequencing adapters. Between every enzymatic step, libraries were purified using AMPure beads (Agencourt). After ligation, in addition to the AMPure bead purification, a DUAL-SPRI size selection was performed to further select for fragments with adapters in the window of 200-400 bp. Sodium bisulfite conversion was performed with EZ DNA Methylation Lightning Kit (Zymo Research), and libraries were amplified using KAPA Hifi HotStart Uracil Tolerant Enzyme (KAPA Biosystems). Library quality was assessed by 2100 Bioanalyzer (Agilent Technologies). Samples showing the desired profile were pooled together in equal mass according to Qubit quantification. We then performed a hybridization using the SeqCap Epi kit (Roche NimbleGen). The sample pool, indexes corresponding to the sequences of the adapters used for library preparation, and repetitive DNA (C0t) were desiccated and then incubated in hybridization buffer with a set of customized probes for 72 hours to select and sequence target regions only. Specifically, DNA methylation data was collected for 33,059 target regions spanning >130,000 CpG sites (mean length = 300 bp; mean number of CpG sites = 5), which is less than 1% of the ~28 million CpGs contained in the human genome. These regions were primarily comprised of MTB-induced differentially methylated regions identified at 18 hours post-infection using whole-genome bisulfite sequencing, as well as other distal regulatory elements in DCs where changes in DNA methylation have been shown to be most likely to occur. Moreover, these candidate regions were nearby differentially expressed genes in response to MTB at 18 hours. Probes targeting a two kilobase region between coordinates 4500 and 6500 bp of the lambda genome (NC_001416.1) are included by default in the SeqCap Epi design, as a control for bisulfite conversion efficiency. Sequencing was performed using the Illumina HiSeq 2500, as per the manufacturer's instructions.
GEO Accession
GSM3231026