GSM1565946: DC_NI_rep4 (Bisulfite-Seq); Homo sapiens; Bisulfite-Seq
DNA from infected and non-infected DCs was extracted using the PureGene DNA extraction kit (Gentra Systems). DNA from infected and non-infected DCs (6ug) was spiked with 30ng of unmethylated cl857 Sam7 Lambda DNA (Promega, Madison, WI) and further sonicated to an average length of ~100bp using a Covaris ultrasonicator under the following settings for 16 cycles: Duty cycle: 10%; Intensity: 5; Cycles/burst: 100. The sonicated product was further subjected to repair of 3’ and 5’ ends followed by the addition of a non-templated dA-tail before ligation to cytosine-methylated adapters provided by Illumina (Illumina, San Diego, CA) as per manufacturerʼs instructions for genomic DNA library construction. Adapter-ligated DNA of 100-200 bp was isolated by 2% agarose gel electrophoresis, and sodium bisulfite conversion was performed on the resulting sample using the MethylCode™ Bisulfite Conversion Kit (Invitrogen) as per manufacturer’s instructions. Half of the bisulfite-converted, adapter-ligated DNA molecules were enriched by six cycles of PCR with the following reaction composition: 2.5 U of uracil-insensitive PfuTurboCx Hotstart DNA polymerase (Agilent), 5 μl 10X PfuTurbo reaction buffer, 25 μM dNTPs, 1 μl PE Primer 1.0 (Illumina), 1 μl PE Primer 2.0 (Illumina) (50 μl final volume). The thermocycling parameters were: 95 ̊C 2 min, 98 ̊C 30 sec, then 6 cycles of 98 ̊C 15 sec, 60 ̊C 30 sec and 72 ̊C 4 min, ending with one 72 ̊C 10 min step. The reaction products were purified using the QIAquick PCR spin column (Qiagen). Two separate PCR reactions were performed on subsets of the adapter-ligated, bisulfite-converted DNA, yielding two independent libraries from the same biological sample.  The quality of the libraries was checked on a Bioanalyzer followed by quantification of the libraries by qPCR using the KAPA Library Quantification Kit prior to sequencing.  Samples were sequenced on an Illumina HiSeq 2000 using 50- and 59-bp single-end reads.