DNMT1 RNA Immunoprecipitated Sequencing in HL60 cells. Homo sapiens strain:HL60 cells

DNA immunoprecipitated with DNMT1 antibody (Abcam cat# ab13537) and IgG (Sigma Aldrich) was processed for sequencing as described with some modifications. Double stranded cDNA was synthesized using the Just cDNA Double-Stranded cDNA Synthesis Kit (Agilent Technology, Santa Clara, CA) according to the manifacturer's instructions. Illumina sequencing libraries were constructed from these cDNA using ChIP-Seq sample preparation kit (cat# IP-102-1001, Illumina, San Diego, CA) with some modifications. Illumina paired-end adaptor and PCR primers were used to replace the single read adaptor and primers in the kit. Constructed libraries were subjected to a final size-selection step on a 10% Novex TBE Gels (Invitrogen, Carlsbad). DNA fragments of 175-200 bp were excised from SYBR-green-stained gel. DNA was recovered from the gel and quantified following Illumina's qPCR quantification protocol. A paired end sequencing of these libraries was then performed on Illumina GA IIx to achieve 2 × 76 bp reads. Paired-End reads were trimmed to 50bp and then aligned to the reference genome hg18 using BWA2 with the following parameters: bwa aln -o 1 -l 25 -k 2; bwa sampe -o 200. The SISSRs (Site Identification from Short Sequence Reads) 3 was used for the peak discovery, multiple identical reads were removed and the background model was built with the IgG control library. Peaks with a P-value threshold (for fold enrichment of DNMT1 reads) below 0.05 and an e-value cut-off of 10 were selected. All peaks were annotated with CEAS 4 build on RefSeq hg18. A peak was considered belonging to a gene if located in the coding region or 3 kb up or downstream the gene (gene loci).