GSM4454698: F4_KO_T0 (input); Homo sapiens; OTHER
We employed the HMCP with reference exogenous genome (ChIP-Rx) method of Orlando et al. for quantitative normalization of HMCP signal according to an exogenous spike-in genome (Orlando et al. 2014). As exogenous control we used the DNA isolated from phage T4gt, which carries mutations in both -agt and -bgt glycosyltransferases and thus the vast majority of cytosines are only hydroxymethylated without glycosylation (Weigele and Raleigh 2016). HMCP was carried out using the 5-hydroxymethylcytosine pull-down assay (Cambridge Epigenetix) according to manufacturer instructions with slight modifications. Exogenous phage T4gt DNA was spiked into the TF1 cell DNA at a 1:50,000 ratio before shearing with a Covaris E220 to an average length of 150 bp. Sheared DNA samples were end-repaired and adapter ligated with barcoded Illumina TruSeq adapters. Libraries were denatured and a new copy strand was synthesized.  DNA fragments containing 5hmC were pulled down using a two-step process where 5hmC were first glycosylated with T4BGT (Thermofisher) using UDP-6-azide-glucose, followed by labeling with PEG (polyethylene glycol)-biotin via copper-free CLICK chemistry. The biotin-containing DNA was then pulled down and purified using streptavidin magnetic beads. Finally, purified libraries were PCR amplified with Kapa real-time library amplification kit and sequenced using single end 50bp protocol on the Illumina NovaSeq.
GEO Accession
GSM4454698