Klenow exo enzyme

Cells were lysed with 0.5% SDS in EB buffer and bisulfite treated with the Imprint DNA Modification Kit (Sigma). The resulting DNA was purified using columns and reagents from the EZ DNA Methylation Direct Kit (Zymo Research). First-strand synthesis was performed with Klenow Exo- enzyme (New England Biolabs) using a customized biotin-conjugated adaptor containing standard Illumina adaptor sequences and 9 bp of random sequences (9N), as previously described (40 (link)). Following exonuclease I (New England Biolabs) treatment and binding to Dynabeads M-280 Streptavidin beads (Thermo Fisher Scientific), second-strand synthesis was performed with Klenow Exo- enzyme (New England Biolabs) using a customized adaptor containing standard Illumina adaptor sequences and 9 bp of random sequences. Ten PCR cycles with Phusion High-Fidelity DNA polymerase (New England Biolabs) were used for library amplification with indexed adaptors. Libraries were multiplexed for 100-bp paired-end sequencing on an Illumina HiSeq 2500.

Steps for RNA extraction and cDNA synthesis followed closely to our previously described methods [32 (link)]. Briefly, 300–400 µl of cell culture supernatant was used for RNA extraction using the AgenCourt RNAdvance Cell V2 kit (Beckman Coulter Genomics, USA) according to the manufacturer’s protocols. First-strand cDNA was synthesized with random hexamers and the Superscript® III First-Strand Synthesis System (Thermofisher, USA). Second-strand cDNA synthesis was performed using Klenow exo-enzyme (New England Biolabs, USA). All cDNA samples were purified using AMPure XP (Beckman Coulter Life Sciences, USA).