HeLa cells expressing FLAG-ORC2-HA, with RNase1 and PNK treatments
Nuclei were isolated from S3 HeLa cells expressing FLAG-ORC2-HA, then Dignam-extracted, Rnase1 and TurboDNase-digested, then ORC complex was purified using FLAG pull-down (elution with FLAG peptide) then HA immuno-precipitation (simultaneously to a PNK and TurboDNase treatment). Complex was eluted using HA peptide, then proteins were digested using proteinase K. RNAs were precipitated, then cDNA libraries were prepared using Clontech’s "Smarter stranded total RNA-seq kit, pico input mammalian"  (setting fragmentation duration to 1 minute). Libraries were sequenced at the MGX facility (Montpellier, France).