HeLa cells expressing FLAG-ORC2-HA, without RNase1 and PNK treatments
Nuclei were isolated from S3 HeLa cells expressing FLAG-ORC2-HA, then Dignam-extracted, TurboDNase-digested, then ORC complex was purified using FLAG pull-down (elution with FLAG peptide) then HA immuno-precipitation (simultaneously to a 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).