Refined method to peer into cancercells' inner working unlocks the interactome IJCResearch NatureComms
) were purified by Miltenyi Biotec CD34-positive selection kit and fluorescence-activated cell sorting from four donations of 15-to 22-week-old human fetal liver and fetal bone marrow as previously describedselection, positive cells were stained for flow cytometry with the following fluorophore-conjugated monoclonal antibodies, all from BD Biosciences, and used at the manufacturer’s recommended concentration: CD34 PECy7 , CD38 FITC , CD19 BV421 and CD33 APC .
In-nucleus ligation of DNA fragments was carried out during 4 h at 16 °C after adding 12.5 µl of 1U/μl T4 DNA ligase , 50 µl of 10× ligation buffer , 5 μl of 10 mg/ml BSA and 170.5 ml of water. Afterward, DNA ligation products were decrosslinked by adding 30 µl of Proteinase K 10 mg/ml and incubating overnight at 65 °C. The following day, an extra decrosslink during two more hours was performed after adding 15 µl of the Proteinase K enzyme.
Biotin removal of the non-ligated ends was skipped. DNA ligation products were sonicated using Covaris M220 focused-ultrasonicator in 130 µl tubes . After shearing, DNA ends were repaired by adding 6.5 µl of T4 DNA polymerase 3U/µl , 6.5 µl of T4 polynucleotide kinase 10U/µl 1.3 µl of Klenow polymerase 5U/µl , 18 µl of dNTP mix 2.5 mM each and 18 µl of 10× ligation buffer and incubating for 30 min at 20 °C.
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