title: GSM4437207: PEO1_16uM_rep3 (Exo-seq); Homo sapiens; OTHER
library_construction_protocol: DNA was isolated from cells using the Gentra PureGene kit (Qiagen). 1µg purified DNA was sheared using Bioruptor Pico (Diagenode) to sizes of ~ 150 bp. Fragment sizes were confirmed using TapeStation (Agilent) .  Fragmented DNA was then incubated at 30˚C for 30 mins, with dNTPs, ATP, T4 DNA polymerase, Polynucleotide Kinase and DNA Polymerase 1 (Klenow fragment) to generate blunt ends before the reaction was stopped by incubation at 75˚C for 20 mins. Fragments were then incubated with the annealed P7 adapters along with ATP, and the T4 DNA ligase. Adapter ligated fragments were purified and incubated with the phi29 DNA polymerase, along with dNTPs, to allow the overhang between the fragment end and the adapter to be filled in. Adapter ligated DNA was incubated with Lambda exonuclease for 1h at 37˚C. After DNA purification, RecJF and single stranded DNA binding protein (SSB) were added with Lambda exonuclease to maximise digestion efficiency. The platinum molecules which were still present in the fragment, adapter ligated, digested DNA were then displaced by incubation with sodium cyanide at 65˚C for 2h. Complementary strand synthesis was carried out using adapters complementary to the P7 adapter sequence, along with the phi29 DNA polymerase and dNTPs. Finally, the P5 adapter was ligated to the purified, double stranded DNA by incubation for 60 min at 25°C and then 10 min at 65°C to inactivate the enzymes. Purified libraries were then amplified by PCR.
attributes.tag: GEO Accession
attributes.value: GSM4437207