title: GSM8714555: K562, daunorubicin 10 µM, 4h, CTCF ChIP-seq, biol rep 1; Homo sapiens; ChIP-Seq
library_construction_protocol: ChIP samples were processed as described in PMID 19275939 and 25961671. The ChIP DNA was cleaned up using  QIAquick PCR Purification Kit (QIAGEN, 28106) and eluted in 35 µl EB. 34 µl ChIP DNA was end-repaired in a 50 µl reaction, with 5 µl 10x Beads were reclaimed and washed once with 70% ethanol without mixing followed by aspirating ethanol.  End-Repair Buffer, 5 µl 2.5 mM dNTP Mix, 5 µl,10 mM ATP, and 1 µl End-Repair Enzyme Mix (Lucigen,14422-5). Incubated at room temperature for 45 mins and purified using the QIAquick PCR Purification Kit (QIAGEN, 28106), eluting in 35 µl of EB. Then the 34 µl end-repaired ChIP DNA was added of 'A' base to 3' Ends in a 50 µl reaction, with 5 µl Klenow buffer (NEB2), 10 µl 1 mM dATP, 1 µl Klenow (3' to 5' exo minus). Incubated for 30 mins at 37°C and purified using the QIAquick PCR Purification Kit (QIAGEN, 28106) eluting in 12 µl of EB. The 12 µl purified DNA was ligated with an adapter in a 30 µl reaction, with 15 µl 2x DNA ligase buffer,1 µl Adapter oligo mix (40 ng/µl), and 2 µl T4 DNA ligase. Incubated at room temperature for 15 minutes and purified using the QIAquick PCR Purification Kit (QIAGEN, 28106), eluting in 25 µl of EB. The 23 µl purified DNA was combined and mixed with 25 μl Phusion High-Fidelity PCR Master Mix (Thermofisher, F531L), 1 µl 10 µM PCR primer 1.1, 1 µl 10 µM PCR primer 2.1 to a total 50 µl reaction volume. PCR procedures were 98 °C for 30 s, 15 cycles of 98 °C for 10 s, 65 °C for 30 s, 72 °C for 30 s, and 72 °C for 5 min (15 cycles could be used for high abundant ChIPed DNA, 17 cycles could be used for low abundant ChIPed DNA). The PCR product was purified using the QIAquick PCR Purification Kit (QIAGEN, 28106), and eluted in 19 µl of EB. The adapter-ligated DNA was run on a 2% Agarose E-gel (ThermoFisher, G402022) and excised DNA of the most abundant band (should be around 300 bp, within the range of 450 bp-600 bp) from the gel with a clean scalpel (be sure to take photos of the gel before and after the gel slices are excised). The cut of gel was purified using MinElute Gel Extraction Kit (QIAGEN, 28606), and eluting in 15 µl of EB. Measure the DNA concentration (ng/µl) using Qbit, and assess the quality of purified libraries using a Bioanalyzer High-Sensitivity DNA Analysis kit (Agilent).