GSM7498147: CT27, Stem, input, rep2; Homo sapiens; ChIP-Seq
ChIP was performed on CT27 human TS cells in three replicates using the SimpleChiP Enzymatic Chromatin IP kit (Cell Signaling Technology. Anti-rabbit AP-2 gamma antibody (1:50, 2320, Cell Signaling) was used in the analysis. In total of 100 ng of fragmented DNA in 25 µL of water was used as input in library preparation. A KAPA HyperPrep Kit (07962347001, Roche) was used for library preparation, following the protocol associated with the kit with the following modifications. During end repair and a-tailing, 35 µL of water was added to the master mix containing 7 µL of end-repair and a-tailing buffer and 3 µL of end-repair and a-tailing enzyme. Forty-five µL of the end-repair and a-tailing master mix was added to each sample and was mixed thoroughly by pipetting. During the adapter ligation step, Illumina TruSeq DNA LT adapters, supplied at 15 µM, were diluted 1:100 in water and 5 µL of diluted adapters were added to the sample. A master mix of 30 µL of ligation buffer and 10 µL of DNA ligase was made and 40 µL of ligation master mix was added to each sample. After adapter ligation, a 0.8x bead clean-up was performed using AMPure XP Beads (A63881, Beckman Coulter) with a 10 min incubation at room temperature to allow the DNA to bind to the beads and a 7 min incubation after two 80% ethanol washes to allow the beads to dry. Once the beads were dry, the beads were resuspended in 25 µL of water and incubated at room temperature for 2 min. Twenty µL of post-ligation sample was then transferred to a new tube to be used in library amplification. The PCR library amplification master mix consisted of 25 µL of 2x KAPA HiFi HotStart ReadyMix, 1.5 µL of 10x PCR primer cocktail, and 3.5 µL of water. Thirty µL of the PCR master mix was added to each post-ligation sample and 13 cycles of PCR were performed. After library amplification, the library underwent a 0.6/0.8x double-sized size selection with 10 min incubations to allow the library to bind to the beads. Two 80% ethanol washes were performed, and the beads were allowed to dry at room temperature for 2 min. Thirty-six µL of water was added to the dried beads and was incubated at room temperature for 2 min to elute the DNA.