title: GSM4420243: EZH2-KO1-MYB-ChIP-seq; Homo sapiens; ChIP-Seq
library_construction_protocol: Exponentially growing cells were washed and Chipmentation protocol adapted from (Schmidl C, et al, Nat Methods, 2015) was carried out. DNA was amplified in the following conditions (25µl KAPA HiFi 2x master mix (pre-activated), 20µl ChIP DNA, Nextera primer Ad1/Ad2.x at 0.75 uM, were Ad2 primers were indexed per sample in a 50µl reaction volume) and cycled to the number of cycles judged from Q-PCR. Input controls were tagmented in the following conditions (2.5ng input DNA, 1µl tagmentation enzyme, 1µl 5x tagmentation buffer (Tris pH8.0 50mM, MgCl2 25mM) in a total volume of 5µl at 55C for 5 mins. Tagmentation was stopped by the addition of 1µl 0.6% SDS at room temperature for 5mins. Inputs were amplified in the following conditions (25µl KAPA HiFi 2x master mix (pre-activated), 1.5µl Ad1 primer, 1.5µl Ad2.x index primer, 25µl, 6µl tagmented DNA in a 50µl reaction) for 12 cycles as above. The resulting PCR products were purified using magnetic SPRI beads (Agencourt AMPure XP Beckman Coulter, Cat. A63881) and after two 80% ethanol washes were resuspended in 50µl water. Size separation was performed by incubation with  SPRI beads, transfer of supernatant to a new tube and then addition of 12.5µl fresh SPRI beads. Following two further 80% ethanol washes the size selected libraries were eluted into 15µl water. Libraries were checked and quantified by QubitTM (Invitrogen) and Tapestation (Agilent). ChIP-seq normalised libraries were pooled and sequenced using a NextSeq® (Illumina) 75bp paired end short read sequencer using standard protocols.
attributes.tag: GEO Accession
attributes.value: GSM4420243