title: GSM6427519: SUM159 pLV-KRAB + all 4-targeting gRNAs, H3K4me3, abcam Ab8580, biol rep 2; Homo sapiens; ChIP-Seq
library_construction_protocol: Changes in histone modification were assessed using a modified Native ChIP protocol adapted from Grzybowski et al, 2019 [10.1038/s41596-019-0218-7] with minor changes from the original protocol as follows. Native ChIPs were performed using Anti-Rabbit IgG isotype (negative control, CST #66362), Anti-H3K4me3 (Abcam ab8580), and Anti-H3K9me3 (Abcam ab8898) in SUM159 All gRNA and No gRNA. Per 6 ChIP reactions, 6 x 106 cells were harvested for nuclei preparation. The M220 Focused-ultrasonicator™ (Covaris) was used to sonicate the chromatin for 3 min at 20% duty cycle, 75W, 200 cycles per burst before proceeding to MNase digestion and HAP purification as per original protocol. Fifteen µL of HAP purified chromatin was set aside as input control. Following incubation in a C1000 Touch Thermal Cycler (Bio-Rad) at 75 °C with a hot lid ≥ 85 °C for 6 min, final clean-up was performed using 1.5x AMpure beads and purified fragment size and quality were determined using High Sensitivity dsDNA Qubit™ and Agilent Tapestation. DNA from ChIP input and pulldown were subjected to ThruPLEX® DNA-Seq (Takara  R400675) library preparation as per manufacturer's instructions.