GSM8721649: control luciferase sgRNA, SU-DIPGXIII-H3K27M-Cas9 cells, rep1; Homo sapiens; RNA-Seq
For CUT&RUN-Rx, cells were harvested using Accutase (STEMCELL Technologies) and counted. Following counting mouse NSCs were spiked-in to human DIPGXIII cell suspensions (1:10 cell number ratio). Cells were fixed in culture media containing 0.1% formaldehyde at room temperature for 1 min. Formaldehyde was quenched with glycine at 0.125 M, followed by a 5-min incubation at room temperature. Fixed cells were washed with PBS. Fixed cells were incubated on ice for 10 min in nuclear extraction buffer (20mM HEPES pH 7.5, 10mM KCL, 0.1% Triton X-100, 20% Glycerol,1X protease inhibitor cocktail and 0.5mM spermidine). Extracted nuclei were collected by centrifugation (4ºC, 600g) and resuspended in cold nuclei extraction buffer (100µL per sample). CUT&RUN was performed following Epicypher CUT&RUN Protocol v1.5.1. CUT&RUN DNA was purified using the Monarch PCR & DNA Cleanup Kit (New England Biolabs) in accordance with the manufacturer's instructions.  For RNA-seq, RNA was extracted from harvested cells (n=2 replicates) using Quick-RNA microprep kit (Zymocat#R1051). CUT&RUN-Rx library preparation was performed using the NEBNext Ultra II DNA Library Kit for Illumina (E7645) according to the manufacturer's protocol. For RNA-seq, poly(A) mRNA enrichment and library preparation were performed by Genewiz.