GSM9569952: DIPGXIII, H3K4me3Q5dopaminyl, untreated, rep2; Homo sapiens; OTHER
Neurospheres were dissociated to single cells with TrypLE Express Enzyme, washed with 1X HBSS, counted, and prepared for CUT&RUN with 500,000 cells/sample. CUT&RUN-enriched DNA was prepared using the Cell Signaling Technologies CUT&RUN Kit (catalog# 86652) following the manufacturer's protocol using the following antibodies: pS2-Pol2 (Anti-RNA polymerase II subunit B1 phospho CTD Ser-2, clone 3E10; MilliporeSigma), H3K4me3 (Anti-Histone H3 tri methyl K4; Abcam, ab8580), H3K4me3Q5dop (Anti-H3K4me3Q5dopaminyl; MilliporeSigma, ABE2590), and IgG (Anti-IgG Rabbit; DIAgenode, cat# C15410206). Yeast spike-in DNA (S. cerevisiae; Cell Signaling Technology) was added at 0.5% to each reaction following chromatin digestion. Sequencing libraries were prepared from 3 ng of enriched DNA using the SMARTer ThruPLEX Plasma-seq kit (Takara Bio, cat# R400681) with dual indexes (Takara Bio, cat# DX34752) according to the manufacturer's protocol. Adapter-ligated libraries were amplified for 11 PCR cycles, purified using AMPure XP beads (Beckman Coulter), and evaluated for fragment size distribution on an Agilent 2100 Bioanalyzer. Library molar concentrations were determined by quantitiative PCR with the KAPA Library Quantification Kit (Roche) before pooling for sequencing on an Illumina NovaSeq 6000 to generate 150-bp paired-end reads.