GSM9248011: dPA_CloneF6_H3K4me3; Mus musculus; OTHER
CUT&RUN was performed on freshly detached NSCs following the protocol detailed in(Janssens and Henikoff, 2019) with some modifications detailed here. Activation buffer and Concanavalin A beads were purchased from Cell Signalling Technologies [#93569]. Digitonin concentration utilised was at 5% across all buffers and MNase digestion time was at 30mins on ice unless otherwise stated. Recovery of DNA fragments using each method was confirmed using Qubit dsDNA HS Assay [Invitrogen, Q32851]. 10ng of this DNA was utilised for generation of CUT&RUN libraries. Libraries were generated using NEBNext® UltraTM II DNA Library Prep Kit for Illumina® [E7645S] and index using NEBNext® Mtiplex Oligos for Illumina® (Index Primers Set 1) [E7335S]. 1µl of 3µM of NEBNext Adaptor was utilised to reduce adaptor dimers. Sera-Mag select beads [Cytiva, 29343052] were used for DNA clean-up and size selection. DNA Library size was measured using Agilent High Sensitivity D1000 ScreenTape [Agilent, 5067- 5585].