title: GSM9232968: RPB1degron_16nM_dTAGv1_IgG_2; Homo sapiens; OTHER
library_construction_protocol: CUT&RUN was performed essentially as per manufacturer's instructions (#86652, Cell Signaling). After 16 hours of dTAGV-1 treatment, cells were trypsinized and fixed for 2 minutes at room temperature with 0.1% formaldehyde. Fixing was stopped by addition of 250mM glycine for 5 minutes, and cells were washed once with 1X wash buffer supplemented with spermidine and protease inhibitor cocktail. Cells for each condition were then aliquoted into 100 thousand cells per Lo-bind microcentrifuge tube, and incubated with activated concanavalin A beads for 5 minutes at room temperate. After addition of antibody binding buffer, cells were incubated overnight at 4C with rabbit anti-HA (4uL per replicate, 3 replicates per dTAGv-1 condition), rabbit anti-Ser5+2P (4uL per replicate, 3 replicates per dTAGv-1 condition), and rabbit anti-IgG (5uL per replicate, 3 replicates per dTAGv-1 condition) antibody. Binding of pAG-MNase and DNA digestion was performed as per manufacturer's instructions, and included the addition of 50pg spike-in DNA. To reverse crosslinks, the eluted DNA was incubated with agitation overnight at 65C in the presence of 0.1% SDS and 133ng/ug proteinase K. Finally, the eluted DNA was purified using DNA Purification Buffers and Spin Columns (#14209 Cell Signaling) 40uL of DNA from each condition was used for library preparation with the NEBNext Ultra DNA Library Prep Kit for Illumina (#E7370L, NEB). Unique dual indices (#E7395S, NEB) were used to facilitate multiplexing and the detection of PCR duplicates. Finally, libraries were pooled and sequenced on an Illumina NextSeq 2000, yielding 50bp paired-end reads.