GSM5605788: MDBK UT pSTAT1 rep2; Bos taurus; OTHER
CUT&RUN pulldowns were generated using a protocol from Meers et al. 2019 (PMID: 31232687, https://dx.doi.org/10.17504/protocols.io.zcpf2vn). All buffers were prepared according to the “High Ca2+/Low Salt” section of the protocol using 0.04% digitonin. 500,000 viable cells were used for each pulldown. The following antibodies were used: rabbit anti-mouse IgG (1:100, Abcam #ab46540), rabbit anti-H3K27ac (1:100, Millipore #MABE647), rabbit anti-pRPB1-Ser5 (1:50, Cell Signalling #13523S), rabbit anti-STAT1 (1:100, Cohesion Biosciences #3322), rabbit anti-pSTAT1-Ser727 (1:100, Active Motif #39634). pA-MNase (gift from Steve Henikoff) was added to each sample following primary antibody incubation at a final concentration of 700 ng/mL. Chromatin digestion, release, and extraction was carried out according to Meers et al. 2019 (PMID: 31232687, https://dx.doi.org/10.17504/protocols.io.zcpf2vn). Pulldown success was determined by Qubit dsDNA High Sensitivity (Life Technologies #Q32851) and TapeStation 4200 HSD5000 (Agilent #50675588) before proceeding with library preparation. Libraries were generated using a modified protocol for use with the KAPA HyperPrep Kit (Roche #KK8502). Briefly, the full volume of each pulldown (~30 uL) was diluted to 50 uL in nuclease-free water, and libraries were generated following the manufacturer's protocol with the following modifications. Freshly diluted 0.200 uM single-index adapters were added to each library at a low concentration (9 nM) to minimize adapter dimer formation. Adapter-ligated libraries were treated with 0.2% SDS and 0.4 mg/mL Proteinase K for 1 h at 37°C and underwent a double-sided 0.8X/1.0X cleanup using KAPA Pure Beads (Roche #07983280001). Purified, adapter-ligated libraries were amplified using the following PCR cycling conditions: 45 s at 98°C, 15x(15 s at 98°C, 10 s at 60°C), 60 s at 72°C. Amplified libraries underwent a double-sided 0.8X/1.0X cleanup. The final libraries were quantified using Qubit dsDNA High Sensitivity and TapeStation 4200 HSD5000. Libraries were pooled and sequenced on an Illumina NovaSeq 6000 (University of Colorado Genomics Core) as 150bp paired-end reads.