GSM6044778: myoLuc_0h_H3K27me3_R1; Myotis lucifugus; 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-pPOLR2A-Ser5 (Cell Signaling #13523S), rabbit anti-STAT1 (1:100, Cohesion Biosciences #3322), rabbit anti-pSTAT1-Ser727 (1:100, Active Motif #39634). pAG-MNase 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 underwent a double-sided 1.0X/1.2X 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 1.1/1.2X 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 (Novogene) as 150bp paired-end reads. CUT&RUN