GSM8829584: A2.2_0nM_H3K27ac_rep2; Homo sapiens; OTHER
The frozen cells were then subjected to the CUT&RUN protocol according to the manufacturer's instructions using the CUTANA CUT&RUN Kit (EpiCypher, 14-1048). H3K4me3 and IgG antibodies served as positive and negative controls, respectively. A K-MetStat panel was added to these samples for quality control. E. coli DNA (0.25 ng) was used as spike-in control for library normalization. After purification, CUT&RUN DNA was quantified using a Qubit 1X dsDNA high sensitivity assay (Invitrogen, Q33230). A maximum of 5 ng DNA served as input for library preparation using CUTANA CUT&RUN Library Prep Kits with primer set 1 and 2 for multiplexing (EpiCypher, 14-1001 and 14-002). Of note, to avoid denaturing small fragments, end repair inactivation was performed at 50°C for 1 hour instead of 65°C for 30 minutes 94. The distribution of DNA fragment sizes was analyzed using High Sensitivity D1000 assay (Agilent, ScreenTape 5067-5548 and Reagents 5067-5585) in Agilent 4200 Tapestation. The molarity of each library was calculated and normalized to 5 nM. Libraries were pooled and sequenced using the NovaSeq 6000 platform (Illumina), generating 50-bp paired-end reads.