title: GSM8632916: iMEF, H3K14ac, rep2; Mus musculus; OTHER
library_name: GSM8632916
library_construction_protocol: Cells were harvested using 2% trypsin and suspended in fresh media for counting CUT&Tag was performed according to Epicypher's CUTANA™ Direct-to-PCR CUT&Tag Protocol with a fixation step adapted from Kaya-Okur et al. (2020) (https://doi.org/10.1038/s41596-020-0373-x). 100,00 MEFs were harvested and rinsed with PBS, then incubated in Nuclear Extraction Buffer for 10 minutes on ice. For some samples, HEK293T cells were added to the MEFs at a 1:5 ratio before nuclei extraction, but this spike-in proved to be unreliable and was ignored for the data analysis. Nuclei were spun down and resuspended in PBS, then fixed by adding formaldehyde to a concentration of 0.1% and incubating 2 min at room temperature. Formaldehyde was quenched with two molar equivalents of glycine and cells were spun down, resuspended in Nuclear Extraction Buffer, and incubated for 10 minutes at room temperature with 10 uL activated Concavalin A beads to allow nuclei to bind to the beads. The nuclei/beads were separated with a magnet to allow removal of the supernatant, suspended in Antibody 150 Buffer with the appropriate dilution of primary antibody and incubated overnight at 4 °C. The next day, the primary antibody was replaced with secondary antibody and nuclei were incubated for 30 min at room temperature. Nuclei were rinsed twice with Digitonin 150 Buffer and incubated for 1 hour with pAG-Tn5 fusion transposase, followed by two rinses with Digitonin 300 Buffer. They were then incubated at 37 °C for 1 hour with 10 mM MgCl2 to activate the Tn5 Transposase. Tagmentation was stopped by rinsing with TAPS Buffer and samples were incubated in SDS Release Buffer at 58 °C for 1 hour to lyse nuclei and release the DNA. SDS was quenched in 0.5% TritonX-100. DNA libraries were amplified using CUTANA High-Fidelity 2X PCR Master Mix with Universal i5 primer and the appropriate barcoded i7 primer, and DNA was extracted using 1.3X AMPure beads.