GSM8829114: MEFs, 24h iMPC medium, IgG antibody, rep2; Mus musculus; OTHER
24 hours after reprogramming initiation, cells were washed 1x with PBS and incubated with Accutase cell detachment solution at 37°C in 5% CO2 for 15 minutes. After inactivation with iMPC medium, cells were centrifuged and resuspended in 2% FBS in PBS. Cells suspensions were manually counted with a hemocytometer and 500k cells per reaction (2-3 reactions per condition) were transferred in a 1.5mL Protein Lo-Bind tube for preparation using the ChIC/CUT&RUN Assay Kit (Active Motif, 53180) according to manufacturer's protocol. Briefly, cells were centrifuged and nuclei isolated with Complete Nuclei Isolation Buffer. Before the washes in Complete Dig-Wash Buffer, 5k Drosophila Spike-In Nuclei per reaction were added to the nuclei solution. Nuclei were then added to activated Concanavalin A Beads (10µL beads per reaction). Before incubation with primary antibody solution, the samples were divided in 2 PCR tubes (3 for one +Myf6+F/R/C sample) and incubated in either anti-HA Tag primary Antibody Buffer (1:100, MA5-27915, Invitrogen) or negative control anti-rabbit IgG primary Antibody Buffer (1:100, Active Motif, 53180, Part# 106138): additionally, one +Myf6+F/R/C sample was incubated in H3K27me3 primary Antibody Buffer (1:100, Active Motif, 39916) as positive control. All antibody buffers were supplemented with 1:50 Spike-In Antibody (Active Motif, 53183, Part# 106426). The samples were then incubated overnight at 4°C on a shaker for cell permeabilization and antibody binding. After washing, pAG-MNase (Active Motif, 53180, Part# 106130) was bound to the antibodies and the chromatin specifically digested. The chromatin fragments were then separated from the beads, purified with DNA Purification Columns SF, eluted in DNA Purification Elution Buffer, and either directly used for library preparation or frozen at -80°C until further use. NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs, E7645) was used for library preparation following the manufacturer's protocol and the modifications suggested in the ChIC/CUT&RUN Assay Kit (Active Motif, 53180) protocol. Concisely, 25µL of chromatin fragments were end-repaired, the 5'-end phosphorylated and dA-tailed by incubating the fragments with the NEB Next Ultra II End Prep Enzyme Mix and Reaction Buffer for 30 minutes at 20°C and 1 hour at 50°C to avoid DNA melting. The non-indexed adaptor was diluted 1:25 in 10mM Tris-HCl with 10mM NaCl before ligation and the solution was then incubated with USER Enzyme. After ligation, a double-sided cleanup (0.4x and 1.6x) was performed with AMPure XP Beads-Based Reagent and the eluted product PCR-amplified using NEBNext Multiplex Oligos for Illumina (Index Primers Set 1, New England Biolabs, E7335) and NEBNext Ultra II Q5 Master Mix for 12 cycles. Following PCR amplification, the DNA fragments were double-side selected (0.4x and 1.2x) with AMPure XP Beads-Based Reagent and eluted in DNA Purification Elution Buffer, analyzed on the 4200 TapeStation System (Agilent Technologies) using the D5000 High Sensitivity ScreenTapes and reagents and stored at -20°C until sequencing.