title: CUT&RUN of histone modifications, chromatin remodelers, and transcription factors in forelimbs, hindlimbs, liver and branchial arches of E10.5 and E11.5 JAX/Swiss mouse embryos
design: CUT&RUN of histone modifications, chromatin remodelers, and transcription factors in forelimbs, hindlimbs, liver and branchial arches of E10.5 and E11.5 JAX/Swiss mouse embryos
library_construction_protocol: Embryo age was determined based on timed mating and vaginal plug observation (0.5 dpc) and confirmed by morphological criteria. Experiments were performed in the afternoons (female sacrifice at approximately 13:00). Pregnant females were sacrificed by cervical dislocation and E10.5 or E11.5 embryos were surgically removed. Tissues were dissected under a dissection stereomicroscope (SZ61, Olympus) and pooled from each litter. Tissue pools were dissociated to single cells via incubation in TrypLE Express Enzyme (12-604-013, Thermo Fisher Scientific) for 15 min at 37°C on a shaker. For liver tissues, dissociation was done with manual pipetting. The cell suspension was resuspended in ice-cold PBS, filtered through a 40 µm cell strainer (KKE3.1, Carl Roth) and further processed for C&R. 3 limbs were used per sample, while the equivalent of 1 liver or 1 set of branchial arches were used per sample. For HEK293T cells, 500,000 cells were used per sample.  C&R LoV-U was performed as described in Zambanini et al., 2022. Filtered cells were washed three times in nuclear extraction (NE) buffer [20 mM HEPES-KOH (pH 8.2), 10 mM KCl, 0.5 mM spermidine, 0.05% IGEPAL, 20% glycerol, Roche Complete Protease Inhibitor EDTA-Free], the resuspended in 40 µl NE per sample and bound to 20 µl magnetic ConA agarose beads (ABIN6952467, antibodies-online) previously equilibrated in 44 ul binding buffer per sample. After incubation, nuclei and beads were resuspended for 5 min in EDTA wash buffer (wash buffer [20 mM HEPES (pH 7.5), 150 mM NaCl, 0.5 mM spermidine in Roche Complete Protease Inhibitor EDTA-Free (COEDTAFRO)] with 2 mM EDTA) and split into PCR tubes. Antibody incubation was performed in 200 µl wash buffer with antibody dilute 1:100 (for antibody and batch information, see Table S1) overnight at 4°C on a rotator. After overnight incubation, samples were washed five times in wash buffer and resuspended in 200 µl of pAG-MN buffer (wash buffer with pAG-MN 120 ng/sample) and incubated for 30 min at 4°C on a rotator. After 5 washes, digestion was performed for 30 min in ice: started by resuspending the samples in 50 µl of wash buffer with 2 mM CaCl2. After 30 min, the digestion buffer was removed and moved to tubes containing 3 µl EDTA/EGTA 250 mM. The digestion reaction on the beads was stopped with 50 µl of 1× Urea STOP buffer (100 mM NaCl, 2 mM EDTA, 2 mM EGTA, 0.5% IGEPAL, 8.8 M urea) and the samples were incubated 1 h at 4°C. Beads were collected on the magnet and liquid transferred to the PCR tube containing the digestion buffer. DNA was purified by two subsequent rounds of bead-based cleanup using Mag-Bind TotalPure NGS beads (M1327, Omega Bio-Tek) at 2×, and then resuspended in 20 µl Tris-HCl (pH 7.5).  Library preparation was performed with KAPA Hyper Prep Kit for Illumina platforms (KK8504, KAPA Biosystems) according to the manufacturer's guidelines with modifications: End repair and A-tailing was performed in 0.4× volume reactions with 20 µl of purified DNA. The thermocycler conditions were set to 12°C for 15 min, 37°C for 15 min and 58°C for 25 min to prevent thermal degradation of the shortest fragments. Adapter ligation was performed in 0.4× volume reactions. KAPA Dual Indexed adapters were used at 0.15 µM. A post-ligation clean-up was performed with Mag-Bind TotalPure NGS beads at 1.2× the sample volume. Resuspension was carried out in 10 mM Tris-HCl (pH 8.0). Library amplification was performed in 0.5× volume reactions. The cycling conditions were set as follows: 45 s initial denaturation at 98°C, 15 s denaturation at 98°C, 10 s annealing/elongation at 60°C, 1 min final extension at 72°C, hold at 4°C, with 13 cycles. After amplification, a post-amplification clean-up was performed using NGS beads at 1.2× sample volume. Libraries were then run on an E-Gel EX 2% agarose gel (G402022, Invitrogen) for 10 min using the E-Gel Power Snap Electrophoresis System (Invitrogen). Bands of interest between 150 and 500 bp were excised and purified using the GeneJET Gel Extraction Kit (K0691, Thermo Fisher Scientific) according to manufacturer's instructions.