GSM9421205: E2C-Ctrl_H3K9me2_spike-in_rep2; Mus musculus; ChIP-Seq
The generated BDF1×PWK hybrid embryos were harvested in 9 µl of Nuclei EZ lysis buffer (Sigma) containing protease inhibitor cocktail and 1 mM PMSF. Sample preparation was conducted by adding 1 µl of lysis buffer, followed by 1 µl of detergent mixture (1% Triton X-100 and 1% deoxycholate) and incubating on ice for 5 min. Chromatin fragmentation was performed using 2 U/µl MNase (NEB) in 1× MNase buffer supplemented with 1% PEG6000 and 2 mM dithiothreitol at 21°C for 7.5 min. Digestion was terminated by the addition of 1:10 volume of 100 mM EDTA and 1:12 volume of detergent mixture, followed by 15 min incubation on ice. The chromatin lysates were supplemented with immunoprecipitation buffer (20 mmol/L Tris-HCl (pH 8.0), 2 mmol/L EDTA, 150 mmol/L NaCl, 0.1% Triton X-100, 1× EDTA-free protease inhibitor cocktail, and 0.1 mmol/L PMSF), with 5% volume reserved for input library construction. To the remaining lysate, 30 ng of annealed I-SceI carrier DNA was added. The forward and reverse strands of the carrier DNA were as follows: /5AmMC6/Gtagggataacagggtaattagggataacagggtaattagggataacagggtaattagggataacagggtaattagggataacagggtaattagggataacagggtaatc/3AmMO/ and /5AmMC6/Gattaccctgttatccctaattaccctgttatccctaattaccctgttatccctaattaccctgttatccctaattaccctgttatccctaattaccctgttatccctac/3AmMO/, respectively, where asterisks represent phosphorothioate bonds. For immunoprecipitation reactions, we utilized 0.5 µg of mouse anti-H3K9me2 (MABI0317 (47)) conjugated to pre-cleared Dynabeads Protein G mixture. Following overnight immunoprecipitation at 4°C, the chromatin-bead complexes were washed sequentially with low-salt buffer (20 mmol/L Tris-HCl (pH 8.0), 0.1% SDS, 1% Triton X-100, 2 mmol/L EDTA, 150 mmol/L NaCl, 1× EDTA-free proteinase inhibitor cocktail, and 0.1 mmol/L PMSF) and high-salt buffer (20 mmol/L Tris-HCl (pH 8.0), 0.1% SDS, 1% Triton X-100, 2 mmol/L EDTA, 500 mmol/L NaCl, 1× EDTA-free proteinase inhibitor cocktail, and 0.1 mmol/L PMSF), and the chromatin was eluted in ChIP elution buffer (100 mmol/L NaHCO3 and 1% SDS) at 65°C for 1 h. DNA was recovered by phenol-chloroform extraction followed by ethanol precipitation. For comparisons between Kdm3a/b-mDKO embryos and control embryos at the E2C stage, we implemented spike-in normalization by adding a constant number of Drosophila S2 cells during cell lysis and 4 pg of Saccharomyces genomic DNA after chromatin elution. Adaptor ligation was performed using NEBNext Ultra II DNA Library Prep kit (NEB) at half-scale, and the libraries were purified by 1.8× SPRIselect beads (Beckman Coulter). The DNA was amplified by KAPA Hifi 2X master PCR mix for 13-15 PCR cycles with dual indexing primers. After purification with 0.9× SPRIselect beads, the samples were digested by I-SceI (5 U/µl) at 37°C for 2 h followed by heat inactivation at 65°C for 20 min and purified by 0.9× SPRIselect beads. DNA libraries were sequenced with paired-end reads on the NextSeq2000 or Novaseq X Plus (illumina).