GSM8182080: Lung_ILC2_ChIPseq_PBS_Mother; Mus musculus; ChIP-Seq
Lung tissues were digested in RPMI enzyme solution containing collagenase D (2 mg/ml), DNase I (0.1 mg/ml), and Dispase II (0.2 mg/ml, Roche 04942078001) at 37°C for 40 min (for adults) or 20 min (for fetuses). Digestion was performed by changing the solution every 20 min. And then, lung tissues were digested in RPMI enzyme solution containing collagenase D (2 mg/ml), DNase I (0.1 mg/ml), Dispase II (0.2 mg/ml) and Liberase TH (0.01 mg/ml, Roche 5401151001) at 37°C for 20 min. The digested samples were filtered through 70 µm strainers and suspended in 36% Percoll (Cytiva, 17-0891-01) diluted with RPMI, 72% Percoll diluted with PBS was injected into the bottom and centrifuged at 800 × g for 20 min at 25°C. Cells were collected in the interlayer between 36% and 72% Percoll, washed with RPMI supplemented with FBS (2% v/v), and stained for flow cytometry. In fetal lung cell collection, ILC2 (CD45+, Lin-, ST2+), immune cells (CD45+) and epithelial cells (EpCAM+) were purified using FACSMelody (BD Biosciences). Snap frozen cell pellets from approximately 3,000 ILC2 with 2-3 µL carryover were lysed by 17 µL of cold Nuclei EZ lysis buffer (Sigma, NUC-101) supplemented with complete EDTA-free protease inhibitor cocktail and 1 mM phenylmethanesulfonyl fluoride. Then, 1 µL of 1% Triton X-100 (Merck 93443) and 1% deoxycholate (Nacalai, 10712-54) mixture solution was added to the samples, and sit on ice for 5 min. The chromatin was fragmented by 4 U/µl MNase (M0247S, NEB) in 1xMNase buffer supplemented with 1% PEG6000 (HAMPTON RESEARCH, HR2-533) and 2 mM DTT (Nacalai) at 37C for 7.5 min. The MNase reaction was stopped by adding 1/10 volume of 100 mM EDTA and 1/12 volume of the 1% Triton X-100 and 1% deoxycholate mixture, and the samples were rest on ice for 15 min. The chromatin lysates were then added with freshly prepared immunoprecipitation buffer (Brind'Amour et al. 2015), and 5% volume was kept for input library construction. To the rest of the lysate, 30 ng of annealed I-SceI carrier DNA was added (Zhu et al. 2021). The forward and reverse strands of the carrier DNA were as follows: /5AmMC6/Gtagggataacagggtaattagggataacagggtaattagggataacagggtaattagggataacagggtaattagggataacagggtaattagggat aacagggtaat*c/3AmMO/ and /5AmMC6/Gattaccctgttatccctaattaccctgttatccctaattaccctgttatccctaattaccctgttatccctaattaccctgttatccctaattaccctgttatcc cta*c/3AmMO/, respectively, where asterisks represent phosphorothioate bonds. The oligos were synthesized by Integrated DNA Technologies. For each immunoprecipitation reaction, 0.25 µL of rabbit anti-H3K4me3 (Active Motif 39159) antibody conjugated to precleared Dynabeads Protein A (Thermo Fisher Scientific, 10006D) and G (Thermo Fisher Scientific, 10007D) mixture was used. After immunoprecipitation at 4C overnight, the chromatin-Dynabeads were washed twice each by the low and high salt wash buffers, and the chromatin was eluted in the freshly prepared ChIP elution buffer at 65C for 1 hr (Brind'Amour et al. 2015). DNA was recovered by phenol-chloroform extraction followed by ethanol precipitation. Adaptor ligation was performed by NEBNext Ultra II DNA Library Prep Kit for Illumina (E7645, NEB) in a half scale of the manufacturer's instruction, and the libraries were purified by 1.8x SPRIselect beads (B23318, Beckman Coulter). The DNA was amplified by KAPA Hifi 2X mater PCR mix (KK2605) for 16 PCR cycles with dual indexing primers (NEBNext Multiplex Oligos for Illumina, E6440). After purification with 0.9x SPRIselect beads, the samples were digested by I-SceI (5 U/µl, NEB, R0694) 37C for 2 hrs followed by heat inactivation at 65C for 20 min and purified by 0.9x SPRIselect beads. The second amplification was not performed.