The present invention discloses a method for extracting corn
endosperm kernels suitable for ATAC-seq sequencing. The method comprises the following steps: (1) removing the seed coat and
embryo from corn seeds, collecting the
endosperm, and storing it at -80°C for a long time; taking approximately 5 g of the
endosperm and manually
grinding it into
powder in
liquid nitrogen; (2) adding 10 mL of
sucrose buffer to each
powder sample, mixing it thoroughly, and then filtering it through a 100 μm pore
sieve; (3) passing the filtrate from step (2) through a 38 μm pore mesh, and then centrifuging the product at 700 × g for 5 minutes to collect the supernatant; (4) filtering the supernatant through a 38 μm
sieve and centrifuging it at 2000 × g for 5 minutes. The cells were centrifuged at 1000 × g for 5 minutes, the supernatant was removed, and the precipitate was resuspended in 10 mL of
sucrose buffer; (5) the cells were filtered through a 38 μm
sieve again, centrifuged at 2000 × g for 5-10 minutes, the supernatant was removed, and the precipitate was resuspended in 5 mL of resuspension buffer; (6) the cells were centrifuged at 1000 × g for 5 minutes, the supernatant was discarded, the precipitate was suspended in 5 mL of PBS solution containing
protease inhibitors, 10-20 μL of the solution was stained with
DAPI at equal volumes for 10 minutes, and the cells were observed under a
fluorescence microscope. The endosperm nuclei prepared by the present invention are numerous and highly active, which can overcome the influence of high
starch content in storage tissues, provide strong
technical support for subsequent single-
cell sequencing,
CUT&TAG, ATAC-seq and other related studies, and the extraction effect will also have an important
impact on the experimental data.