GSM8894842: ∆stemin, 10-14 h, ATAC; Physcomitrium patens; ATAC-seq
Nuclei were isolated by chopping collected tissue in 3 mL of chopping buffer (1% [w/v] BSA; 10 mM NaCl; 10 mM Tris-HCl, pH 7.5; 3 mM MgCl2; 1 mM DTT; 0.5 mM spermine; 0.5 mM spermidine; 1 U/µL Protector RNase Inhibitor, 1x cOmplete Mini, EDTA-free Protease Inhibitor Cocktail [Roche], 1% [w/v] PVP40; 0.3% [v/v] NP-40; 0.1 % [v/v] Tween 20 in RNase free water) for 3 minutes using a double-edged safety razor (FEATHER) in a 5 mL culture dish on ice. After a 2-minute incubation, the tissue was strained through a 20 µm CellTrics filter (Sysmex). The filter was rinsed with 1 mL of rinsing buffer (1% [w/v] BSA; 10 mM NaCl; 10 mM Tris-HCl, pH 7.5; 3 mM MgCl2; 1 mM DTT; 0.5 mM spermine; 0.5 mM spermidine; 1 U/µL Protector RNase Inhibitor, 1×cOmplete Mini, EDTA-free Protease Inhibitor Cocktail [Roche], 0.5 µg/mL 4',6-diamidino-2-phenylindole [DAPI]). The DAPI stained nuclei were isolated by fluorescence-activated nuclei sorting (FANS) using a using a BD FACSMelody fitted with a 100 µm sorting nozzle. Nuclei were sorted into a collection tube filled with 500 µL of rinsing buffer kept at 4˚C. To ensure equal representation across subsamples, nuclei were pooled during sorting, collecting 20,000 nuclei for each of three subsamples, resulting in a total of 60,000 nuclei per sample. For the two gametophore samples (T00), 60,000 nuclei for each line were used repectively. Collected nuclei were centrifuged at 500×g for 3 minutes with the centrifuge brake turned off in a swing-bucket centrifuge. Excess buffer was carefully removed, leaving a pellet of nuclei in approximately 10 µL of buffer. Nuclei were gently mixed in the remaining buffer by pipetting with a wide bore tip. Library construction was done in accordance to the user guide (CG000338 Rev E) tailored for the Chromium Next GEM Single Cell Multiome ATAC + Gene Expression Kit.