GSM8286088: scATAC_mEBs_d21_crispri_rep2_lane_b; Mus musculus; ATAC-seq
Single-nuclei preparation for scATAC-seq were prepared from day 21 mEB as follows: At day 21 of mEB cultures, mEBs (2X 10cm suspension plates) are collected into a 50 mL conical tube and washed 2X with 1X PBS (w/o Ca, Mg). After consecutive PBS washes, mEBs are treated with 1.5mL of 0.25% trypsin and incubated in 37C bath with gentle agitation (steady concentric swirls of 50mL concial) for 3 minutes. For further dissociation, mEBs are then gently titurated 10X with a p1000 pipette. Again, incubate at 37C for 3 minutes with gentle agitation. After second incubation, mEBs are gently titurated 10X with a p1000 pipette. Inactivate trypsin digestion with CA medium and filter single cell suspension through a 100um filter into new 50mL conical. Count single cell suspension and aim for >>100K total cells. Spin 50mL conical tube at 300g for 5 minutes. After removing supernatant, wash 1X with 1mL of 1X PBS + 0.04% BSA and gently pipette mix 5X. Transfer to a 1.5 mL tube and spin at 300g for 5 min at 4C. Wash again with 1mL of 1X PBS + 0.04% BSA. Again,  spin at 300g for 5 min at 4C. Proceed with 10x "Nuclei isolation for Single Cell ATAC Sequencing" protocol. scATAC-seq libraries were prepared following 10x Genomics protocol in "Chromium Next GEM Single Cell ATAC Reagent Kits v1.1".  Read structure: read 1 50 cycles, index 1 8 cycles, read 2 50 cycles, index 2 16 cycles