GSM8809082: Multiome, Ctrl-GEX; Mus musculus; RNA-Seq
Lumbar segments from the P100~P120 Sun1sfGFP; ChAT-Cre; Mettl14floxed (cKO) and Sun1sfGFP; ChAT-Cre (control) spinal cord were dissected based on morphology and dissociated two at a time in 2 mL pre-chilled lysis buffer with a 7 mL Dounce Homogenizer (pestle A: eight strokes; pestle B: 5 strokes). An additional 3 mL lysis buffer was added, and the entire homogenate was filtered through a 100 µm strainer. Then, 5 mL of 50 % iodixanol was added to create a 10 mL suspension of 25 % iodixanol. The suspension was inverted to mix well before equally splitting into two 15 mL tubes. Iodixanol (40 %, 2 mL) was layered gently at the bottom of the homogenate and centrifuged at 1,000 g for 12 minutes at 4 oC, with a swing bucket centrifuge (brand). The interphase nuclei were transferred to a new 15 mL tube and resuspended with 1:1 volume of 10 X wash buffer (10 mM Tris-HCl pH7.4, 10 mM NaCl, 3 mM MgCl2, 1 % BSA, 0.1 % Tween 20, 1 mM DTT, and 1 U/µL RNase inhibitor), followed by centrifuging at 500 g for 10 minutes at 4 oC. The supernatant was discarded and the pellet was resuspended in 800 µL diluted nuclei buffer (20X Nuclei Buffer from Chromium Next GEM Single Cell Multiome ATAC + Gene Expression Kit supplemented with 1 mM DTT, and 1 U/µL RNase inhibitor), before conducting FANS (fluorescence-activated nuclei sorting) using a BD FACS Aria III cell sorter (BD BioSciences, USA). GFPon nuclei were collected in diluted nuclei buffer and centrifuged at 500 g for 7 minutes at 4 oC. Nuclei were adjusted to an appropriate concentration following the manufacturer's protocol (10x Genomics, 1000285) with diluted nuclei buffer. Then the sorted nuclei from Sun1sfGFP; ChAT-Cre; Mettl14floxed (cKO) and Sun1sfGFP; ChAT-Cre (control) were procced for 10x Genomics Chromium Next GEM single Cell Multiome ATAC+Gene expression experiment. single-nucleus multiome libraries were prepared following the Chromium Next GEM Single Cell Multiome ATAC + Gene Expression protocol CG000338 Rev F (10X Genomics, Pleasanton, CA).