GSM6958411: HB16hpf_GEX; Danio rerio; RNA-Seq
Tissue was collected in 1XPBS, dissociated by repeated pipetting through a p1000 tip, and centrifuged at 4℃ at 400G for 5 minutes followed by resuspension of the pellet in 500 μl of protease (10 mg/ml BI protease (Sigma, P5380), 125 U/ml DNase, 2.5 mM EDTA in PBS) for 15minutes on ice. Samples were centrifuged at 4℃ at 400G for 5 minutes, resuspended in 1ml HBSS + FBS (2%), filtered through a 20 μm cell strainer (pluriSelect, KL-071912), recentrifuged at 4℃ at 400G for 5 minutes, resuspended in 500 μl of HBSS + FBS (2%) and again filtered through a 20 μm cell strainer. Following centrifugation at 4℃ at 400G for 5 minutes, the cells were resuspended in 200 μl PBS. For nuclei isolation, we followed the 10X Genomics recommended protocol with minor modifications. The dissociated cells were centrifuged at 900G at 4℃ for 5 minutes, resuspended in 100 μl of 0.1X lysis buffer (1 mM Tris-HCl pH7.4, 1 mM NaCl, 0.3 mM MgCl2, 0.1% BSA, 0.01 % Tween-20, 0.01 % NP40, 0.001 % Digitonin (Invitrogen, BN2006), 0.1 mM DTT, 0.1 U/μl RNase inhibitor, in nuclease-free water) and incubated on ice for 5 min. Samples were then washed three times by centrifugation at 4℃ at 900G for 10 min and resuspension in 1 ml of chilled wash buffer (10 mM Tris-HCl pH7.4, 10 mM NaCl, 3 mM MgCl2, 1% BSA, 0.1 % Tween-20, 1 mM DTT, 1 U/μl RNase inhibitor in nuclease-free water). Finally, nuclei were counted and resuspended in 1X nuclei buffer (provided in the 10x Genomics Single Cell Multiome ATAC Kit A; 1 mM DTT, 1 U/μl RNase inhibitor in nuclease free water) at a final concentration of approximately 2,000-3,000 nuclei/μl for sequencing on the 10X scMultiome platform. 10x Genomics Single Cell Multiome ATAC Kit A was used with the standard protocol Targets of 100,000 reads each GEX and ATAC per cell and  >= 2,000 cells per sample.