GSM6997754: Control1, snATAC-seq; Homo sapiens; ATAC-seq
Frozen human liver samples were homogenized in 0.1X lysis buffer (10mM Tris-HCl, 10mM NaCl, 3mM MgCl2, 1% BSA, 1mM DTT, 0.1% Tween-20, 0.1% NP-40, 0.01% Digitonin, 1U/μl RNase inhibitor) using a Dounce Homogenizer (Active Motif, 40401), followed by 5 min of incubation on ice.  Nuclei quality was examined under microscope after this step.  Next, nuclei were centrifuged at 500 rcf for 5 min at 4°C.  Supernatant was carefully disregarded, and nuclei was resuspended in 500ul pre-chilled wash buffer containing 10mM Tris-HCl, 10mM NaCl, 3mM MgCl2, 1% BSA, 1mM DTT, 0.1% Tween-20, and 1U/μl RNase inhibitor.  The washing step was repeated twice.  After washing, all nuclei passed through 40um Flowmi cell strainer (Bel-Art, H13680-0040). Library was performed according to the manufacture's instructions (Single Cell Multiome ATAC + Gene Expression, 10x Genomics). Briefly, Nuclei were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). Incubation of GEMs produces Barcoded transposed DNA (for ATAC) which contains an illumina P5 sequence, a 16 nt 10x Barcode and a Spacer sequence. In the same partition, cDNA (for GEX) from poly-A mRNA contains illumine R1 primer sequence, 16 nt 10x Barcode, 12 nt unique molecular identifier (UMI). During the ATAC library construction, P7 and a sample index i7 were added to transposed DNA (for ATAC). The pooled barcoded cDNA (for GEX) was then cleaned up with Silane DynaBeads, amplified by PCR and the appropriated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the GEX library construction illumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and sample indexes i5 and i7 were added.