title: GSM6745651: 10x scATAC-seq on the mouse liver (0d3236); Mus musculus; ATAC-seq
library_construction_protocol: For scATAC-seq we used a modified protocol from the Nuclei Isolation for Single Cell ATAC Sequencing (CG000169) from 10x Genomics. In brief, 200 mg of fresh mouse liver tissue was minced and transferred to a Dounce homogenizer cylinder containing 1 mL of ice-cold homogenization buffer (10 mM NaCl, 10 mM Tris-HCl (pH 7.4), 3 mM MgCl2, 0.1% Tween-20, 0.1% IGEPAL CA-63, 0.01% Digitonin, 1% BSA) and incubated for 5 min on ice. Next the tissue was homogenized with 15 strokes of pestle A and 15 strokes of pestle B until a homogeneous nuclei suspension was achieved. The resulting homogenate was filtered through a 70-μm cell strainer (Corning). The tissue material was spun down at 500 x g for 5 min and the supernatant was discarded. The tissue pellet was resuspended in 1 mL wash buffer (20 mM NaCl, 20 mM Tris-HCl (pH 7.4), 6 mM MgCl2, 1% BSA). The wash step was repeated one more time and the resulting final pellet was resuspended in 100 µl Diluted Nuclei Buffer (10x Genomics scATAC kit). 9 μL of sample was mixed with 1 μL of arginine orange/propidium iodide (AO/PI) stain, loaded onto a LUNA-FL slide and visualized with the LUNA-FL Automated cell counter for nuclei yield, morphology, and presence of clumps/debris. Single-nuclei libraries were generated using the 10x Chromium Single-Cell Instrument and Single Cell ATAC v1 kit (10x Genomics) according to the manufacturer's protocol. In brief, the single mouse liver nuclei were incubated for 60 min at 37°C with a transposase that fragments the DNA in open regions of the chromatin and adds adapter sequences to the ends of the DNA fragments. After generation of nanolitre-scale gel bead-in-emulsions (GEMs), GEMs were incubated in a C1000 Touch Thermal Cycler (Bio-Rad) under the following program: 72°C for 5 min; 98°C for 3 s; 12 cycles of 98°C for 10 s, 59°C for 30 s, 72°C for 1 min; 72°C for 1 min; and hold at 4°C. Incubation of the GEMs produced 10x barcoded DNA from the transposed DNA. Next, single-cell droplets were dissolved, and the transposed DNA was isolated using Cleanup Mix containing Silane Dynabeads. Illumina P7 sequence and a sample index were added to the single-strand DNA during ATAC library construction via PCR: 98°C for 45 s; 9 cycles of 98°C for 20 s, 67°C for 30 s, 72°C for 20 s; 72°C for 1 min; and hold at 4°C. The sequencing-ready ATAC-seq library was cleaned up with SPRIselect beads (Beckman Coulter). Before sequencing, the fragment size of every library was analyzed using the Bioanalyzer high-sensitivity chip and were sequenced on NextSeq500 instruments (Illumina) with the following sequencing parameters: 50 bp read 1 – 8 bp index 1 (i7) – 16 bp index 2 (i5) - 49 bp read 2.