title: GSM8248521: Reprogrammed bile duct cells, DDC diet, Tomato positive; Mus musculus; ATAC-seq
library_construction_protocol: Nuclei isolation was performed using the Omni-ATAC protocol for isolation of nuclei from frozen tissues. Nuclei were resuspended in Whole Cell Tagmentation Mix containing 0.1% Tween-20, 0.01% Digitonin, 1X PBS supplemented with 0.1% BSA, ATAC Tagmentation Buffer and ATAC Tagmentation Enzyme (parts of the SureCell ATAC-Seq Library Prep Kit - 17004620, Bio-Rad). Cells were mixed and agitated on a ThermoMixer for 30 min at 37°C. Tagmented cells were kept on ice prior to encapsulation. Tagmented nuclei were loaded onto a ddSEQ Single-Cell Isolator (12004336, Bio-Rad). Single-cell ATAC-Seq libraries were prepared using the SureCell ATAC-Seq Library Prep Kit (17004620, Bio-Rad) and SureCell ddSEQ Index Kit (12009360, Bio-Rad). Bead barcoding and sample indexing was performed in a C1000 Touch™ Thermal cycler with a 96-Deep Well Reaction Module (1851197, Bio-Rad): 37°C for 30 min, 85°C for 10 min, 72°C for 5 min, 98°C for 30 sec, 8 cycles of 98°C for 10 sec, 55°C for 30 sec, and 72°C for 60 sec, and a single 72°C extension for 5 min to finish. Emulsions were broken and products cleaned up using Ampure XP beads (A63880, Beckman Coulter). Barcoded ATAC amplicons were further amplified using a C1000 Touch™ Thermal cycler with a 96-Deep Well Reaction Module: 98°C for 30 sec, 6-9 cycles (cycle number depending on the cell input, Section 4 Table 3 of the User Guide) of 98°C for 10 sec, 55°C for 30 sec, and 72°C for 60 sec, and a single 72°C extension for 5 min to finish. PCR products were purified using Ampure XP beads and quantified on an Agilent Bioanalyzer (G2939BA, Agilent) using the High-Sensitivity DNA kit (5067-4626, Agilent). Libraries were loaded at 1.5 pM on a NextSeq 550 (SY-415-1002, Illumina) using the NextSeq High Output Kit (150 cycles; 20024907, Illumina) and sequencing was performed using the following read protocol: Read 1 118 cycles, i7 index read 8 cycles, and Read 2 40 cycles. A custom sequencing primer is required for Read 1 (16005986, Bio-Rad; included in the kit).