title: 10XmtATAC_C57BL6_mtPWD_hepatocytes_mixing_2
design: Frozen hepatocytes were thawed, washed with PBS (Gibco, 10010-023) and fixed in 1% PFA for 10 min at RT. After fixation PFA was quenched with glycine (125 mM final concentration) and washed with cold PBS supplemented with 1% BSA (Sigma, A1953). Next, hepatocytes were permeabilized. To this end, 1 million fixed cells were resuspended in 200 ul of lysis solution (0.5% n-Dodecyl β-D-maltoside, 45 mM NaCl, 10 mM Tris-HCl pH 8.0, 5 mM MgCl2, 10% dimethylformamide) and incubated on ice for 5 min. Permeabilization was stopped by adding 1.8 ml of wash buffer (45 mM NaCl, 10 mM Tris-HCl pH 8.0, 5 mM MgCl2, 1% BSA). Permeabilized cells from C57BL6/J and mtPWD mice were divided in two aliquots and each was labeled with unique lipid modified oligo (MULTI-ATAC; Conrad et al., in preparation). To this end, Lignoceric Anchor oligo (2 uM; Sigma, LMO001A) was mixed with a unique barcode oligo (1 uM; BC) and reverse primer (1 uM; BE) at 1:1:1 molar ratio to form Anchor-BC-BE Complex (20x, 1 uM). Note that BC contains an 8-nucleotide-long stretch of random nucleotides to serve as a unique molecular identifier (UMI) to enable barcode counting. Permeabilized hepatocytes were resuspended at 10^6 cells/mL in cold PBS and Anchor-BC-BE Complex was added to cell suspension (final 1x, 50 nM) followed by incubation on ice for 5 min. To stabilize labeling, Palmitic Co-anchor oligo (2uM, 20x; Sigma, LMO001B) was added to the cell suspension (final 1x, 50 nM) followed by additional incubation on ice for 5 min. After MULTI-ATAC barcoding, unbound complexes were washed away with PBS supplemented with 2% BSA, hepatocytes labeled with different MULTI-ATAC barcodes were pooled together, resuspended in diluted nuclei buffer, passed through 35-40um cell strainer, and used to prepare 10X-mtATAC libraries using Chromium Next GEM Single Cell ATAC Reagent kit (10X Genomics, PN-1000176 and PN-1000406) according to the manufactures protocol (CG000209 Rev F and CG000496 Rev B) with 2 minor modifications. First, after step 3.2o 1 ul of the sample was used to prepare the MULTI-ATAC barcode library (described below). Second, the remaining 39 ul were used in step 4.1 where SI-PCR Primer B concentration was increased to 100 uM. Before permeabilization, after permeabilization and after MULTI-ATAC barcoding cells were pelleted by centrifugation at 100g for 3 min, 300g for 3 min and 500g for 5 min, respectively. To prepare MULTI-ATAC barcode libraries, 1ul of sample from 3.2o step was amplified in a PCR reaction: 1 ul of sample, 500nM SI-PCR-B primer, 500nM TruSeq primer, 1x Kapa HiFi HotStart ReadyMix (Roche, KK2601). The reaction mixture was thermocycled using the following conditions: 5 min polymerase activation at 95°C; 14 cycles of denaturation at 98°C for 20 sec, annealing at 67°C for 30 sec and extension at 72°C for 20 sec; incubation at 72°C for 1 min. To quality control and quantify libraries, 1 ul of 1:5 diluted 10X-mtATAC and MULTI-ATAC barcode libraries were run on Bioanalyzer. 10X-mtATAC library was sequenced on NovaSeq6000, S1 200 as 101x12x24x101. MULTI-ATAC barcode library was sequenced on NovaSeq6000, S4 200 as 101x12x24x101. For optimal demultiplexing we aimed to obtain 5,000 MULTI-ATAC barcode reads per cell.
library_name: EKPOF06 and EKPOF10