title: GSM8630440: HepG2, 0.1% FA + Cryopreservation, DirectATAC; Homo sapiens; ATAC-seq
library_construction_protocol: Cells were pelleted and resuspended in 1 ml of fixative buffer (0.1% Formaldehyde in DMEM) and incubated at room temperature for 5 minutes. Afterwards, cells were pelleted and washed in DMEM + 0.1% BSA, pelleted again, and either snap-frozen by submerging the tubes in liquid nitrogen or cryopreserved. Cells and nuclei were permeabilized in lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% Igepal CA-630, 0.1% Tween-20, 0.01% Digitonin, 1% BSA) and incubated for 5 minutes on ice. Ice-cold wash buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% Tween-20, 1% BSA) was added and mixed by inversion. Nuclei were pelleted and resuspended inin 1x Nuclei Dilution Buffer (10x Genomics). Transposition mix for multiplexed scATAC (comprising 7.5 μl of 2x Diagenode Tn5 reaction buffer and 1.5 μl of 10x Genomics 1x Nuclei Dilution Buffer) was added. To each transposition reaction, 1 μl of custom barcoded Tn5 at a 1:4 dilution was added, ensuring the selection of Tn5 barcodes to avoid overlap after pooling. All reactions were mixed by pipetting and incubated at 37°C for 30 minutes.Transposition was quenched by adding 1 μl of 300 mM EDTA, followed by incubation at 37°C for 15 minutes. The reactions were then cooled and pooled on ice. The pooled cells were centrifuged, washed and resuspended with 1x Nuclei Dilution Buffer. 15 μl of the diluted cell suspension were aliquoted into a PCR tube strip on ice for loading into the Chromium Controller, with a total of 15,600 cells loaded according to 10x Genomics recommendations. The single-cell ATAC v2 protocol was then continued from step 2.1 of the manufacturer's manual (Chromium Next GEM Single Cell ATAC Reagent Kits v2 User Guide, CG000496 rev B).  For final PCR amplification at step 4.1 of the User Guide, the "individual single index set N" was replaced with 2.5 μl of 25 μM custom oligo (MUXscATAC_i7_n, sequences in Additional File 1), selecting library indices appropriately to prevent equal index assignment. Library amplification was performed using 9 PCR cycles.