GSM7102973
Cryopreserved human PBMCs from a male and female donor were puchased from AllCells and (AllCells, Lot #3030456 and 3030663). Cryopreserved PBMC were thawed according to the 10x Genomics demonstrated protocol CG00039 ("Fresh Frozen Human Peripheral Blood Mononuclear Cells for Single Cell RNA Sequencing"). Briefly, 1 mL of frozen cells was rapidly thawed in a 37 °C water bath and transferred to a 50 mL tube using a 1000 µL wide bore tip. Next, 1 mL of 37 °C pre-warmed medium supplemented with 10% FBS (Thermo Fisher Scientific) was added drop-wise while gently swirling the sample. After 1 minute of incubation at room temperature, 2, 4, 8 and 16 mL of medium with 10% FBS were added drop-wise with 1 minute of incubation at room-temperature inbetween. The cell suspension was then centrifuged at 300 g for 5 min. at room temperature. The pellet was resuspended in 10 mL of medium with 10% FBS, and cells were counted. Unless specified otherwise in technology-specific methods sections, nuclei isolation was performed according to the 10x Genomics demonstrated protocol “Nuclei Isolation for Single Cell ATAC Sequencing”. Briefly, 1 million cells from the cell mix were transferred to a 1.5 mL microcentrifuge tube and centrifuged at 500 rcf for 5 min at 4 °C. The supernatant was removed without disrupting the cell pellet and 100 µL of chilled Lysis Buffer (10 mM Tris-HCl (pH 7.4); 10 mM NaCl; 3 MgCl2; 0.1% Tween-20; 0.1% Nonidet P40 Substitute; 0.01% Digitonin and 1% BSA) were added and pipette mixed 10 times. Samples were then incubated on ice for 3 min. Following lysis, 1 mL of chilled Wash Buffer (10 mM Tris-HCl (pH 7.4); 10 mM NaCl; 3 MgCl2; 0.1% Tween-20 and 1% BSA) was added and pipette mixed. Nuclei were centrifuged at 500 rcf for 5 min at 4 °C and the supernatant removed without disrupting the nuclei pellet. Based on the starting number of cells and assuming a 50% loss during the procedure, nuclei were resuspended into the appropriate volume of chilled Diluted Nuclei Buffer (10x Genomics) in order to achieve a nuclei concentration of 925-2300 nuclei/µL, suitable for a Target Nuclei Recovery of 3000. HyDrop was performed as previously described (De Rop et al. 2022), but with an updated barcoded hydrogel bead design and minor improvements in nuclei handling. Briefly, barcoded hydrogel beads were produced as described previously, but using 384 x 384 combinations of primers instead of the original method's 96 x 96 x 96, resulting in a barcode sequence of 30 instead of 50 base pairs. 1 million PBMCs was counted, pelleted and resuspended in 200 μL of ATAC Lysis Buffer (1% BSA, 10 mM Tris-HCl pH 7.5, 10 mM NaCl, 0.1% Tween-20, 0.1% NP-40, 3 mM MgCl2, 70 μM Pitstop in DMSO, 0.01% Digitonin) for 5 min. on ice. 1 mL of ATAC Nuclei Wash Buffer (1% BSA, 10 mM Tris-HCl pH 7.5, 0.1% Tween-20, 10 mM NaCl, 3 mM MgCl2) was added and the nuclei were pelleted at 500 xg, 4 °C for 5 minutes. The resulting pellet was resuspended in 100 μL of ice-cold PBS and filtered with a 40 μm strainer (Flowmi). 25 000 PBMC nuclei were resuspended in 25 μL of ATAC Reaction Mix (10% DMF, 10% Tris-HCl pH 7.4, 5 mM MgCl2, 5 ng/μL Tn5, 70 μM Pitstop in DMSO, 0.1% Tween-20, 0.01% Digitonin) and incubated at 37 °C for 1 hour without shaking. To recover a targeted 3000 nuclei, 5625 tagmented nuclei were added to 48 μL of PCR mix (1.3X Phusion HF Buffer, 15% Optiprep, 1.3 mM dNTPs, 39 mM DTT, 0.065 U/μL Phusion HF Polymerase, 0.065 U/μL Deep Vent Polymerase, 0.013 U/μL ET SSB). PCR mix was co-encapsulated with 35 μL of freshly thawed HyDrop-ATAC beads in HFE-7500 Novac oil with EA-008 surfactant (RAN Biotech) on the Onyx microfluidics platform (Droplet Genomics). The resulting emulsion was collected in aliquots of 25 μL total volume and thermocycled according to the linear amplification program (72 °C 15 min., 98 °C 3 min., 12 amplification cycles of [98 °C 10 s, 63 °C 30 s, 72 °C 1 min.], followed by a final hold on 4 °C). 125 μL of recovery agent (20% PFO in HFE), 55 μL of GITC Buffer (5 M GITC, 25 mM EDTA, 50 mM Tris-HCl pH 7.4) and 5 μL of 1 M DTT was added to each separate aliquot of 50 μL thermocycled emulsion and incubated on ice for 5 minutes. 5 μL of Dynabeads was added to the aqueous phase and incubated for 10 minutes. Dynabeads were pelleted on a Nd magnet and washed twice with 80% EtOH. Elution was performed in 50 μL of EB-DTT-Tween (10 mM DTT, 0.1% Tween-20 in EB (10 mM Tris-HCl, pH 8.5)). A 1x Ampure bead purification was performed according to manufacturer's recommendations. Elution was performed in 30 μL of EB-DTT (10 mM DTT in EB). Eluted library was further amplified in 100 μL of PCR mix (1x KAPA HiFi, 1 μM index i7 primer, 1 μM index i5 primer). Final library was purified in a 0.4x-1.2x double-sided Ampure purification and eluted in 25 μL of EB-DTT (10 mM DTT in EB) and quality-controlled on an Agilent Bioanalyzer High Sensitivity chip (Agilent Technologies).