title: GSM5343843: HYA__932b0e__20210309_mouse_cortex_MCF-7_PC3_1-1_S2; Homo sapiens; Mus musculus; ATAC-seq
library_construction_protocol: Cells were washed in PBS and dissociated into single cell suspensions using 1.5 mL of 0.05% Trypsin. The single-cell suspension was centrifuged at 500 rcf for 5 min at 4°C and the resulting pellet was resuspended in PBS. This PBS wash was repeated once more. A pellet of 1 million dissociated cells or fewer was incubated on ice 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 uM Pitstop, 0.01% Digitonin) for 5 to 7 minutes. 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 Flowmi strainer. 50 000 nuclei were resuspended in 50 μL of ATAC Reaction Mix (10% DMF, 10% Tris-HCl pH 7.4, 5 mM MgCl2, 5 ng/uL Tn5, 70 uM Pitstop, 0.1% Tween-20, 0.01% Digitonin) and incubated at 37 °C for 1 hour without shaking. 100 μL of 0.1% BSA in PBS was added and the nuclei were pelleted at 500 xg, 4 °C for 5 minutes and resuspended in 40 μL of 0.1% BSA in PBS. Tagmented nuclei were added to 100 μL of PCR mix (1.3X Phusion HF Buffer, 15% Optiprep, 1.3 mM dNTPs, 39 mM DTT, 0.065 U/uL Phusion HF Polymerase, 0.065 U/uL Deep Vent Polymerase, 0.013 U/uL ET SSB). PCR mix was co-encapsulated with 35 μL of freshly thawed HyDrop-ATAC beads in RAN oil on the Onyx microfluidics platform. The resulting emulsion was collected in aliquots of 50 μL total volume and thermocycled according to the PCR1 program (72 °C 15 min., 98 °C 3 min., 13 PCR 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). 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 PCR2 mix (1X KAPA HiFi, 1 uM index i7 primer, 1 uM 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).
attributes.tag: GEO Accession
attributes.value: GSM5343843