title: GSM5343849: HYA__combined__20210323_cortex_phu_dv_etssb_1-5_S5; Mus musculus; ATAC-seq
library_construction_protocol: Cortex was separated from female P56 BL/6 mouse brains, immediately snap-frozen in liquid nitrogen and preserved in -80 °C. A ~1 cm3 frozen piece of mouse cortex tissue was transferred to 1 mL of ice-cold homogenisation buffer (320 mM Sucrose, 10 mM NaCl, 10 mM Tris-HCl pH 7.5, 3 mM MgAcetate, 1 mM DTT, 0.1 mM EDTA, 0.1% IGEPAL, 1X cOmplete Protease Inhibitor Cocktail) in a Dounce homogenizer mortar and thawed for 3 minutes. The tissue was homogenised with 10 strokes of pestle A and 5 strokes of pestle B until a homogeneous cell suspension was achieved. The resulting homogenate was filtered through a 40 μm Flowmi strainer. The mortar was washed with 1.65 mL of ice-cold homogenisation buffer to collect any remaining cells. 2.65 mL of ice-cold gradient medium was added to 1.65 mL of homogenate. 4 mL of 29% iodoxanol cushion (129.2 mM Sucrose, 77.5 mM KCl, 15.5 mM MgCl, 31 mM Tris-HCl pH 7.5, 29% Iodoxanol) was loaded into an ultracentrifuge. 5.3 mL of sample was gently layered on top of the 29% iodoxanol cushion without mixing both phases. Sample was centrifuged at 10 000 xg, 4°C for 30 minutes and the supernatant was gently removed without disturbing the nuclei pellet. Nuclei were resuspended in 100 μL of Nuclei buffer. 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: GSM5343849