GSM8885705: mouse_cortex_HyDrop_v2_D_beads_4; Mus musculus; ATAC-seq
Mouse: The nuclei were extracted from mouse cortex samples that were snap-frozen in liquid nitrogen and stored at -80 °C. We transferred ~1 cm3 of frozen mouse cortex to 500 μL of ice-cold homogenization buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 3 mM MgCl2, 320 mM sucrose, 0.1 mM EDTA, 0.5% BSA, 0.1% IGEPAL CA-630, 1× complete protease inhibitor, and 1 mM DTT) in a Dounce Homogenizer. The tissue was left to thaw for 2 min before the tissue was homogenized with 10 strokes of the loose pestle and 5 strokes of the tight pestle. Before the end of a 5-minute incubation, the homogenate was filtered through a 70 μm cell strainer (Corning), and the homogenizer and strainer were washed with homogenization buffer to obtain a final volume of 1 mL. The filtrate was transferred to a 1.5 mL DNA LoBind tube (Eppendorf) and centrifuged at 500 xg for 5 minutes. The supernatant was discarded, and the pellet was topped up to 520 μL with wash buffer 1 (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 3 mM MgCl2, 320 mM sucrose, 0.1 mM EDTA, 0.5% BSA, 1× complete protease inhibitor, and 1 mM DTT) and resuspended. Next, we added 520 μL gradient medium (50% Optiprep, 10 mM Tris-HCl pH 7.5, 1 mM CaCl2, 5 mM MgCl2, 75 mM sucrose, 1× complete protease inhibitor, and 1 mM DTT) and mixed the entire volume by gentle pipetting. In a 2 mL DNA LoBind tube we layered 790 μL Optiprep cushion (29% Optiprep, 31 mM Tris-HCl pH 8, 77.5 mM KCl, 15.5 mM MgCl2, 129.2 mM sucrose) at the bottom and 1040 μL of the sample on top, gently without disrupting the cushion. The tube was then centrifuged at 9000 xg for 20 minutes at 4 °C. After the centrifugation the debris on top was carefully removed with a 1 mL tip and the rest of the supernatant was also carefully removed until 50 μL was left in the tube. The pellet was gently resuspended before we mixed it with 50 μL 0.2× permeabilization buffer (20 mM Tris-HCl pH 7.5, 20 mM NaCl, 6 mM MgCl2, 0.2% Tween-20, 0.02% IGEPAL CA-630, 0.02% Digitonin, 2% BSA, and 2 mM DTT) and left the nuclei suspension for 2 minutes on ice before we stopped the permeabilization by mixing it with 1 mL of wash buffer 2 (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 3 mM MgCl2, 0.1% Tween-20, 1% BSA). The nuclei were pelleted by centrifugation at 500 xg for 5 minutes at 4 °C. The nuclei were resuspended in PBS with 0.04% BSA. For Drosophila, the nuclei were isolated by using an adapted protocol based on the nuclei isolation protocol for single-cell ATAC sequencing (10x Chromium CG000169-RevE).  Similar to mouse, for the barnyard, the cell lines were mixed to equal parts and a pellet of one 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 μM Pitstop in DMSO, 0.01% digitonin) for 5 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 strainer (Flowmi). Nuclei, resuspended in PBS (supplemented with 0.04% BSA), were tagmented for one hour at 37 °C in 15 μL ATAC reaction mix (10% DMF, 10 mM Tris-HCl pH 7.4, 5 mM MgCl2, 5 ng/µL Tn5, 70 µM Pitstop in DMSO, 0.1% Tween-20, 0,01% Digitonin). Tagmented nuclei were placed on ice and 95 µL of PCR mix (1.25× Phusion HF buffer, 15% Optiprep, 1.25 mM dNTPs, 75 mM DTT, 0.0625 U/µL Phusion HF polymerase, 0.0625 U/μL Deep Vent polymerase, 0.0125 U/μL ET SSB) was added. Next, 110 μL of nuclei mixed in PCR mix was co-encapsulated with 45 μL of HyDrop-ATACv2 beads in HFE-7500 Novac oil with EA-008 surfactant (RAN Biotech) using the Onyx microfluidics platform (Droplet Genomics). The resulting emulsion was split over two PCR tubes and was placed in a thermocycler with the following program: 72 °C – 15 mins, 98 °C – 3 mins, 12 cycles of 98 °C – 10 s; 55 °C – 30 s; 72 °C – 1 min; 72 °C – 5 mins, and finally the amplification products were cooled to 4 °C. To break the emulsion and remove excess oil, 125 μL of recovery agent (20% PFO in HFE) was added and the PCR tubes were inverted 10 times to mix before the oily phase was removed. Next, we added 180 μL GITC mix (5M GITC, 25 mM EDTA, 50 mM Tris-HCl pH 7.4), 10 μL Dynabeads, and 10 μL 2.4 M DTT to each of the aliquots, and mixed everything by pipetting up and down 10 times and incubating for 10 minutes. Dynabeads were pelleted on a magnet and washed twice with 80% ethanol. Elution was done in 50 μL of EB-DTT-Tween (24 mM DTT, 0.1% Tween-20 in EB (10 mM Tris-HCl pH 8.5)). Next, a 1.2× Ampure bead purification was performed according to manufacturer's recommendations. Elution was done with 40 μL of EB-DTT (10 mM DTT in EB). The library was completed by amplifying the eluate in a total volume of 100 μL PCR mix (1× KAPA HiFi, 1 μM index i7 primer, 1 μM Universal P5 primer) with the following program: 95 °C – 3 mins, 12 cycles of 98 °C – 10 s; 63°C – 30 s; 72 °C – 1 min; 72 °C – 1 min, and finally a hold stage at 4 °C. The final libraries were subjected to 0.4×–1.2× doublesided Ampure purification and eluted in 20 μL elution buffer.