GSM5738222: Lane3-mixed sample [AdiposeVSG.L3]; Mus musculus; ATAC-seq
Whole adipose depots were finely minced on dry ice. Then 80mg of adipose tissue was transferred to a pre-chilled Dounce homogenizer (Wheaton #357546) with 30 mL of HB (320 mM sucrose, 0.1 mM EDTA, 0.1% NP40, 5 mM CaCl2, 3 mM Mg(Ac)2, 10mM Tris pH7.4, protease inhibitors (Pierce #88666), 0.016 mM PMSF). Samples were processed in groups of two (one SHAM and one VSG) to ensure any variation due to library preparation is equally applied to both SHAM and VSG. All steps were performed on ice or at 4°C. The tissue was first homogenized with 10-15 strokes using a loose pestle then an additional 10 strokes with the tight pestle. The homogenized tissue was then filtered through a 70 µm cell strainer (BD Biosciences #352350) and centrifuged for 5 mins at 500g in a centrifuge with a swinging bucket rotor. The supernatant was removed, the cell pellet washed with ATAC-RSB (10 mM Tris-HCL pH 7.4, 10 mM NaCl, 3 mM MgCl2, H2O), then centrifuged for 10 minutes at 750g. After removing the supernatant, nuclei were resuspended in 1 mL of OMNI-ATAC buffer (10 mM Tris pH7.4, 5 mM MgCl2, 10% DMF, 33% 1x PBS(no Ca++ or Mg++), 0.1% Tween-20, 0.01% Digitonin (ThermoFisher #BN2006)), counted on a hemacytometer, and the concentration was adjusted to 100,000 nuclei/mL. The snATAC library preparation protocol was based on Preissl et al. and Spektor et al. with some modifications (62,63). For tagmentation with Tn5 transposase, 8 µL of the nuclei suspension was added to each well of a 96 well plate. Then, 1 µL of ME-C barcoded transposome loaded Tn5 and 1 µL of ME-D barcoded transposome loaded Tn5 were added to each well. Tagmentation was carried out at 50°C for 30mins. 10 µL of 40 µM EDTA was added to each well, gently vortexed, and incubated for 15 minutes at 37°C to inactivate Tn5 transposase. 20µL of sorting buffer (2% BSA, 2mM EDTA in PBS (no Ca++ or Mg++)) was added to each well, and all wells were combined. For the freeze-thaw test, nuclei were centrifuged for 5 mins at 500g and resuspended in 500 µL of freeze buffer (10% DMSO, 90% sort buffer) and slowly frozen and stored at -80°C. Nuclei were stained with Draq7(1:100; Abcam #ab109202), filtered through a 30 µm filcon cell strainer (BD Biosciences #340597), and 25 nuclei were sorted into each well of a 96 well plate containing 16.5 µL of sort EB (10mM Tris pH 8, 2% BSA, H2O) per well using a BD FACSAria Fusion. For PCR amplification, 2 µL of 0.2% SDS was added to each well and incubated for 7 minutes at 55°C. Next, 2.5 µL of 10% Triton X-100, 2 µL of 25 µM Primer i5, 2 µL of 25 µM Primer i7, and 25 µL of 2X PCR mix with Q5 DNA polymerase (NEB #M0491S) (Q5 5X buffer, dNTP, Q5 enzyme, GC enhancer, H2O) was added to each well. All PCR reactions were run in the same thermocycler with the following program: 72°C 5 minutes, 98°C 30 seconds, 15 cycles of (98°C 10 seconds, 63°C 30 seconds, 72°C 1 minute), and 72°C 5 minutes. After PCR, all wells were pooled, then 24 mL buffer PB (5:1, Qiagen #19066) and 1.2mL NaOAc were added sequentially. Samples were each run through a separate MinElute column (Qiagen #28004) using a QIAvac apparatus (Qiagen #19413), then washed twice with 750 mL Buffer PE (Qiagen #19065). The sample was then eluted in 40 uL of Buffer EB (10 mM Tris pH 8) and centrifuged at max speed on a tabletop centrifuge for 2 minutes. The following size selection steps were performed at room temperature. First, large fragments were removed with 0.5X vol AMPure XP (Beckman Coulter #A63880) purification, and then size selected 3 times with 1.5X vol AMPure XP consecutively to remove PCR primers according to the manufacturer's protocol.