GSM8526529: smart-Seq3 transcriptomic data; Mus musculus; RNA-Seq
To purify nuclei, we used a modified density gradient centrifugation protocol similar to those previously published.142–144 “Buffer A” consisted of OptiPrep™ 60% iodixanol (Sigma-Aldrich, D1556). “Buffer B” consisted of 900 mM KCl (Invitrogen, AM9640G), 30 mM MgCl2 (Invitrogen, AM9530G), 120 mM Tricine-KOH (pH = 7.8) (Thermo Scientific, 15426389) in UltraPure™ water (Invitrogen, 10977015). “Buffer C” consisted of 5 volumes of Buffer A, 1 volume of Buffer B, 0.5 mM spermidine (Thermo Scientific, A19096.03), 0.15 mM spermine (Sigma-Aldrich, S3256), 1 mM DTT (Thermo Scientific, R0861), and 20 units/mL SUPERase•In™ RNase inhibitor (Invitrogen, AM2694), and 1 tablet per 10 mL of  cOmplete™ EDTA-free Protease Inhibitor Cocktail (Roche, 11836170001). “Buffer D” consisted of 0.25 M sucrose (Thermo Scientific, AC419762500), 150 mM KCl (Invitrogen, AM9640G), 5 mM MgCl2 (Invitrogen, AM9530G), 20 mM Tricine-KOH (pH 7.8) (Thermo Scientific, 15426389), 0.5 mM spermidine (Thermo Scientific, A19096.03), 0.15 mM spermine (Sigma-Aldrich, S3256), 1 mM DTT (Thermo Scientific, R0861), 20 units/mL  SUPERase•In™ RNase inhibitor (Invitrogen, AM2694), and 40 units/mL RNAsin Plus RNase inhibitor (Promega, N2611). “Buffer E” consisted of 1.17 parts Buffer C to 1 part Buffer D. In each buffer, DTT, spermine, and spermidine were added immediately before commencing purification. Buffer E was also mixed immediately before purification. Tissue pieces for each replicate were added to 4.5 mL of Buffer D in a dounce homogenizer (DWK Life Sciences, 357542) at 4° C, dounced 40 times on ice with the loose (0.089 - 0.14 mm clearance range), and then 40 times with the tight (0.025 - 0.076 mm clearance range) pestles, taking care not to cause bubbles. The result was placed in a 15 mL protein LoBind® conical tube (Eppendorf, 0030122216). The total liquid was brought to 5 mL, and 4.6 mL Buffer C was added. The mixture was inverted ten times. 2 mL of Buffer E was added to a 13.2 mL thin-wall clear centrifuge tube (Beckman Coulter Life Sciences, 344059), and the 9.6 mL of solution was carefully layered on top 1 mL at a time. The solution was spun at 10,000 rpm (max acceleration, level 7 deceleration, max 17,136 g, average 12,320 g, K-factor 2,089) at 4°C in a swinging bucket rotor (SW41) for 30 minutes in a Beckman Coulter XL-70 ultracentrifuge. The supernatant was emptied from the tube by inverting it and “flicking” the liquid out. The nuclei were resuspended in 1 mL of Buffer D with 1:10,000 DAPI and passed through a cell strainer (Corning Life Sciences, 352235) into a 1.5 mL protein LoBind® tube (Eppendorf, 022431064) before being quantified on a Countess II FL Cell Counter (Thermo Fisher Scientific).