Fraction recovery system

OTX2-HeLa cells (2 × 10⁷) were scrapped from the culture plates and resuspended in phosphate-buffered saline (PBS; Gibco, 10010023) to separate the nucleus from cytoplasmic fraction using NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific, 78833). Cytoplasmic fractions were then centrifuged at 500rcf for 10 min at 4 °C to collect the supernatant (S1). Next, the S1 fractions were centrifuged at 2000 rcf for 20 min at 4 °C. The resultant S2 samples were carefully loaded on top of sucrose solution forming the gradients (5–45%), which were generated by Linear Gradient Makers (C.B.S. Scientific, GM-20) according to manufacturer’s instructions, prior to ultracentrifugation at 257,000rcf for 18 h at 4 °C. After ultracentrifugation, each fraction was collected in an equal volume using the Fraction Recovery System (Beckman Coulter, 270-331580) from bottom to top.

DNA samples were prepared for density gradient centrifugation according to previously defined protocol for qSIP [23 ]. In brief, density gradient centrifugations were carried out in a TLN-100 Optima MAX-TL ultracentrifuge (Beckman Coulter, Brea, CA, USA) near-vertical rotor at 18 °C for 72 h at 165,000 × g. In all, 50 µl of DNA spanning from 0.5 to 1.5 µg [24 ] was added to a solution of cesium chloride (CsCl) and gradient buffer (0.1 M Tris, 0.1 M KCl and 1 mM EDTA) in order to achieve a starting density of 1.70 g mL^-1 in a 3.3-mL polyallomer OptiSeal tubes (Beckman Coulter, Brea, CA, USA). After ultracentrifugation, the density gradients were fractionated into 15 equal fractions of 200 µl from the bottom of polyallomer OptiSeal tubes by using a syringe pump and fraction recovery system (Beckman Coulter, Brea, CA, USA). The density of these fractions was measured with an AR200 digital refractometer (Reichert Analytical Instruments, Depew, NY, USA). DNA was precipitated from the fractions using two volumes of polyethylene glycol with 2 µl (10 mg mL^-1) glycogen and precipitated overnight at room temperature. DNA was pelleted by centrifugation (13,000 × g; 40 min), washed with 70% ethanol, and resuspended with 30 µl molecular-grade (DEPC-treated) water. DNA was quantified fluorometrically using a Qubit 4 fluorometer (Thermo Scientific).

The 84-31 and 84-31 mNIPP1 cells were grown on 100 mm plates, then lysed with 0.5 mL of whole cell lysis buffer (50 mM Tris-HCl, pH 7.5, 0.5 M NaCl, 1% NP-40, 0.1% SDS) supplemented with a cocktail of protease inhibitors (Sigma-Aldrich). Cell lysates were clarified by centrifugation for 30 min at 10,000× g and loaded on top of 10% to 30% glycerol gradient. The glycerol gradient buffer contained 20 mM HEPES-KOH, pH 7.9, 150 mM KCl, 0.2 mM EDTA. The gradients were spun in a Sorvall XL-90 Ultracentrifuge (Beckman Coulter, Brea, CA, USA) with a SW41 Ti rotor at 38,000 rpm for 18 h. Twenty fractions were collected through a needle inserted to the bottom of the tube using fraction recovery system (Beckman Coulter). Proteins were precipitated with 70% trichloroacetic acid (TCA), resolved in 10% SDS-PAGE and analyzed by immunoblotting as indicated.

DNA extraction was conducted on two soil samples from each treatment that were incubated for 3, 6, and 9 days, using a Powersoil DNA extraction kit (MO BIO Laboratories, Inc. Carlsbad, CA, USA) following the manufacturer’s instructions. The DNA content was quantified with an ND-2,000 UV-Vis spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). Thereafter, about 10,000-ng DNA was added to Quick-Seal polyallomer tubes (13 × 51 mm, 5.1 mL, Beckman Coulter, Pasadena, CA, USA) and spun in Tris-EDTA (TE, pH 8.0)/CsCl solution. Prior to sealing the tubes with one cordless quick-seal tube topper (Beckman Coulter), the average buoyant density (BD) of all prepared gradients was confirmed with a digital refractometer (model AR200, Leica Microsystems Inc., Buffalo Grove, IL, USA), and adjusted by adding small volumes of CsCl solution or Tris-EDTA buffer. The tubes were transferred to an ultracentrifuge (Optima L-100XP, Beckman Coulter). Centrifugation was performed at 45,000 g (20°C) for 48 h. Subsequently, the centrifuge tubes were placed onto a fraction recovery system (Beckman Coulter) and fractions (150 μL for each) were collected. The BD of each fraction was then measured, and CsCl was removed by introducing concentrated ethanol [30 (link)].

¹²C- and ¹³C-labeled DNA were separated on a CsCl gradient. The CsCl solution was prepared in 0.1 M Tris-EDTA (TE) buffer, resulting in a buoyant density of about 1.7742 g/mL as measured by an AR200 digital refractometer (Reichert Technologies, NY), and was distributed into 4.7-mL OptiSeal polypropylene ultracentrifuge tubes (Beckman Coulter, CA). All the genomic DNA (gDNA) from the gas-phase incubations and 1 μg of the gDNA of the liquid-phase incubations of the air enrichment were added to the CsCl gradients. The buoyant densities of the gradients were then adjusted to be between 1.7231 and 1.7340 g/mL by the addition of more of the CsCl solution or TE buffer. The gradients were centrifuged at 86,700 × g at 28°C for at least 48 h in an Optima Max TL ultracentrifuge (Beckman Coulter, CA). After centrifugation, the samples were fractionated with a fraction recovery system (Beckman Coulter, CA), with the aid of a syringe pump, and 24 fractions of 200 μL were collected. The DNA was precipitated using a glycogen-assisted ethanol precipitation. After precipitation, DNA was resuspended in 20 μL of nuclease-free water.