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MB Sample ID: SA289188

Local Sample ID:MED1 PO Condensate Sample 6
Subject ID:SU002852
Subject Type:Mammal
Subject Species:Mus musculus
Taxonomy ID:10090

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Subject:

Subject ID:SU002852
Subject Type:Mammal
Subject Species:Mus musculus
Taxonomy ID:10090

Factors:

Local Sample IDMB Sample IDFactor Level IDLevel ValueFactor Name
MED1 PO Condensate Sample 6SA289188FL035658condensatefraction
MED1 PO Condensate Sample 6SA289188FL035658No heat stepExtraction from condensate

Collection:

Collection ID:CO002845
Collection Summary:Mouse metabolites were collected from the liver of female mice using methanol extraction. After euthanizing a mouse, the liver was immediately frozen in liquid nitrogen. We then used cold 80% methanol to extract metabolites. First, 1 ml of 80% methanol was added to the liver and incubated for 10 min at -20oC. Glass beads were added to the liver and then the liver was lysed by bead-beating for 45 s using a Tissuelyser cell disrupter (Qiagen). The lysate was incubated for 10 min at -20oC and centrifuged (13200 rpm, 5 min) to separate metabolites from macromolecules. The supernatant was collected and 200 µl of 80% methanol was added to the pellet. The incubation, shaking and centrifugation steps were repeated twice to extract more metabolites from the pellet. The three supernatants were combined and centrifuged (14000 rpm, 10 min) to separate any remaining macromolecules from the metabolites. The combined supernatants were dried using a SpeedVac Concentrator (Savant, SPD131DDA) at 25oC and the dried metabolite samples were stored at -80oC. The amount of protein in the pellet was measured using the Quick Start Bradford assay to calculate the metabolites’ protein equivalent mass. Mouse metabolites were initially re-suspended in condensate buffer (50 mM NH4HCO3 pH 7.5, 50 mM NaCl, 1 mM DTT) to a protein equivalent concentration of 938 g/l. The chosen final concentration of metabolites is slightly lower than the 200-300 g/l protein concentration observed in cells. Metabolites that were not fully soluble in condensate buffer were removed by centrifugation (2x5 min, 16,000 g each), in which only the supernatant was retained. Purified mCherry tagged MED1 low-complexity domain (37.5 μM) was centrifuged (1 min, 1,000 g) to disrupt any existing condensates and to remove any precipitated proteins. The MED1 (final concentration, 30 μM) was then combined with metabolites (final concentration, 150 g/l protein equivalent) and then phage lambda RNA (final concentration, 0.15 μM) in a total volume of 300 µl. An input sample (10 µl) was saved and then the sample was allowed to incubate for 10 min at 25oC. Condensates were then separated from the aqueous environment by centrifugation (10 min, 12,500 g, 25oC). The aqueous phase was removed from the condensate phase and then equal volumes (usually ~ 2 µl) of the aqueous fraction, condensate fraction and input sample were processed for metabolomics using identical approaches as described below. First the samples were diluted in ammonium bicarbonate buffer (50 mM NH4HCO3 pH 7.5) and briefly heated (2 min, 65oC) to disrupt condensates before being added immediately to 4x volume of ice-cold 100% methanol to precipitate protein and RNA. This heating step was excluded for some samples where noted. Protein and RNA were separated from metabolites by vortexing the samples (2 min), followed by incubation at -25oC (10 min) and then centrifugation (5 min, 13,000 rpm). The supernatant was saved and the process was repeated on the pellet two more times after adding 200 µl of 80% methanol each time to the pellet. The three supernatants were combined and centrifuged (10 min, 14000 rpm) to remove any additional macromolecules. The final supernatant was collected and dried using a SpeedVac Concentrator run at 25oC. Notably, in one subset of experiments, metabolites were added to MED1 condensates after the 10 min incubation rather than prior to the incubation.
Sample Type:Liver
Collection Method:80% methanol
Storage Conditions:-80℃

Treatment:

Treatment ID:TR002861
Treatment Summary:Mouse liver metabolites were combined with the condensate-forming low-complexity domain of MED1. Condensates were stimulated with 150 nM RNA and then incubated for 10 min. In a subset of samples, RNA addition occurred 10 min before metabolite addition. Next, condensates were centrifuged to the bottom of a 600 ul tube. Equal fractions from the input sample, aqueous phase and condensate phases were collected separately. Metabolites were extracted from each fraction using 80% methanol in steps that involved disrupting condensates with heat. In a subset of samples, this heat step was omitted.

Sample Preparation:

Sampleprep ID:SP002858
Sampleprep Summary:Dried-down extracts were reconstituted in 150 µl 70% acetonitrile, at a relative protein concentration of ~ 2 µg/µl. These were stored until used at -20C. SPLASH® LIPIDOMIX® Quantitative Mass Spec Standard was diluted 1/30 or 1/200, combined with the reconstituted extract and then either 3 µl (for PIs samples) or 5 µl (all other samples) were injected for LC/MS-based targeted metabolite profiling.
Processing Storage Conditions:-80℃
Extract Storage:-20℃

Combined analysis:

Analysis ID AN004451 AN004452
Analysis type MS MS
Chromatography type Reversed phase Reversed phase
Chromatography system Agilent Model 1290 Infinity II liquid chromatography system Agilent Model 1290 Infinity II liquid chromatography system
Column Cogent Diamond Hydride (150 × 2.1 mm, 4um) Cogent Diamond Hydride (150 × 2.1 mm, 4um)
MS Type ESI ESI
MS instrument type Triple quadrupole Triple quadrupole
MS instrument name Agilent 6460 QQQ Agilent 6460 QQQ
Ion Mode POSITIVE POSITIVE
Units Ion abundance (peak area) Ion abundance (peak area)

Chromatography:

Chromatography ID:CH003343
Chromatography Summary:Chromatography of metabolites utilized reversed phase chromatography on a Agilent ZORBAX Eclipse Plus C18, 100 × 2.1 mm, 1.8 μm. Mobile phases consisted of (A) 10 mM ammonium formate with 5 μM Agilent deactivator additive in 5:3:2 water:acetonitrile:2-propanol and (B) 10 mM ammonium formate in 1:9:90 water:acetonitrile:2-propanol. Column temperature was set at 60°C and autosampler temperature was at 20°C. The flow rate was 0.4 mL/min. The following gradient was applied: 0 min, 15% B; 0-2.5 min, to 50% B; 2.5-2.6 min, to 57%, 2.6-9 min, to 70% B; 9-9.1 min, to 93% B; 9.1-11.1 min, to 96%; 11.1- 15min, 100% B; 15-20 min, 15% B.
Instrument Name:Agilent Model 1290 Infinity II liquid chromatography system
Column Name:Cogent Diamond Hydride (150 × 2.1 mm, 4um)
Column Temperature:60
Flow Gradient:0 min, 15% B; 0-2.5 min, to 50% B; 2.5-2.6 min, to 57%, 2.6-9 min, to 70% B; 9-9.1 min, to 93% B; 9.1-11.1 min, to 96%; 11.1- 15min, 100% B; 15-20 min, 15% B.
Flow Rate:0.4 mL/min
Solvent A:50% water/30% acetonitrile/20% isopropanol;10 mM ammonium formate with 5 µM Agilent deactivator additive
Solvent B:1% water/9% acetonitrile/90% isopropanol;10 mM ammonium formate
Chromatography Type:Reversed phase

MS:

MS ID:MS004198
Analysis ID:AN004451
Instrument Name:Agilent 6460 QQQ
Instrument Type:Triple quadrupole
MS Type:ESI
MS Comments:(PE, PC, SM, others)LC/MS-based targeted metabolite profiling. MS mode was a Agilent Jet Stream ESI. To quantify phospholipid concentrations, we used stable isotope standards and dynamic multiple reaction monitoring (dMRM). The transitions for lipid standards were monitored as recommended by the supplier.
Ion Mode:POSITIVE
  
MS ID:MS004199
Analysis ID:AN004452
Instrument Name:Agilent 6460 QQQ
Instrument Type:Triple quadrupole
MS Type:ESI
MS Comments:(PI) LC/MS-based targeted metabolite profiling. MS mode was a Agilent Jet Stream ESI. To quantify phospholipid concentrations, we used stable isotope standards and dynamic multiple reaction monitoring (dMRM). The transitions for lipid standards were monitored as recommended by the supplier.
Ion Mode:POSITIVE
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