Assay:	_A_TransientCompLPPSCPPS-GCMS
Short Name:	TransientCompLPPSCPPS-GCMS
Assay Class:	WET
Assay Type:	GCMS
Title:	Transient expression of LPPS and CPPS genes in Nicotiana benthamiana plants
Description:	The purpose of this assay is to check the transient expression of the LPPS and CPPS genes in Nicotiana benthamiana plants. Two different Agrobacterium strains were assayed and compared the levels of production between them. Volatile monoterpenoid levels were measured by GCMS of samples.
pISA Assay creation date:	2021-12-03
pISA Assay creator:	RMF
Lab manager:	DO
Sample collection protocol:	Between 5 and 6 disks were collected 5 days postinfiltration using a 1.5-2 cm corkborer and snap frozen in liquid nitrogen.
Extraction protocol:	50mg of frozen, ground leaf samples were weighed in a 10mL headspace screw-cap vial and stabilized by adding 1mL of 5M CaCl2 and 150 æL of 500mM EDTA (pH = 7.5), after which they were sonicated for 5 minutes. Volatile compounds were captured by means of headspace solid phase microextraction (HS-SPME) with a 65 æm polydimethylsiloxane/divinylbenzene (PDMS/DVB) SPME fiber (Supelco, Bellefonte, PA, USA). Volatile extraction was performed automatically by means of a CombiPAL autosampler (CTC Analytics). Vials were first incubated at 50øC for 10 minutes with 500 rpm agitation. The fiber was then exposed to the headspace of the vial for 20 min under the same conditions of temperature and agitation. Desorption was performed at 250øC for 1 minute (splitless mode) in the injection port of a 6890N gas chromatograph coupled to PEGASUS 4D mass spectrometer (LECO). After desorption, the fiber was cleaned in a SPME fiber conditioning station (CTC Analytics) at 250øC for 5 min under a helium flow.
Chromatography protocol:	Chromatography was performed on a BPX-35 (30 m, 0.32 mm, 0.25 æm) capillary column (SGE) with helium as the carrier gas at a constant flow of 2mLxmin-1. The oven conditions started with an initial temperature of 40øC for 2 min, 5øCmin-1 ramp until 250øC, and a final hold at 250øC for 5 minutes.
Mass spectrometry protocol:	Electron impact ionization (EI), 70 eV ionization energy, m/z range 35-300 at 20 scans/s. 
Phenodata:	../../phenodata_20200720.txt
Featuredata:	
Creation date:	2020-07-20
Extract ID:	$_extr
Extraction Method:	HS-SPME
Date Extraction:	2020-07-20
Derivatization or Labelling:	none
Date Derivatization or Labelling:	2020-07-20
Derivatized or labeled Extract ID:	$_extrD
Other Post Extraction Procedures:	
Storage:	
Date GC-MS Run:	2020-07-20
GC Instrument:	6890N gas chromatograph (Agilent Technologies)
GC Autosampler Model:	CombiPAL autosampler (CTC Analytics)
GC Column model:	BPX-35 (30 m, 0.32 mm, 0.25 æm) capillary column (SGE)
GC Column type:	capillary column
Guard Column:	
MS Scan polarity:	positive
MS Scan mz range:	35-300
MS Instrument:	PEGASUS 4D mass spectrometer (LECO)
MS Ion source:	electron ionization (EI)
Mass analyzer:	time-of-flight
Operator:	RMF
Notes:	Chromatograms were proccesed by means of the ChromaTOF software (LECO). Quantification was made by integrating peak area for the corresponding ion. Ions selected for each compound were the following: QI 69 for lavandulol, QI 81 for chrysanthemol, QI 41 for artemisia alcohol, QI 43 for santolina/yomogi alcohol, QI 43 for santolinatriene/isomyrcenyl acetate.
Data:	10.5281/zenodo.6554066