#METABOLOMICS WORKBENCH jliu381_20181024_223224 DATATRACK_ID:1554 STUDY_ID:ST001086 ANALYSIS_ID:AN001771 PROJECT_ID:PR000725
VERSION             	1
CREATED_ON             	October 31, 2018, 11:48 am
#PROJECT
PR:PROJECT_TITLE                 	Targeted metabolomics of SETD2 isogenic cell lines
PR:PROJECT_SUMMARY               	SETD2, the histone methyltransferase responsible for the trimethylation of
PR:PROJECT_SUMMARY               	H3K36, is inactivated in approximately 10-32% of clear renal cell carcinoma
PR:PROJECT_SUMMARY               	(ccRCC) cases. To reveal the impact of SETD2 loss on metabolic alterations in
PR:PROJECT_SUMMARY               	ccRCC. In this study, SETD2 null isogenic 38E/38F clones derived from 786-O
PR:PROJECT_SUMMARY               	cells were generated by zinc finger nucleases, and the cellular metabolic
PR:PROJECT_SUMMARY               	changes were analyzed by GC-MS-based targeted metabolomics.
PR:INSTITUTE                     	Arizona State University
PR:DEPARTMENT                    	College of Health Solutions
PR:LAST_NAME                     	Liu
PR:FIRST_NAME                    	Jingping
PR:ADDRESS                       	13208 E. Shea Blvd, Scottsdale, AZ
PR:EMAIL                         	jliu381@asu.edu
PR:PHONE                         	4802078529
#STUDY
ST:STUDY_TITLE                   	Targeted GC-MS of SETD2 isogenic cell lines
ST:STUDY_SUMMARY                 	In this study, SETD2 null isogenic 38E/38F clones derived from 786-O cells were
ST:STUDY_SUMMARY                 	generated by zinc finger nucleases, and the cellular metabolic changes of 786-O
ST:STUDY_SUMMARY                 	(WT) and 38E/38F isogenic cell lines (n=3 per group) were analyzed by
ST:STUDY_SUMMARY                 	GC-MS-based targeted metabolomics.
ST:INSTITUTE                     	Arizona State University
ST:DEPARTMENT                    	College of Health Solutions
ST:LAST_NAME                     	Liu
ST:FIRST_NAME                    	Jingping
ST:ADDRESS                       	13208 E. Shea Blvd, Scottsdale, AZ
ST:EMAIL                         	jliu381@asu.edu
ST:PHONE                         	4802078529
#SUBJECT
SU:SUBJECT_TYPE                  	Cultured cells
SU:SUBJECT_SPECIES               	Homo sapiens
SU:TAXONOMY_ID                   	9606
#SUBJECT_SAMPLE_FACTORS:         	SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data
SUBJECT_SAMPLE_FACTORS           	-	786-O rep1	Genotype:Wild-type	
SUBJECT_SAMPLE_FACTORS           	-	786-O rep2	Genotype:Wild-type	
SUBJECT_SAMPLE_FACTORS           	-	786-O rep3	Genotype:Wild-type	
SUBJECT_SAMPLE_FACTORS           	-	38-E rep1	Genotype:SETD2-mutation	
SUBJECT_SAMPLE_FACTORS           	-	38-E rep2	Genotype:SETD2-mutation	
SUBJECT_SAMPLE_FACTORS           	-	38-E rep3	Genotype:SETD2-mutation	
SUBJECT_SAMPLE_FACTORS           	-	38-F rep1	Genotype:SETD2-mutation	
SUBJECT_SAMPLE_FACTORS           	-	38-F rep2	Genotype:SETD2-mutation	
SUBJECT_SAMPLE_FACTORS           	-	38-F rep3	Genotype:SETD2-mutation	
#COLLECTION
CO:COLLECTION_SUMMARY            	Quickly aspirate cell culture medium from 3.5 cm plates, gently dispense 2 mL of
CO:COLLECTION_SUMMARY            	PBS to rinse cells, shake the plate back and forth for 2 s and then quickly
CO:COLLECTION_SUMMARY            	remove the PBS. Immediately add 1 mL 8:2 methanol:H2O (on dry ice) into the
CO:COLLECTION_SUMMARY            	plates placed on dry ice to quench cell metabolism, store samples at -80℃.
CO:SAMPLE_TYPE                   	Cultured cells
#TREATMENT
TR:TREATMENT_SUMMARY             	The cell lines including 786-O and 38E/38F were cultured in RPMI 1640 medium
TR:TREATMENT_SUMMARY             	supplemented with 10% FBS, 1% penicillin/streptomycin, and 2 mM L-glutamine.
TR:TREATMENT_SUMMARY             	After culture at 37 °C, 5% CO2 condition for 24 h, the cells were collected for
TR:TREATMENT_SUMMARY             	GC-MS analysis.
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	Sampleprep Summary: Scrape all the cells from culture dishes on dry ice, and
SP:SAMPLEPREP_SUMMARY            	transfer them into a 2 mL Eppendorf, spin the mixture at 14000 rpm for 10 min at
SP:SAMPLEPREP_SUMMARY            	4°C. Remove all the soluble extract into a vial and completely dry samples. The
SP:SAMPLEPREP_SUMMARY            	extracted samples were incubated with 30 μL O-methylhydroxylamine hydrochloride
SP:SAMPLEPREP_SUMMARY            	solution in pyridine (Sigma-Aldrich, St. Louis, MO, USA) at 60°C for 90 min.
SP:SAMPLEPREP_SUMMARY            	Next, 70 μL of N-tert-Butyldimethylsilyl-N-methyltrifluoroacetamide (MTBSTFA)
SP:SAMPLEPREP_SUMMARY            	were added and placed at 60 °C for 30 min. After centrifuge at 14000 rpm for 10
SP:SAMPLEPREP_SUMMARY            	min, 70 μL of supernatant was transferred to a clean glass GC insert tube.
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_TYPE           	GC
CH:INSTRUMENT_NAME               	Agilent 7890A
CH:COLUMN_NAME                   	Agilent DB5-MS (30m × 0.25mm, 0.25um)
CH:FLOW_RATE                     	0.5 mL/min
CH:INJECTION_TEMPERATURE         	250°C
CH:TRANSFERLINE_TEMPERATURE      	290°C
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
#MS
MS:MS_COMMENTS                   	-
MS:INSTRUMENT_NAME               	Agilent 5975C
MS:INSTRUMENT_TYPE               	Single quadrupole
MS:MS_TYPE                       	EI
MS:ION_MODE                      	POSITIVE
MS:ION_SOURCE_TEMPERATURE        	230°C
MS:IONIZATION                    	70 eV
MS:SCANNING_RANGE                	50-600 m/z
#MS_METABOLITE_DATA
MS_METABOLITE_DATA:UNITS         	pmole/ug
MS_METABOLITE_DATA_START
Samples	786-O rep1	786-O rep2	786-O rep3	38-E rep1	38-E rep2	38-E rep3	38-F rep1	38-F rep2	38-F rep3
Factors	Genotype:Wild-type	Genotype:Wild-type	Genotype:Wild-type	Genotype:SETD2-mutation	Genotype:SETD2-mutation	Genotype:SETD2-mutation	Genotype:SETD2-mutation	Genotype:SETD2-mutation	Genotype:SETD2-mutation
Lactate	58.10607	61.61175	43.46	31.06999	41.29203	46.43195	29.888	38.20992	25.59586
Succinate	0.564642	1.185374	0.813217	1.283617	1.730575	1.291269	0.866918	0.978369	0.740564
Fumarate	0.254165	0.325853	0.2337	0.291722	0.334525	0.460743	0.436472	0.476206	0.336574
Oxaloacetate	0	0	0	0	0	0	0	0	0
α-Ketoglutarate	0.100781	0.095843	0.042723	0.178187	0.135907	0.183634	0.372147	0.224725	0.158176
Malate	1.051511	1.77141	1.170534	1.564412	1.945175	2.998501	2.537524	2.765253	1.9131
Aspartate	1.125651	1.336688	1.072446	2.034551	3.183649	2.989099	2.296467	2.84556	1.735897
2-Hydroxyglutarate	0.139388	0.149611	0.082812	0.123103	0.133739	0.132376	0.11567	0.117894	0.067892
Glutamate	23.12394	40.85939	28.81189	35.63481	44.51025	44.30002	38.73059	38.98803	36.35525
cis-Aconitic Acid	0.01216	0.016236	0.009817	0.01672	0.014724	0.021761	0.015894	0.020102	0.012276
Citrate	0.960231	1.159267	0.565582	1.318814	1.241837	2.168567	1.476703	1.159748	0.916632
Isocitrate	0.021495	0.030921	0.014977	0.028633	0.024717	0.04327	0.032398	0.025001	0.020415
MS_METABOLITE_DATA_END
#METABOLITES
METABOLITES_START
metabolite_name	rtimes (min)	quantified m/z	PubChem ID	KEGG ID
Lactate	12.79	261.1	107689	C00186
Succinate	15.91	289.1	1110	C00042
Fumarate	16.29	287.1	444972	C00122
Oxaloacetate	17.14	332.1	970	C00036
α-Ketoglutarate	18.39	346.2	51	C00026
Malate	19.39	419.3	222656	C00149
Aspartate	19.77	418.3	5960	C00049
2-Hydroxyglutarate	20.36	433.2	439391	C01087
Glutamate	20.87	432.2	33032	C00025
cis-Aconitic Acid	21.78	459.3	643757	C00417
Citrate	23.56	591.4	311	C00158
Isocitrate	23.65	591.4	1198	C00311
METABOLITES_END
#END