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MB Sample ID: SA090822
| Local Sample ID: | KO_6DFA_01 |
| Subject ID: | SU001314 |
| Subject Type: | Cultured cells |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU001314 |
| Subject Type: | Cultured cells |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| KO_6DFA_01 | SA090822 | FL013085 | KO HADHA | Genoytpe |
| KO_6DFA_01 | SA090822 | FL013085 | Control | Treatment |
Collection:
| Collection ID: | CO001308 |
| Collection Summary: | hESC and hiPSC and cardiac differentiation: The hESC line RUES2 (NIHhESC-09-0013) and hiPSC line WTC #11, previously derived in the Conklin laboratory 83, were cultured on Matrigel growth factor-reduced basement membrane matrix (Corning) in mTeSR media (StemCell Technologies). A monolayer-based directed differentiation protocol was followed to generate hESC-CMs and hiPSC-CMs, as done previously 25. hiPSC-CM cardiolipin assay was done with a small molecule monolayer-based directed differentiation protocol, as done previously 84. 15 days after differentiation hPSC-CMs were enriched for the cardiomyocyte population using a lactate selection process 85. We generated cardiomyocyte populations ranging from 40-60% that were then enriched to 75-80% cardiomyocytes after 4 days of lactate enrichment. METABOLITE EXTRACTION: 1 million cells were extracted with 225 µl of methanol, after enzymatic lifting from cell culture plates. |
| Sample Type: | Cultured cells |
Treatment:
| Treatment ID: | TR001329 |
| Treatment Summary: | HADHA Line Creation: Using LentiCrisprV2 plasmid 91 (lentiCRISPRV2 was a gift from Feng Zhang (Addgene plasmid # 52961) two different gRNAs targeted to Exon 1 of HADHA were designed using CRISPRScan 92. Sequences for the gRNAs can be found in Supplemental Table S12. The gRNA and Cas9 expressing plasmids were transiently transfected into the WTC line using GeneJuice (EMD Millipore). 24 hours after transfection, WTCs were puromycin selected for two days and then clonally expanded. DNA of the clones was isolated, the region around the targeting guides was PCR amplified (see guides in Supplemental Table S12) and sequenced to determine the insertion and deletion errors generated by CRISPR-Cas9 system in exon 1 of HADHA. Western analysis was performed to determine the levels of HADHA protein in HADHA mutants. 31 clones were sent for sequencing from gRNA1 experiment, 6 clones (19%) had no mutations while 25 clones (81%) were found to have mutations. 24 clones were sent for sequencing from gRNA2, 1 clone had no mutations (4%) while 23 clones (96%) were found to have mutations. Two of the mutant lines were analyzed further in this study. Glucose and Fatty Acid Media: The base media which we are calling Glucose Media, is RPMI supplemented with B27 with insulin. The fatty acid media is the glucose media with oleic acid conjugated to BSA (Sigma O3008): 12.7μg/mL, linoleic acid conjugated to BSA (Sigma L9530): 7.05μg/mL, sodium palmitate (Sigma P9767) conjugated to BSA (Sigma A8806): 52.5μM and L-carnitine: 125μM. Fatty acid (FA) experiments used this B27 + insulin + the three FAs (oleic acid, linoleic acid and palmitatic acid), in RPMI media. This media was changed every other day during the 6-days or 12-days of treatment. |
Sample Preparation:
| Sampleprep ID: | SP001322 |
| Sampleprep Summary: | 1 million cells were extracted with 225 µl of methanol at −20°C containing an internal standard mixture of PE(17:0/17:0), PG(17:0/17:0), PC(17:0/0:0), C17 sphingosine, ceramide (d18:1/17:0), SM (d18:0/17:0), palmitic acid-d3, PC (12:0/13:0), cholesterol-d7, TG (17:0/17:1/17:0)-d5, DG (12:0/12:0/0:0), DG (18:1/2:0/0:0), MG (17:0/0:0/0:0), PE (17:1/0:0), LPC (17:0), LPE (17:1), and 750 µL of MTBE (methyl tertiary butyl ether) (Sigma Aldrich) at −20°C containing the internal standard cholesteryl ester 22:1. Cells were vortexed for 20 sec, sonicated for 5 min and shaken for 6 min at 4°C with an Orbital Mixing Chilling/Heating Plate (Torrey Pines Scientific Instruments). Then 188 µl of LC-MS grade water (Fisher) was added. Samples were vortexed, centrifuged at 14,000 rcf (Eppendorf 5415D). The upper (non-polar, organic) phase was collected in two 350 µL aliquots and evaporated to dryness. |
Combined analysis:
| Analysis ID | AN002070 |
|---|---|
| Analysis type | MS |
| Chromatography type | Reversed phase |
| Chromatography system | Thermo Q Exactive HF Hybrid Quadrupole-Orbitrap |
| Column | Waters Acquity CSH C18 (100 x 2.1mm,1.7um) |
| MS Type | ESI |
| MS instrument type | Orbitrap |
| MS instrument name | Thermo Q Exactive HF hybrid Orbitrap |
| Ion Mode | UNSPECIFIED |
| Units | ng |
Chromatography:
| Chromatography ID: | CH001507 |
| Chromatography Summary: | Re-suspended samples were injected at 3 µL and 5 µL for ESI positive and negative modes respectively, onto a Waters Acquity UPLC CSH C18 (100 mm length × 2.1 mm id; 1.7 µm particle size) with an additional Waters Acquity VanGuard CSH C18 pre-column (5 mm × 2.1 mm id; 1.7 µm particle size) maintained at 65°C was coupled to a Vanquish UHPLC System. To improve lipid coverage, different mobile phase modifiers were used for positive and negative mode analysis 103. For positive mode 10 mM ammonium formate and 0.1% formic acid were used and 10 mM ammonium acetate (Sigma–Aldrich) was used for negative mode. Both positive and negative modes used the same mobile phase composition of (A) 60:40 v/v acetonitrile:water (LC-MS grade) and (B) 90:10 v/v isopropanol:acetonitrile. The gradient started at 0 min with 15% (B), 0–2 min 30% (B), 2–2.5 min 48% (B), 2.5–11 min 82% (B), 11–11.5 min 99% (B), 11.5–12 min 99% (B), 12–12.1 min 15% (B), and 12.1–15 min 15% (B). A flow rate of 0.6 mL/min was used. |
| Instrument Name: | Thermo Q Exactive HF Hybrid Quadrupole-Orbitrap |
| Column Name: | Waters Acquity CSH C18 (100 x 2.1mm,1.7um) |
| Column Temperature: | 65 |
| Flow Gradient: | 0 min with 15% (B), 0-2 min 30% (B), 2-2.5 min 48% (B), 2.5-11 min 82% (B), 11-11.5 min 99% (B), 11.5-12 min 99% (B), 12-12.1 min 15% (B), and 12.1-15 min 15% (B) |
| Flow Rate: | 0.6ml/min |
| Solvent A: | Pos mode:60% acetonitrile/40% water; 0.1% formic acid; 10 mM ammonium formate, Neg mode:60% acetonitrile/40% water; 0.1% formic acid; 10 mM ammonium acetate |
| Solvent B: | Pos mode:90% isopropanol/10% acetonitrile; 0.1% formic acid; 10 mM ammonium formate,Neg mode:90% isopropanol/10% acetonitrile; 0.1% formic acid; 10 mM ammonium acetate |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS001921 |
| Analysis ID: | AN002070 |
| Instrument Name: | Thermo Q Exactive HF hybrid Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | For data acquisition a Q-Exactive HF Hybrid Quadrupole-Orbitrap Mass Spectrometer was used with the following parameters: mass range, m/z 100-1200; MS1 resolution 60,000: data-dependent MS2 resolution 15,000; NCE 20, 30, 40; 4 targets/MS1 scan; gas temperature 369°C, sheath gas flow (nitrogen), 60 units, aux gas flow 25 units, sweep gas flow 2 units; spray voltage 3.59 kV. LC-MS data processing using MS-DIAL and statistics: Untargeted lipidomic data processing was performed using MS-DIAL 104 for deconvolution, peak picking, alignment, and identification. In house m/z and retention time libraries were used in addition to MS/MS spectra databases in msp format 105. Features were reported when present in at least 50% of samples in each group. Statistical analysis was done by first normalizing data using the sum of the knowns, or mTIC normalization, to scale each sample. Normalized peak heights were then submitted to R for statistical analysis. ANOVA analysis was performed with FDR correction and post hoc testing. |
| Ion Mode: | UNSPECIFIED |
