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MB Sample ID: SA239047
| Local Sample ID: | Z22 |
| Subject ID: | SU002487 |
| Subject Type: | Fish |
| Subject Species: | Danio rerio |
| Taxonomy ID: | 7955 |
| Genotype Strain: | wildtype and tango2bwh211 |
| Age Or Age Range: | 4 weeks |
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Subject:
| Subject ID: | SU002487 |
| Subject Type: | Fish |
| Subject Species: | Danio rerio |
| Taxonomy ID: | 7955 |
| Genotype Strain: | wildtype and tango2bwh211 |
| Age Or Age Range: | 4 weeks |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| Z22 | SA239047 | FL029790 | tango2 Mutant | Genotype |
| Z22 | SA239047 | FL029790 | 26 | Weight (mg) |
Collection:
| Collection ID: | CO002480 |
| Collection Summary: | Fish were bred and maintained using standard methods as described (Westerfield, 2000). All procedures were approved by the Brigham and Women’s Hospital Animal Care and Use Committee. tango2bwh210 and tango2bwh211 zebrafish lines were created in our laboratory by CRISPR-Cas9 approach. Zebrafish embryonic (0-2 days post fertilization) and larval stages (3-45 dpf), juvenile stage (45 dpf-3months) and adults (3 months) have been defined as described previously (Kimmel, Ballard, Kimmel, Ullmann, & Schilling, 1995). All studies presented in this work were performed on tango2bwh211 mutants obtained from heterozygous parents unless specified. Myofibers were isolated from control or tango2 larval zebrafish (45 dpf) as described previously with minor modifications (Ganassi, Zammit, & Hughes, 2021). Skinned zebrafish muscle samples were treated with collagenase for 90 minutes and triturated to release the myofibers. Myofibers were centrifuged at 1000g for 60 sec, washed and resuspended in DMEM media. Myofibers were plated on laminin coated 8 chamber permanox slides (Thermofisher Scientific) for further analysis. Fixed cells were blocked in 10% goat serum/0.3% Triton, incubated in primary antibody overnight at 4ºC, washed in PBS, incubated in secondary antibody for 1 h at room temperature (RT), washed in PBS, then mounted with Vectashield Mounting Medium (Vector Laboratories, Burlingame, CA, USA). Primary antibodies used were anti Tango2 (1:250, 27846-1-AP, Proteintech), mouse monoclonal anti sarcomeric -actinin (1:100, A7732, Millipore Sigma), mouse monoclonal anti Ryr1 (1:100, R129-100UL, Millipore Sigma), anti Tomm20 (1:100, MABT166, Millipore Sigma) and Alexa fluor 568-phalloidin (1:100, Thermo Fisher Scientific, A12380). After washing in PBS several times, samples were incubated with anti-mouse Alexa Fluor (1:100, A-11005) secondary antibody (Thermofisher Scientific). Imaging was performed using a Nikon Ti2 spinning disk confocal microscope. |
| Sample Type: | Muscle |
| Collection Method: | Muscle Dissection |
| Storage Conditions: | Described in summary |
Treatment:
| Treatment ID: | TR002499 |
| Treatment Summary: | Fish were bred and maintained using standard methods as described (Westerfield, 2000). All procedures were approved by the Brigham and Women’s Hospital Animal Care and Use Committee. tango2bwh210 and tango2bwh211 zebrafish lines were created in our laboratory by CRISPR-Cas9 approach. Zebrafish embryonic (0-2 days post fertilization) and larval stages (3-45 dpf), juvenile stage (45 dpf-3months) and adults (3 months) have been defined as described previously (Kimmel, Ballard, Kimmel, Ullmann, & Schilling, 1995). All studies presented in this work were performed on tango2bwh211 mutants obtained from heterozygous parents unless specified. |
Sample Preparation:
| Sampleprep ID: | SP002493 |
| Sampleprep Summary: | Control and tango2 mutant myofibers (4 weeks, n=5 each) were homogenized with 1 ml of MBTE. 300 µL of methanol with internal standard was added and samples were mixed for 10 minutes. 200 µL of water was added to facilitate phase separation. The extracts were centrifuged at 20,000 rcf for 10 minutes. The top layer was removed, dried and reconstituted in 150 µl of IPA for analysis. |
| Processing Method: | Liquid/Liquid Partition |
| Processing Storage Conditions: | -80℃ |
| Extraction Method: | Liquid/Liquid Partition |
| Extract Cleanup: | Centrifugation (20,000 rcf for 10 min) |
| Extract Storage: | -80℃ |
| Sample Resuspension: | 150 µL of IPA |
| Sample Derivatization: | none |
| Sample Spiking: | Spiked with 1.5 µg/mL of deuterated internal standards (Avanti EquiSplash mix) |
Combined analysis:
| Analysis ID | AN003905 |
|---|---|
| Analysis type | MS |
| Chromatography type | Reversed phase |
| Chromatography system | Waters Acquity H-Class |
| Column | Waters ACQUITY UPLC BEH C18 (100 x 2.1mm,1.7um) |
| MS Type | ESI |
| MS instrument type | Orbitrap |
| MS instrument name | Thermo Q Exactive HF-X Orbitrap |
| Ion Mode | POSITIVE |
| Units | peak area |
Chromatography:
| Chromatography ID: | CH002891 |
| Chromatography Summary: | Analysis was performed using a Thermo Q Exactive Plus coupled to a Waters Acquity H-Class LC. A 100 mm x 2.1 mm, 2.1 µm Waters BEH C18 column was used for separations. The following mobile phases were used: A- 60/40 ACN/H20 B- 90/10 IPA/ACN; both mobile phases contained 10 mM Ammonium Formate and 0.1% Formic Acid. A flow rate of 0.2 mL/minutes was used. Starting composition was 32% B, which increased to 40% B at 1 minute (held until 1.5 minutes) then 45% B at 4 minutes. This was increased to 50% B at 5 minutes, 60% B at 8 minutes, 70% B at 11 minutes, and 80% B at 14 minutes (held until 16 minutes). At 16 minutes the composition switched back to starting conditions (32% B) and was held for 4 minutes to re-equilibrate the column. Samples were analyzed in positive/negative switching ionization mode with top 5 data dependent fragmentation. |
| Instrument Name: | Waters Acquity H-Class |
| Column Name: | Waters ACQUITY UPLC BEH C18 (100 x 2.1mm,1.7um) |
| Column Pressure: | 4220 psi |
| Column Temperature: | 45 |
| Flow Gradient: | Starting composition was 32% B, which increased to 40% B at 1 minute (held until 1.5 minutes) then 45% B at 4 minutes. This was increased to 50% B at 5 minutes, 60% B at 8 minutes, 70% B at 11 minutes, and 80% B at 14 minutes (held until 16 minutes). At 16 minutes the composition switched back to starting conditions (32% B) and was held for 4 minutes to re-equilibrate the column. |
| Flow Rate: | 0.2 mL/min |
| Injection Temperature: | 10 |
| Internal Standard: | Avanti EquiSplash mix |
| Sample Injection: | 5 µL |
| Solvent A: | 40% water/60% acetonitrile; 10 mM ammonium formate |
| Solvent B: | 90% isopropanol/10% acetonitrile; 10 mM ammonium formate |
| Capillary Voltage: | 3.50 KV |
| Running Voltage: | 3.50 KV |
| Weak Wash Solvent Name: | 90:10 Water:Acetontrile |
| Weak Wash Volume: | 300 µL |
| Strong Wash Solvent Name: | 1:1 Methanol:Isopropanol |
| Strong Wash Volume: | 50 µL |
| Sample Loop Size: | 10 µL |
| Sample Syringe Size: | 100 µL |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS003644 |
| Analysis ID: | AN003905 |
| Instrument Name: | Thermo Q Exactive HF-X Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | Analysis was performed using a Thermo Q Exactive Plus coupled to a Waters Acquity H-Class LC. A 100 mm x 2.1 mm, 2.1 µm Waters BEH C18 column was used for separations. The following mobile phases were used: A- 60/40 ACN/H20 B- 90/10 IPA/ACN; both mobile phases contained 10 mM Ammonium Formate and 0.1% Formic Acid. A flow rate of 0.2 mL/minutes was used. Starting composition was 32% B, which increased to 40% B at 1 minute (held until 1.5 minutes) then 45% B at 4 minutes. This was increased to 50% B at 5 minutes, 60% B at 8 minutes, 70% B at 11 minutes, and 80% B at 14 minutes (held until 16 minutes). At 16 minutes the composition switched back to starting conditions (32% B) and was held for 4 minutes to re-equilibrate the column. Samples were analyzed in positive/negative switching ionization mode with top 5 data dependent fragmentation. Raw data were analyzed by LipidSearch 4.2. Lipids were identified by MS2 fragmentation (mass error of precursor=5 ppm, mass error of product=8 ppm). The identifications were generated individually for each sample and then aligned by grouping the samples (OxPAPC=C, HF=S1, Con=S2). Normalization was performed using EquiSplash from Avanti. Samples were normalized and biological replicates were averaged. P-value and fold change were calculated as instructed as previously described (Aguilan, Kulej, & Sidoli, 2020). P-value was set to 0.05. |
| Ion Mode: | POSITIVE |
| Capillary Temperature: | 300 |
| Capillary Voltage: | 3.50 KV |
| Collision Energy: | 25, 35, 45 V stepped collision energy |
| Fragmentation Method: | HCD |
| Ion Source Temperature: | 400 |
| Ion Spray Voltage: | 3.50 KV |
| Ionization: | HESI |
| Mass Accuracy: | 5 ppm |
| Reagent Gas: | Nitrogen |
| Source Temperature: | 400 |
| Spray Voltage: | 3.5 KV |
