{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST003108","ANALYSIS_ID":"AN005089","VERSION":"1","CREATED_ON":"February 27, 2024, 10:57 am"},

"PROJECT":{"PROJECT_TITLE":"Complete absence of GLUT1 does not impair human terminal erythroid differentiation","PROJECT_SUMMARY":"The Glucose transporter 1 (GLUT1) is one of the most abundant proteins within the erythrocyte membrane and is required for glucose and dehydroascorbic acid (Vitamin C precursor) transport. It is widely recognized as a key protein for red cell structure, function, and metabolism. Previous reports highlighted the importance of GLUT1 activity within these uniquely glycolysis-dependent cells, in particular for increasing antioxidant capacity needed to avoid irreversible damage from oxidative stress in humans. However, studies of glucose transporter roles in erythroid cells are complicated by species-specific differences between humans and mice. Here, using CRISPRmediated gene editing of immortalized erythroblasts and adult CD34+ hematopoietic progenitor cells, we generate committed human erythroid cells completely deficient in expression of GLUT1. We show that absence of GLUT1 does not impede human erythroblast proliferation, differentiation, or enucleation. This work demonstrates for the first-time generation of enucleated human reticulocytes lacking GLUT1. The GLUT1-deficient reticulocytes possess no tangible alterations to membrane composition or deformability in reticulocytes. Metabolomic analyses of GLUT1-deficient reticulocytes reveal hallmarks of reduced glucose import, downregulated metabolic processes and upregulated AMPK-signalling, alongside alterations in antioxidant metabolism, resulting in increased osmotic fragility and metabolic shifts indicative of higher oxidant stress. Despite detectable metabolic changes in GLUT1 deficient reticulocytes, the absence of developmental phenotype, detectable proteomic compensation or impaired deformability comprehensively alters our understanding of the role of GLUT1 in red blood cell structure, function and metabolism. It also provides cell biological evidence supporting clinical consensus that reduced GLUT1 expression does not cause anaemia in GLUT1 deficiency syndrome.","INSTITUTE":"University of Colorado","LAST_NAME":"Stephenson","FIRST_NAME":"Daniel","ADDRESS":"Research 1 South L18-1303 12801 E. 17th Ave., Aurora, Colorado, 80045, USA","EMAIL":"daniel.stephenson@cuanschutz.edu","PHONE":"303-724-3339"},

"STUDY":{"STUDY_TITLE":"Complete absence of GLUT1 does not impair human terminal erythroid differentiation","STUDY_SUMMARY":"The Glucose transporter 1 (GLUT1) is one of the most abundant proteins within the erythrocyte membrane and is required for glucose and dehydroascorbic acid (Vitamin C precursor) transport. It is widely recognized as a key protein for red cell structure, function, and metabolism. Previous reports highlighted the importance of GLUT1 activity within these uniquely glycolysis-dependent cells, in particular for increasing antioxidant capacity needed to avoid irreversible damage from oxidative stress in humans. However, studies of glucose transporter roles in erythroid cells are complicated by species-specific differences between humans and mice. Here, using CRISPRmediated gene editing of immortalized erythroblasts and adult CD34+ hematopoietic progenitor cells, we generate committed human erythroid cells completely deficient in expression of GLUT1. We show that absence of GLUT1 does not impede human erythroblast proliferation, differentiation, or enucleation. This work demonstrates for the first-time generation of enucleated human reticulocytes lacking GLUT1. The GLUT1-deficient reticulocytes possess no tangible alterations to membrane composition or deformability in reticulocytes. Metabolomic analyses of GLUT1-deficient reticulocytes reveal hallmarks of reduced glucose import, downregulated metabolic processes and upregulated AMPK-signalling, alongside alterations in antioxidant metabolism, resulting in increased osmotic fragility and metabolic shifts indicative of higher oxidant stress. Despite detectable metabolic changes in GLUT1 deficient reticulocytes, the absence of developmental phenotype, detectable proteomic compensation or impaired deformability comprehensively alters our understanding of the role of GLUT1 in red blood cell structure, function and metabolism. It also provides cell biological evidence supporting clinical consensus that reduced GLUT1 expression does not cause anaemia in GLUT1 deficiency syndrome.","INSTITUTE":"University of Colorado","LAST_NAME":"Stephenson","FIRST_NAME":"Daniel","ADDRESS":"Research 1 South L18-1303 12801 E. 17th Ave., Aurora, Colorado, 80045, USA","EMAIL":"daniel.stephenson@cuanschutz.edu","PHONE":"303-724-3339"},

"SUBJECT":{"SUBJECT_TYPE":"Cultured cells","SUBJECT_SPECIES":"Homo sapiens","TAXONOMY_ID":"9606"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"Donor 1",
"Sample ID":"DS1-095-001+",
"Factors":{"Sample source":"cultured reticulocytes","Assay":"04CF29"},
"Additional sample data":{"RAW_FILE_NAME(Raw File Name)":"DS1-095-001+.mzXML","Pellet size":"10 Million","Analysis":"Metabolomics","Polarity":"Positive"}
},
{
"Subject ID":"Donor 1",
"Sample ID":"DS1-095-002+",
"Factors":{"Sample source":"cultured reticulocytes","Assay":"04CF29"},
"Additional sample data":{"RAW_FILE_NAME(Raw File Name)":"DS1-095-002+.mzXML","Pellet size":"10 Million","Analysis":"Metabolomics","Polarity":"Positive"}
},
{
"Subject ID":"Donor 4",
"Sample ID":"DS1-095-003+",
"Factors":{"Sample source":"cultured reticulocytes","Assay":"04CF29"},
"Additional sample data":{"RAW_FILE_NAME(Raw File Name)":"DS1-095-003+.mzXML","Pellet size":"10 Million","Analysis":"Metabolomics","Polarity":"Positive"}
},
{
"Subject ID":"Donor 4",
"Sample ID":"DS1-095-004+",
"Factors":{"Sample source":"cultured reticulocytes","Assay":"04CF29"},
"Additional sample data":{"RAW_FILE_NAME(Raw File Name)":"DS1-095-004+.mzXML","Pellet size":"10 Million","Analysis":"Metabolomics","Polarity":"Positive"}
},
{
"Subject ID":"Donor 5",
"Sample ID":"DS1-095-005+",
"Factors":{"Sample source":"cultured reticulocytes","Assay":"04CF29"},
"Additional sample data":{"RAW_FILE_NAME(Raw File Name)":"DS1-095-005+.mzXML","Pellet size":"10 Million","Analysis":"Metabolomics","Polarity":"Positive"}
},
{
"Subject ID":"Donor 5",
"Sample ID":"DS1-095-006+",
"Factors":{"Sample source":"cultured reticulocytes","Assay":"04CF29"},
"Additional sample data":{"RAW_FILE_NAME(Raw File Name)":"DS1-095-006+.mzXML","Pellet size":"10 Million","Analysis":"Metabolomics","Polarity":"Positive"}
},
{
"Subject ID":"Donor A",
"Sample ID":"DS1-095-007+",
"Factors":{"Sample source":"cultured reticulocytes","Assay":"01CF19"},
"Additional sample data":{"RAW_FILE_NAME(Raw File Name)":"DS1-095-007+.mzXML","Pellet size":"10 Million","Analysis":"Metabolomics","Polarity":"Positive"}
},
{
"Subject ID":"Donor C",
"Sample ID":"DS1-095-008+",
"Factors":{"Sample source":"cultured reticulocytes","Assay":"01CF19"},
"Additional sample data":{"RAW_FILE_NAME(Raw File Name)":"DS1-095-008+.mzXML","Pellet size":"10 Million","Analysis":"Metabolomics","Polarity":"Positive"}
},
{
"Subject ID":"Donor D",
"Sample ID":"DS1-095-009+",
"Factors":{"Sample source":"cultured reticulocytes","Assay":"01CF19"},
"Additional sample data":{"RAW_FILE_NAME(Raw File Name)":"DS1-095-009+.mzXML","Pellet size":"10 Million","Analysis":"Metabolomics","Polarity":"Positive"}
},
{
"Subject ID":"Donor A",
"Sample ID":"DS1-095-010+",
"Factors":{"Sample source":"cultured reticulocytes","Assay":"01CF19"},
"Additional sample data":{"RAW_FILE_NAME(Raw File Name)":"DS1-095-010+.mzXML","Pellet size":"10 Million","Analysis":"Metabolomics","Polarity":"Positive"}
},
{
"Subject ID":"Donor C",
"Sample ID":"DS1-095-011+",
"Factors":{"Sample source":"cultured reticulocytes","Assay":"01CF19"},
"Additional sample data":{"RAW_FILE_NAME(Raw File Name)":"DS1-095-011+.mzXML","Pellet size":"10 Million","Analysis":"Metabolomics","Polarity":"Positive"}
},
{
"Subject ID":"Donor D",
"Sample ID":"DS1-095-012+",
"Factors":{"Sample source":"cultured reticulocytes","Assay":"01CF19"},
"Additional sample data":{"RAW_FILE_NAME(Raw File Name)":"DS1-095-012+.mzXML","Pellet size":"10 Million","Analysis":"Metabolomics","Polarity":"Positive"}
},
{
"Subject ID":"Donor A",
"Sample ID":"DS1-095-013+",
"Factors":{"Sample source":"cultured reticulocytes","Assay":"01CF19"},
"Additional sample data":{"RAW_FILE_NAME(Raw File Name)":"DS1-095-013+.mzXML","Pellet size":"10 Million","Analysis":"Metabolomics","Polarity":"Positive"}
},
{
"Subject ID":"Donor C",
"Sample ID":"DS1-095-014+",
"Factors":{"Sample source":"cultured reticulocytes","Assay":"01CF19"},
"Additional sample data":{"RAW_FILE_NAME(Raw File Name)":"DS1-095-014+.mzXML","Pellet size":"10 Million","Analysis":"Metabolomics","Polarity":"Positive"}
},
{
"Subject ID":"Donor D",
"Sample ID":"DS1-095-015+",
"Factors":{"Sample source":"cultured reticulocytes","Assay":"01CF19"},
"Additional sample data":{"RAW_FILE_NAME(Raw File Name)":"DS1-095-015+.mzXML","Pellet size":"10 Million","Analysis":"Metabolomics","Polarity":"Positive"}
},
{
"Subject ID":"Donor A",
"Sample ID":"DS1-095-016+",
"Factors":{"Sample source":"Red blood cells","Assay":"01CF19"},
"Additional sample data":{"RAW_FILE_NAME(Raw File Name)":"DS1-095-016+.mzXML","Pellet size":"10 Million","Analysis":"Metabolomics","Polarity":"Positive"}
},
{
"Subject ID":"Donor C",
"Sample ID":"DS1-095-017+",
"Factors":{"Sample source":"Red blood cells","Assay":"01CF19"},
"Additional sample data":{"RAW_FILE_NAME(Raw File Name)":"DS1-095-017+.mzXML","Pellet size":"10 Million","Analysis":"Metabolomics","Polarity":"Positive"}
},
{
"Subject ID":"Donor D",
"Sample ID":"DS1-095-018+",
"Factors":{"Sample source":"Red blood cells","Assay":"01CF19"},
"Additional sample data":{"RAW_FILE_NAME(Raw File Name)":"DS1-095-018+.mzXML","Pellet size":"10 Million","Analysis":"Metabolomics","Polarity":"Positive"}
},
{
"Subject ID":"Donor 1",
"Sample ID":"DS1-095-001-",
"Factors":{"Sample source":"cultured reticulocytes","Assay":"04CF29"},
"Additional sample data":{"RAW_FILE_NAME(Raw File Name)":"DS1-095-001-.mzXML","Pellet size":"10 Million","Analysis":"Metabolomics","Polarity":"Negative"}
},
{
"Subject ID":"Donor 1",
"Sample ID":"DS1-095-002-",
"Factors":{"Sample source":"cultured reticulocytes","Assay":"04CF29"},
"Additional sample data":{"RAW_FILE_NAME(Raw File Name)":"DS1-095-002-.mzXML","Pellet size":"10 Million","Analysis":"Metabolomics","Polarity":"Negative"}
},
{
"Subject ID":"Donor 4",
"Sample ID":"DS1-095-003-",
"Factors":{"Sample source":"cultured reticulocytes","Assay":"04CF29"},
"Additional sample data":{"RAW_FILE_NAME(Raw File Name)":"DS1-095-003-.mzXML","Pellet size":"10 Million","Analysis":"Metabolomics","Polarity":"Negative"}
},
{
"Subject ID":"Donor 4",
"Sample ID":"DS1-095-004-",
"Factors":{"Sample source":"cultured reticulocytes","Assay":"04CF29"},
"Additional sample data":{"RAW_FILE_NAME(Raw File Name)":"DS1-095-004-.mzXML","Pellet size":"10 Million","Analysis":"Metabolomics","Polarity":"Negative"}
},
{
"Subject ID":"Donor 5",
"Sample ID":"DS1-095-005-",
"Factors":{"Sample source":"cultured reticulocytes","Assay":"04CF29"},
"Additional sample data":{"RAW_FILE_NAME(Raw File Name)":"DS1-095-005-.mzXML","Pellet size":"10 Million","Analysis":"Metabolomics","Polarity":"Negative"}
},
{
"Subject ID":"Donor 5",
"Sample ID":"DS1-095-006-",
"Factors":{"Sample source":"cultured reticulocytes","Assay":"04CF29"},
"Additional sample data":{"RAW_FILE_NAME(Raw File Name)":"DS1-095-006-.mzXML","Pellet size":"10 Million","Analysis":"Metabolomics","Polarity":"Negative"}
},
{
"Subject ID":"Donor A",
"Sample ID":"DS1-095-007-",
"Factors":{"Sample source":"cultured reticulocytes","Assay":"01CF19"},
"Additional sample data":{"RAW_FILE_NAME(Raw File Name)":"DS1-095-007-.mzXML","Pellet size":"10 Million","Analysis":"Metabolomics","Polarity":"Negative"}
},
{
"Subject ID":"Donor C",
"Sample ID":"DS1-095-008-",
"Factors":{"Sample source":"cultured reticulocytes","Assay":"01CF19"},
"Additional sample data":{"RAW_FILE_NAME(Raw File Name)":"DS1-095-008-.mzXML","Pellet size":"10 Million","Analysis":"Metabolomics","Polarity":"Negative"}
},
{
"Subject ID":"Donor D",
"Sample ID":"DS1-095-009-",
"Factors":{"Sample source":"cultured reticulocytes","Assay":"01CF19"},
"Additional sample data":{"RAW_FILE_NAME(Raw File Name)":"DS1-095-009-.mzXML","Pellet size":"10 Million","Analysis":"Metabolomics","Polarity":"Negative"}
},
{
"Subject ID":"Donor A",
"Sample ID":"DS1-095-010-",
"Factors":{"Sample source":"cultured reticulocytes","Assay":"01CF19"},
"Additional sample data":{"RAW_FILE_NAME(Raw File Name)":"DS1-095-010-.mzXML","Pellet size":"10 Million","Analysis":"Metabolomics","Polarity":"Negative"}
},
{
"Subject ID":"Donor C",
"Sample ID":"DS1-095-011-",
"Factors":{"Sample source":"cultured reticulocytes","Assay":"01CF19"},
"Additional sample data":{"RAW_FILE_NAME(Raw File Name)":"DS1-095-011-.mzXML","Pellet size":"10 Million","Analysis":"Metabolomics","Polarity":"Negative"}
},
{
"Subject ID":"Donor D",
"Sample ID":"DS1-095-012-",
"Factors":{"Sample source":"cultured reticulocytes","Assay":"01CF19"},
"Additional sample data":{"RAW_FILE_NAME(Raw File Name)":"DS1-095-012-.mzXML","Pellet size":"10 Million","Analysis":"Metabolomics","Polarity":"Negative"}
},
{
"Subject ID":"Donor A",
"Sample ID":"DS1-095-013-",
"Factors":{"Sample source":"cultured reticulocytes","Assay":"01CF19"},
"Additional sample data":{"RAW_FILE_NAME(Raw File Name)":"DS1-095-013-.mzXML","Pellet size":"10 Million","Analysis":"Metabolomics","Polarity":"Negative"}
},
{
"Subject ID":"Donor C",
"Sample ID":"DS1-095-014-",
"Factors":{"Sample source":"cultured reticulocytes","Assay":"01CF19"},
"Additional sample data":{"RAW_FILE_NAME(Raw File Name)":"DS1-095-014-.mzXML","Pellet size":"10 Million","Analysis":"Metabolomics","Polarity":"Negative"}
},
{
"Subject ID":"Donor D",
"Sample ID":"DS1-095-015-",
"Factors":{"Sample source":"cultured reticulocytes","Assay":"01CF19"},
"Additional sample data":{"RAW_FILE_NAME(Raw File Name)":"DS1-095-015-.mzXML","Pellet size":"10 Million","Analysis":"Metabolomics","Polarity":"Negative"}
},
{
"Subject ID":"Donor A",
"Sample ID":"DS1-095-016-",
"Factors":{"Sample source":"Red blood cells","Assay":"01CF19"},
"Additional sample data":{"RAW_FILE_NAME(Raw File Name)":"DS1-095-016-.mzXML","Pellet size":"10 Million","Analysis":"Metabolomics","Polarity":"Negative"}
},
{
"Subject ID":"Donor C",
"Sample ID":"DS1-095-017-",
"Factors":{"Sample source":"Red blood cells","Assay":"01CF19"},
"Additional sample data":{"RAW_FILE_NAME(Raw File Name)":"DS1-095-017-.mzXML","Pellet size":"10 Million","Analysis":"Metabolomics","Polarity":"Negative"}
},
{
"Subject ID":"Donor D",
"Sample ID":"DS1-095-018-",
"Factors":{"Sample source":"Red blood cells","Assay":"01CF19"},
"Additional sample data":{"RAW_FILE_NAME(Raw File Name)":"DS1-095-018-.mzXML","Pellet size":"10 Million","Analysis":"Metabolomics","Polarity":"Negative"}
},
{
"Subject ID":"Donor 1",
"Sample ID":"DS1-095-001L+",
"Factors":{"Sample source":"cultured reticulocytes","Assay":"04CF29"},
"Additional sample data":{"RAW_FILE_NAME(Raw File Name)":"DS1-095-001L+.RAW","Pellet size":"10 Million","Analysis":"Lipidomics","Polarity":"Positive"}
},
{
"Subject ID":"Donor 1",
"Sample ID":"DS1-095-002L+",
"Factors":{"Sample source":"cultured reticulocytes","Assay":"04CF29"},
"Additional sample data":{"RAW_FILE_NAME(Raw File Name)":"DS1-095-002L+.RAW","Pellet size":"10 Million","Analysis":"Lipidomics","Polarity":"Positive"}
},
{
"Subject ID":"Donor 4",
"Sample ID":"DS1-095-003L+",
"Factors":{"Sample source":"cultured reticulocytes","Assay":"04CF29"},
"Additional sample data":{"RAW_FILE_NAME(Raw File Name)":"DS1-095-003L+.RAW","Pellet size":"10 Million","Analysis":"Lipidomics","Polarity":"Positive"}
},
{
"Subject ID":"Donor 4",
"Sample ID":"DS1-095-004L+",
"Factors":{"Sample source":"cultured reticulocytes","Assay":"04CF29"},
"Additional sample data":{"RAW_FILE_NAME(Raw File Name)":"DS1-095-004L+.RAW","Pellet size":"10 Million","Analysis":"Lipidomics","Polarity":"Positive"}
},
{
"Subject ID":"Donor 5",
"Sample ID":"DS1-095-005L+",
"Factors":{"Sample source":"cultured reticulocytes","Assay":"04CF29"},
"Additional sample data":{"RAW_FILE_NAME(Raw File Name)":"DS1-095-005L+.RAW","Pellet size":"10 Million","Analysis":"Lipidomics","Polarity":"Positive"}
},
{
"Subject ID":"Donor 5",
"Sample ID":"DS1-095-006L+",
"Factors":{"Sample source":"cultured reticulocytes","Assay":"04CF29"},
"Additional sample data":{"RAW_FILE_NAME(Raw File Name)":"DS1-095-006L+.RAW","Pellet size":"10 Million","Analysis":"Lipidomics","Polarity":"Positive"}
},
{
"Subject ID":"Donor A",
"Sample ID":"DS1-095-007L+",
"Factors":{"Sample source":"cultured reticulocytes","Assay":"01CF19"},
"Additional sample data":{"RAW_FILE_NAME(Raw File Name)":"DS1-095-007L+.RAW","Pellet size":"10 Million","Analysis":"Lipidomics","Polarity":"Positive"}
},
{
"Subject ID":"Donor C",
"Sample ID":"DS1-095-008L+",
"Factors":{"Sample source":"cultured reticulocytes","Assay":"01CF19"},
"Additional sample data":{"RAW_FILE_NAME(Raw File Name)":"DS1-095-008L+.RAW","Pellet size":"10 Million","Analysis":"Lipidomics","Polarity":"Positive"}
},
{
"Subject ID":"Donor D",
"Sample ID":"DS1-095-009L+",
"Factors":{"Sample source":"cultured reticulocytes","Assay":"01CF19"},
"Additional sample data":{"RAW_FILE_NAME(Raw File Name)":"DS1-095-009L+.RAW","Pellet size":"10 Million","Analysis":"Lipidomics","Polarity":"Positive"}
},
{
"Subject ID":"Donor A",
"Sample ID":"DS1-095-010L+",
"Factors":{"Sample source":"cultured reticulocytes","Assay":"01CF19"},
"Additional sample data":{"RAW_FILE_NAME(Raw File Name)":"DS1-095-010L+.RAW","Pellet size":"10 Million","Analysis":"Lipidomics","Polarity":"Positive"}
},
{
"Subject ID":"Donor C",
"Sample ID":"DS1-095-011L+",
"Factors":{"Sample source":"cultured reticulocytes","Assay":"01CF19"},
"Additional sample data":{"RAW_FILE_NAME(Raw File Name)":"DS1-095-011L+.RAW","Pellet size":"10 Million","Analysis":"Lipidomics","Polarity":"Positive"}
},
{
"Subject ID":"Donor D",
"Sample ID":"DS1-095-012L+",
"Factors":{"Sample source":"cultured reticulocytes","Assay":"01CF19"},
"Additional sample data":{"RAW_FILE_NAME(Raw File Name)":"DS1-095-012L+.RAW","Pellet size":"10 Million","Analysis":"Lipidomics","Polarity":"Positive"}
},
{
"Subject ID":"Donor A",
"Sample ID":"DS1-095-013L+",
"Factors":{"Sample source":"cultured reticulocytes","Assay":"01CF19"},
"Additional sample data":{"RAW_FILE_NAME(Raw File Name)":"DS1-095-013L+.RAW","Pellet size":"10 Million","Analysis":"Lipidomics","Polarity":"Positive"}
},
{
"Subject ID":"Donor C",
"Sample ID":"DS1-095-014L+",
"Factors":{"Sample source":"cultured reticulocytes","Assay":"01CF19"},
"Additional sample data":{"RAW_FILE_NAME(Raw File Name)":"DS1-095-014L+.RAW","Pellet size":"10 Million","Analysis":"Lipidomics","Polarity":"Positive"}
},
{
"Subject ID":"Donor D",
"Sample ID":"DS1-095-015L+",
"Factors":{"Sample source":"cultured reticulocytes","Assay":"01CF19"},
"Additional sample data":{"RAW_FILE_NAME(Raw File Name)":"DS1-095-015L+.RAW","Pellet size":"10 Million","Analysis":"Lipidomics","Polarity":"Positive"}
},
{
"Subject ID":"Donor A",
"Sample ID":"DS1-095-016L+",
"Factors":{"Sample source":"Red blood cells","Assay":"01CF19"},
"Additional sample data":{"RAW_FILE_NAME(Raw File Name)":"DS1-095-016L+.RAW","Pellet size":"10 Million","Analysis":"Lipidomics","Polarity":"Positive"}
},
{
"Subject ID":"Donor C",
"Sample ID":"DS1-095-017L+",
"Factors":{"Sample source":"Red blood cells","Assay":"01CF19"},
"Additional sample data":{"RAW_FILE_NAME(Raw File Name)":"DS1-095-017L+.RAW","Pellet size":"10 Million","Analysis":"Lipidomics","Polarity":"Positive"}
},
{
"Subject ID":"Donor D",
"Sample ID":"DS1-095-018L+",
"Factors":{"Sample source":"Red blood cells","Assay":"01CF19"},
"Additional sample data":{"RAW_FILE_NAME(Raw File Name)":"DS1-095-018L+.RAW","Pellet size":"10 Million","Analysis":"Lipidomics","Polarity":"Positive"}
},
{
"Subject ID":"Donor 1",
"Sample ID":"DS1-095-001L-",
"Factors":{"Sample source":"cultured reticulocytes","Assay":"04CF29"},
"Additional sample data":{"RAW_FILE_NAME(Raw File Name)":"DS1-095-001L-.RAW","Pellet size":"10 Million","Analysis":"Lipidomics","Polarity":"Negative"}
},
{
"Subject ID":"Donor 1",
"Sample ID":"DS1-095-002L-",
"Factors":{"Sample source":"cultured reticulocytes","Assay":"04CF29"},
"Additional sample data":{"RAW_FILE_NAME(Raw File Name)":"DS1-095-002L-.RAW","Pellet size":"10 Million","Analysis":"Lipidomics","Polarity":"Negative"}
},
{
"Subject ID":"Donor 4",
"Sample ID":"DS1-095-003L-",
"Factors":{"Sample source":"cultured reticulocytes","Assay":"04CF29"},
"Additional sample data":{"RAW_FILE_NAME(Raw File Name)":"DS1-095-003L-.RAW","Pellet size":"10 Million","Analysis":"Lipidomics","Polarity":"Negative"}
},
{
"Subject ID":"Donor 4",
"Sample ID":"DS1-095-004L-",
"Factors":{"Sample source":"cultured reticulocytes","Assay":"04CF29"},
"Additional sample data":{"RAW_FILE_NAME(Raw File Name)":"DS1-095-004L-.RAW","Pellet size":"10 Million","Analysis":"Lipidomics","Polarity":"Negative"}
},
{
"Subject ID":"Donor 5",
"Sample ID":"DS1-095-005L-",
"Factors":{"Sample source":"cultured reticulocytes","Assay":"04CF29"},
"Additional sample data":{"RAW_FILE_NAME(Raw File Name)":"DS1-095-005L-.RAW","Pellet size":"10 Million","Analysis":"Lipidomics","Polarity":"Negative"}
},
{
"Subject ID":"Donor 5",
"Sample ID":"DS1-095-006L-",
"Factors":{"Sample source":"cultured reticulocytes","Assay":"04CF29"},
"Additional sample data":{"RAW_FILE_NAME(Raw File Name)":"DS1-095-006L-.RAW","Pellet size":"10 Million","Analysis":"Lipidomics","Polarity":"Negative"}
},
{
"Subject ID":"Donor A",
"Sample ID":"DS1-095-007L-",
"Factors":{"Sample source":"cultured reticulocytes","Assay":"01CF19"},
"Additional sample data":{"RAW_FILE_NAME(Raw File Name)":"DS1-095-007L-.RAW","Pellet size":"10 Million","Analysis":"Lipidomics","Polarity":"Negative"}
},
{
"Subject ID":"Donor C",
"Sample ID":"DS1-095-008L-",
"Factors":{"Sample source":"cultured reticulocytes","Assay":"01CF19"},
"Additional sample data":{"RAW_FILE_NAME(Raw File Name)":"DS1-095-008L-.RAW","Pellet size":"10 Million","Analysis":"Lipidomics","Polarity":"Negative"}
},
{
"Subject ID":"Donor D",
"Sample ID":"DS1-095-009L-",
"Factors":{"Sample source":"cultured reticulocytes","Assay":"01CF19"},
"Additional sample data":{"RAW_FILE_NAME(Raw File Name)":"DS1-095-009L-.RAW","Pellet size":"10 Million","Analysis":"Lipidomics","Polarity":"Negative"}
},
{
"Subject ID":"Donor A",
"Sample ID":"DS1-095-010L-",
"Factors":{"Sample source":"cultured reticulocytes","Assay":"01CF19"},
"Additional sample data":{"RAW_FILE_NAME(Raw File Name)":"DS1-095-010L-.RAW","Pellet size":"10 Million","Analysis":"Lipidomics","Polarity":"Negative"}
},
{
"Subject ID":"Donor C",
"Sample ID":"DS1-095-011L-",
"Factors":{"Sample source":"cultured reticulocytes","Assay":"01CF19"},
"Additional sample data":{"RAW_FILE_NAME(Raw File Name)":"DS1-095-011L-.RAW","Pellet size":"10 Million","Analysis":"Lipidomics","Polarity":"Negative"}
},
{
"Subject ID":"Donor D",
"Sample ID":"DS1-095-012L-",
"Factors":{"Sample source":"cultured reticulocytes","Assay":"01CF19"},
"Additional sample data":{"RAW_FILE_NAME(Raw File Name)":"DS1-095-012L-.RAW","Pellet size":"10 Million","Analysis":"Lipidomics","Polarity":"Negative"}
},
{
"Subject ID":"Donor A",
"Sample ID":"DS1-095-013L-",
"Factors":{"Sample source":"cultured reticulocytes","Assay":"01CF19"},
"Additional sample data":{"RAW_FILE_NAME(Raw File Name)":"DS1-095-013L-.RAW","Pellet size":"10 Million","Analysis":"Lipidomics","Polarity":"Negative"}
},
{
"Subject ID":"Donor C",
"Sample ID":"DS1-095-014L-",
"Factors":{"Sample source":"cultured reticulocytes","Assay":"01CF19"},
"Additional sample data":{"RAW_FILE_NAME(Raw File Name)":"DS1-095-014L-.RAW","Pellet size":"10 Million","Analysis":"Lipidomics","Polarity":"Negative"}
},
{
"Subject ID":"Donor D",
"Sample ID":"DS1-095-015L-",
"Factors":{"Sample source":"cultured reticulocytes","Assay":"01CF19"},
"Additional sample data":{"RAW_FILE_NAME(Raw File Name)":"DS1-095-015L-.RAW","Pellet size":"10 Million","Analysis":"Lipidomics","Polarity":"Negative"}
},
{
"Subject ID":"Donor A",
"Sample ID":"DS1-095-016L-",
"Factors":{"Sample source":"Red blood cells","Assay":"01CF19"},
"Additional sample data":{"RAW_FILE_NAME(Raw File Name)":"DS1-095-016L-.RAW","Pellet size":"10 Million","Analysis":"Lipidomics","Polarity":"Negative"}
},
{
"Subject ID":"Donor C",
"Sample ID":"DS1-095-017L-",
"Factors":{"Sample source":"Red blood cells","Assay":"01CF19"},
"Additional sample data":{"RAW_FILE_NAME(Raw File Name)":"DS1-095-017L-.RAW","Pellet size":"10 Million","Analysis":"Lipidomics","Polarity":"Negative"}
},
{
"Subject ID":"Donor D",
"Sample ID":"DS1-095-018L-",
"Factors":{"Sample source":"Red blood cells","Assay":"01CF19"},
"Additional sample data":{"RAW_FILE_NAME(Raw File Name)":"DS1-095-018L-.RAW","Pellet size":"10 Million","Analysis":"Lipidomics","Polarity":"Negative"}
}
],
"COLLECTION":{"COLLECTION_SUMMARY":"For flow cytometry, cells were fixed (1% paraformaldehyde, 0.0075% glutaraldehyde) to prevent antibody agglutination, except for assays using GLUT1.RBD. Samples were labelled with primary antibodies in PBSAG (PBS with 1% (w/v) Glucose and 0.5%(w/v) |Bovine Serum Albumin (BSA)) supplemented with extra 1%(w/v) BSA for 30min, in the dark. METAFORA’s GLUT1.RBD was incubated at 37°C and all remaining antibodies incubated at 4°C. Cells were washed 2x PBSAG and, if required, incubated with appropriate secondary antibody under the same conditions as described for primary. Cells were washed 2x with PBSAG and analysed on a Miltenyi MACSQuant 10 flow cytometer. Data was analysed using FlowJo v10.7 (FlowJo). Reticulocytes were identified by gating on the Hoechst-negative population. 4 Cells were sorted using a BD Influx Cell Sorter (BD Biosciences). BEL-A CRISPR edited populations were single cell sorted based on viability (DRAQ7 negativity). Primary GLUT1 KO cultures were sorted on days 6 or 7 of differentiation using DRAQ7 and the eGFP-fused GLUT1.RBD to purify the negative population with a gate based on non-targeting guide control cells.","SAMPLE_TYPE":"Cultured cells"},

"TREATMENT":{"TREATMENT_SUMMARY":"Filtered reticulocytes (1-2 x105/well) were incubated in decreasing NaCl concentrations (0.9–0%) for 10min at 37°C. Lysis was stopped by adding 4x volume of PBSAG. Live cells, considered as having a normal FSC/SSC profile as defined by the 0.9% NaCl control, were counted by flow cytometry using the MACSQuant10"},

"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"Extraction of metabolites and lipids from red cell pellets was as follows: 36 µL of cold MeOH:MeCN:H2O (5:3:2, v:v:v) was added to all pellets (pellet size pre-determined to be 10 million prior to extraction). Samples were vortexed at 4°C for 30 minutes. Insoluble material was pelleted by centrifugation (18000g, 10 min) and supernatants were isolated for analysis by UHPLC-MS for metabolomics and lipidomics."},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_SUMMARY":"Lipidomics Positive","CHROMATOGRAPHY_TYPE":"Reversed phase","INSTRUMENT_NAME":"Thermo Vanquish","COLUMN_NAME":"Phenomenex Kinetex C18 (30 x 2.1mm, 1.7um)","SOLVENT_A":"75:25 H2O:ACN 5mM NH4OAc","SOLVENT_B":"90:10 iPrOH:ACN 5mM NH4OAc","FLOW_GRADIENT":"0 min - 0.3ml/min - 30%B, 3 min - 0.3ml/min - 100%B, 4.2min - 0.3ml/min - 100%B, 4.3min - 0.4ml/min - 30%B, 4.9min - 0/4ml/min - 30%B, 5 min - 0.3ml/min 30%B","FLOW_RATE":"0.3-0.4ml/min","COLUMN_TEMPERATURE":"50"},

"ANALYSIS":{"ANALYSIS_TYPE":"MS"},

"MS":{"INSTRUMENT_NAME":"Thermo Q Exactive Orbitrap","INSTRUMENT_TYPE":"Orbitrap","MS_TYPE":"ESI","ION_MODE":"POSITIVE","MS_COMMENTS":"Lipidsearch used for data analysis"},

"MS_METABOLITE_DATA":{
"Units":"area",

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