Computational protocol: The human ankyrin 1 promoter insulator sustains gene expression in a β-globin lentiviral vector in hematopoietic stem cells

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Protocol publication

[…] Secondary mice were bled 11 weeks post-transplant. One microliter of peripheral blood per was lysed as follows. RBC were pelleted and lysed in Hemolysate reagent (Helena Laboratories, Beaumont, TX) for 5–10 minutes at room temperature. After centrifugation at 20,800g for 10 minutes at 4°C to remove cellular debris, RBC lysates were stored frozen at −80°C. Upon thawing, cell lysates were diluted 1:40 in mobile phase A and characterized by HPLC (Infinity 1260, Agilent) using a weak cation-exchange column (PolyCAT A, PolyLC, Coulmbia, MD). FASC Reference Material (Trinity Biotech, Wicklow, Ireland) was used to define the elution time of common human hemoglobin forms (HbF, HbA, HbS, and HbC). Analysis and peak integration were performed using OpenLAB CDS Chemstation software (Agilent, Santa Clara, CA). The relative percentage of HbAS3 produced for each sample was calculated based on the sum total of areas under the curve for each of the primary hemoglobin peaks, which included βmajor and βminor. […]

Pipeline specifications

Software tools PolyCat, Trinity
Application RNA-seq analysis
Organisms Homo sapiens, Mus musculus
Diseases Leukemia, Erythroblastic, Acute, Hemoglobinopathies