Lentiviral-based i53 variant libraries were transduced at an MOI of ~0.2–0.5 (aiming for ~ 30% transduction and a coverage of >500 cells per library member in mCherry + /GFP+ cell population for each replicate tested). Three days after transduction, cells were edited in triplicate or quadruplet at HBB (or NPM1) as described above, using HBB-UbC-GFP donor AAV6 (or NPM1-GFP donor AAV6) at an MOI of 2.5 × 104 vector genomes/cell. Three days post editing, cells were pelleted and resuspended in media with DAPI (Miltenyi Biotec). Single, live, mCherry + /GFP+ and mCherry + /GFP- cells were collected using a FACSAria cell sorter (Becton Dickinson); purity of populations was confirmed by post-sort purity checks. Post sort, genomic DNA was harvested from each sorted cell population using a Quick-DNA 96 Plus Kit (Zymo Research). The DNA concentration of each sample was measured using a Qubit 1X dsDNA BR assay kit (ThermoFisher).
Qubit 1x dsdna br assay kit
Manufactured by Thermo Fisher Scientific
The Qubit 1X dsDNA BR assay kit is a fluorometric-based solution for the quantification of double-stranded DNA (dsDNA) in samples. The kit provides a sensitive and accurate method to measure dsDNA concentrations.
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Lab products found in correlation
2 protocols using qubit 1x dsdna br assay kit
1
Lentiviral-based i53 variant library editing
2
Fetal Tissue Collection and DNA Extraction
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A total of 261 fetal tissue specimens from miscarriage and stillbirth were collected from Deyang People's Hospital, China, between 2018 and 2021. The chorionic villus or fetal muscle tissue was removed from uterine cavity by the clinical routine uterine curettage, and the blood on the tissue surface was washed with sterile normal saline. Chorionic villus would be sent for histological analysis and genetic testing simultaneously. Genetic testing is carried out only if the specimen is confirmed to be of fetal origin. About 10 mg of fetal tissue was minced into pieces and frozen overnight at −20°C for DNA extraction. Total genomic DNA was extracted from fetal specimens and parental peripheral blood using the TIANamp Genomic DNA Kit (TIANGEN Biotech) according to the user's manual. The concentration of genomic DNA was measured using Qubit 1X dsDNA BR Assay Kit (Thermo Fisher Scientific Inc.) with a Qubit 4 Fluorometer (Thermo Fisher Scientific).
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