GSM9801835: Grp7, mCherry quartile sort, lower quartile; synthetic construct; OTHER
Genomic DNA was extracted using a Dneasy Blood and Tissue Kit (Qiagen, 69506) and DNA concentration was calculated using a Qubit 4 fluorometer (Thermo Fisher, Q33238) using a Qubit Broad Range dsDNA assay kit (Thermo Fisher, Q33265) Extracted genomic DNA was used as a template to produce a 1564 nucleotide amplicon spanning the genomic integration junction.  Samples were run with gel electrophoresis, the appropriate band was extracted using a Bio-Rad Freeze 'N Squeeze DNA Gel Extraction Spin Column (Bio-Rad, 7326165) and cleaned using a Zymo DNA Clean and Concentrator-5 kit (Zymo Research, D4004).  The resulting DNA was used as the template for to produce a 299 nudleotide amplicon surrounding the DNA sequence of interest using distinct indices for each quartile to be run on the same Illumina NextSeq 550 kit run.  Samples were again run with gel electrophoresis, extracted, and cleaned.  DNA concentration was calculated using a Qubit 4 fluorometer (Thermo Fisher, Q33238) using a Qubit Broad Range dsDNA assay kit (Thermo Fisher, Q33265).  DNA amplicons were pooled to a final concentration of 10-20ng/uL in 21uL of nuclease free water using 15-20ng of DNA from each quartile prior to running the Illumina NextSeq 550 kit.
GEO
GSM9801835_r1