GPR68
Homo sapiens
GPR68-p2a-puroR deep mutational library was delivered a genomic landing pad using BxbI recombinase in HEK293T cells, expressed, and assayed for receptor activation and surface expression using Fluorescence Activated Cell Sorting (FACS). Genomic DNA (gDNA), was isolated using the Zymo Quick-DNA Microprep Plus kit. The library was PCR amplified using all gDNA from each sample using TakaraBio PrimeStar GXL, with a final forward and reverse primer concentration of 0.3uM using the following parameters: initial denaturation at 98C for 30s, 24x cycles of denaturation at 98C for 10 sec, annealing at 60C for 15 sec, extension at 68C for 14 sec, and a final extension at 68C for 1 min. The purified libraries were gel purified (1% agarose, 1X TBE) with the Zymo Gel DNA Recovery kit and quantified by a Qubit dsDNA HS assay. Amplicons were  prepared for deep sequencing with the Nextera XT DNA Library Prep and indexed using IDT for Nextera Unique Dual Indexes (Set C). Indexed samples were quantified with TapeStation HS D5000 reagents, normalized, pooled, and paired-end sequenced on a NovaSeq6000.
isolate
N/A
age
N/A
biomaterial_provider
Coyote-Maestas Lab, UCSF, 1700 4th Street, Byers Hall Room 403ESan Francisco, CA 94158
collection_date
2022-11/2022-12
geo_loc_name
USA: San Francisco, CA
sex
N/A
tissue
N/A
cell_line
HEK293T
BioSampleModel
Human