Genemapper id v3

CTC cultures were grown at 37 ⁰C under hypoxic conditions (4% O2) in ultra-low attachment flasks (Corning) in CTC media comprising of RPMI-1640, 1X antibiotic/antimycotic supplemented with bFGF (20 ng/ml), EGF (20 ng/ml), and 1X B27 (Life Technologies). Cultures were routinely checked for mycoplasma with the MycoAlert, Lonza Kit. Cell cultures and patient blood were tested for authentication via STR profiling by Genetica DNA Laboratories (a LabCorp brand; Burlington, NC) using the commercially available PowerPlex^® 16HS amplification kit (Promega Corporation; mouse marker included) and GeneMapper ID v3.2.1 software (Applied Biosystems). Brx50 and Brx68 have been described previously by Yu et al [16 (link)]. Brx211, Brx250, and Brx394 have been previously described by Brett et al [30 (link)]. Mutational screening for 98 known cancer genes was performed using the MGH SNaPshot-NGS clinical assay as described elsewhere [31 (link)]. The assay includes single-nucleotide variants (SNVs) within ESR1 as well as ESR gene fusions [32 (link)].

DNA was extracted using the Chelex-100 as described in previous protocol15 (link). Multiplexed PCR amplifications of 60 variants were co-amplified in 5 fluorescence-based reaction using Expressmarker mtDNA-SNP 60 kit (nt10398, 9 bp, 10873, 3010, 709, 7196, 12705, 3970, 13104, 10310, 5178, 13928, 6446, 8414, 8793, 8794, 15043, 16311, 16126, 16129, 8701, 8697, 4883, 10400, CA, 1719, 14668, 12811, 9824, 9123, 7028, 11719, 8584, 11251, 8020, 5460, 2706, 11215, 4216, 12372, 16362, 9698, 1541, 8684, 9477, 4491, 1811, 16316, 16319, 9545, 152, 14569, 8964, 10397, 3348, 4833, 7600, 5417, 5442, 15784, AGCU ScienTech Incorporation, Jiangsu, Wuxi). Briefly, PCR amplification system (25 μl) contained 1 μl genomic DNA, 10 μl reaction mix, 5 μl primers, 1μl tag DNA polymerase (5U) and 8 μl sdH₂O. The cycling parameters were set up according to the manufacturer’s protocol. The PCR production of 1 μl was mixed with 0.5 μl AGCUMarkerSIZ-500 and 12 μl Hi-Di formamide. Electrophoresis was performed by the ABI Prism 3130XL Genetic analyzer and fragment sizing was analyzed by GeneMapper ID v3.2.1 software (Applied Biosystems, USA). Control DNA 9947A was used as the positive control in our experiment.

DNA amplification of all the individuals was performed using the AmpFLSTR Yfiler^TM Kit (Thermo Fisher Scientific, USA), the AmpFLSTR Yfiler^TM Plus Kit (Thermo Fisher Scientific, USA) and the AGCU Y SUPP (AGCU ScienTech Incorporation, China) on a ProFlex^TM 96-well PCR System (Thermo Fisher Scientific, USA) according to the manufacturer’s recommendations. The PCR products were separated by CE on the ABI 3500xl Genetic Analyzer (Applied Biosystems, USA) and all haplotypes were determined with GeneMapper ID v3.2 software (Thermo Fisher Scientific). Four samples failed to produce genotypes.

An ancestry analysis was performed as described by Ramos et al. [17 (link)] using 61 autosomal ancestry informative markers (AIMs) in three multiplex PCR reactions, aiming to accurately estimate the individual and global interethnic mix [14 (link)]. Amplicons were analyzed using an ABI Prism 3130 sequencer (Thermo Fisher Scientific, Waltham, MA, USA) and Gene Mapper ID v.3.2 software (Thermo Fisher Scientific, Waltham, MA, USA). The proportions of individual genetic ancestors were estimated using the STRUCTURE v.2.3.3 software (Stanford University, Stanford, CA, USA), assuming three parental populations. This analysis was performed to control a possible population substructure, as the investigated population was highly mixed.

Genomic DNA from HOMC cell lines was isolated using the PureLink Genomic DNA Mini Kit (Thermo Fisher Scientific). Fifteen STR sites (D3S1358, TH01, D21S11, D18S51, Penta E, D5S818, D13S317, D7S820, D16S539, CSF1PO, Penta D, vWA, D8S1179, TPOX, and FGA) and amelogenin were analyzed using PowerPlex 16 HS System (Promega, Madison, WI) and GeneMapper ID v3.2 Software (Thermo Fisher Scientific). Fifty-two SNP array analyses were performed identical.