Powerplex 18d kit

Seventeen short tandem repeat (STR) loci plus the gender determining locus, Amelogenin, were amplified using the commercially available PowerPlex 18D Kit from Promega. The cell line sample was processed using the ABI Prism 3500xl Genetic Analyzer. Data were analyzed using GeneMapper ID-X v1.2 software (Applied Biosystems). Appropriate positive and negative controls were run and confirmed for each sample submitted.’

A detailed procedure for iPSC generation was previously published [11 (link)]. Briefly, fibroblasts were cultured in Dulbecco’s Modified Eagle medium (DMEM) and transfected with Yamanaka factors using a non-integrating CytoTune-iPS 2.0 sendai reprogramming kit to produce iPSCs. Two iPSC clones of each fibroblast line were picked and expanded in Matrigel pre-coated six-well plates. The pluripotency protein markers were examined by immunofluorescence staining. Karyotyping was performed by Wicell Cytogenetics (Madison, WI). Short tandem repeat analysis was conducted by the Johns Hopkins University Genetic Resources Core Facility using a PowerPlex 18D kit (Promega).

Genomic DNA was isolated from all cell lines and sent to the Johns Hopkins Fragment Analysis Facility for STR profiling on all cell lines except MCC13 and MCC26 using the AmpFt’STR Identifiler PCR Amplification Kit (Applied Biosystems), which profiles 16 STR markers (Table S7). MCC13 and MCC26 were profiled using the Promega PowerPlex 18D Kit, which profiles the same 16 markers plus two others.

All cell lines were maintained in a humidified incubator at 37 °C with 5% CO2. PC9, HCC827, H3122, A549, A375, GR4, WZR12, PC9R, MGH134, MGH006, MGH1109, and MOLM13 were cultured in RPMI 1640 medium with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin. MIA PaCa-2 and SW1573 were cultured in DMEM/F-12 medium with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin. MGH119 were cultured in DMEM medium with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin. 293FT cells were cultured in DMEM high glucose medium with 10% FBS, 1% penicillin–streptomycin, 1% sodium pyruvate, 1% nonessential amino acids and 1% GlutaMax. All cell lines were purchased from American Type Culture Collection or Duke University Cell Culture Facility except for MGH lines, which were obtained from Dr. Aaron Hata. Cell lines were authenticated using the Promega PowerPlex 18D kit. Drugs were purchased from ApexBio (Osimertinib, Gefitinib, SCH772984, Ceritinib, Quizartinib, PLX4720, AMG510, Lorlatinib), Cayman Chemical (Olaparib), and SelleckChem (AZD0156, AZD1390, Q-VD-Oph, Cycloheximide).