GM12878 cells were obtained from Coriel Institute for Medical Research. Species of origin of the cell line was confirmed by PCR targeting the glucose-6-phosphate dehydrogenase gene. Donor subject has a single bp (G-to-A) transition at nucleotide 681 in exon 5 of the CYP2C19 gene (CYP2C19*2) which creates an aberrant splice site. Donor origin of the cell line was confirmed using PCR against the point mutation. The cell line was tested for mycoplasma contamination using ABI MycoSEQ mycoplasma detection assay (Applied Biosystems). Cells were propagated in RPMI 1640 (11875-093, Gibco) plus 15% fetal bovine serum (26140-079, Gibco), 100 U penicillin (15140-122, Gibco), and 100mg/ml streptomycin (15140-122, Gibco) at 37°C in a humidified incubator containing 5% CO2. Cells were sub-cultured every two days to maintain them in exponential growth phase.
Abi mycoseq mycoplasma detection assay
Manufactured by Thermo Fisher Scientific
The ABI MycoSEQ mycoplasma detection assay is a real-time PCR-based assay designed to detect the presence of mycoplasma contamination in cell culture samples. The assay utilizes primers and probes targeting conserved regions of the mycoplasma 16S rRNA gene, allowing for the sensitive and specific detection of a wide range of mycoplasma species.
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Lab products found in correlation
Rpmi 1640, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Prl tk, by Promega (1 mentions)
Dual luciferase reporter assay kit, by Promega (1 mentions)
In fusion hd cloning kit, by Takara Bio (1 mentions)
Transit jurkat reagent, by Mirus Bio (1 mentions)
3 protocols using abi mycoseq mycoplasma detection assay
1
Characterization of GM12878 Cell Line
2
Characterization of GM12878 Cell Line
3
Enhancer Activity Assay in Jurkat Cells
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Jurkat cells were purchased from ATCC (TIB-152). The cell line was tested for mycoplasma contamination using ABI MycoSEQ mycoplasma detection assay (Applied Biosystems). Enhancer sequences containing predicted risk eSNPs were cloned using In-Fusion HD Cloning Kit (Clontech, Cat # 639648) into a luciferase reporter construct pGL3-HS in which expression of the luciferase gene is driven by a minimal heat-shock promoter. Sanger sequencing was used to determine the alleles of the risk eSNPs. Two control regions of ~2 kb without either H3K4me1 or H3K27ac signals were cloned into the same plasmid as negative controls. Reporter constructs were transfected into Jurkat cells using TransIT-Jurkat Reagent (Mirus Bio, MIR 2120). As an internal control, a plasmid containing Renilla luciferase (pRL-TK from Promega) was co-transfected at a molar ratio of 1:10 for Renilla vs firefly luciferases. Cells were collected 48 h post transfection and luciferase reporter levels were measured and compared to Renilla luciferase reporter activity using the Dual-Luciferase Reporter Assay kit (Promega, cat # E1910). Primer sequences for cloning enhancers and mutagenesis are listed in Supplementary Tables 7 and 8 .
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