Escherichia Coli (E.Coli, STBL3, Stellar) was used for transformation and streak plated on selective antibiotic-containing (ampicillin, 100μg/mL, or kanamycin, 25μg/mL) on Luria Bertani (LB)-agar plates then incubated for 16 hours at 37oC. Single clones were then inoculated in LB broth with noted antibiotic and cultured (16 hours, 32oC, 185 rpm orbital shaking). Clones were stored in 1:1 glycerol/water at -80oC. CRISPR plasmid constructs containing mosaic gRNAs were cloned by T4 ligase oligonucleotide insertion (Table S1 ) in digested px333 (Addgene #64073) or pLentiCRISPR-RFP657 (Addgene #75162) and transformed into STBL3 E. coli. The px333 used was a gift from Andrea Ventura (Addgene plasmid #64073) http://n2t.net/addgene:64073 ; (Addgene #64073) [28] . In-Fusion HD Cloning (Takara #638920) was utilized to synthesize tat mutants on pHIV-1NL4-3-Δnef-eGFP background. Maxiprep purified plasmids were transfected into HEK293FT cells using PEI or into ACH2 / U1 leukocytes via TransIT-Jurkat reagent (MirusBio #2120) or TransIT-X2 reagent (MirusBio #6000). Supernatants were collected 72 hours after media replacement (containing PEI) or plasmid transfection (TransIT-Jurkat or TransIT-X2).
Transit jurkat reagent
Manufactured by Mirus Bio
The TransIT-Jurkat reagent is a transfection reagent designed for the efficient delivery of nucleic acids, such as plasmid DNA, into Jurkat T cells. It is optimized to facilitate high transfection efficiency and cell viability in this specific cell line.
Automatically generated - may contain errors
Lab products found in correlation
In fusion hd cloning, by Takara Bio (1 mentions)
Px333, by Addgene (1 mentions)
Transit x2 reagent, by Mirus Bio (1 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)
Abi mycoseq mycoplasma detection assay, by Thermo Fisher Scientific (1 mentions)
2 protocols using transit jurkat reagent
1
CRISPR Plasmid Construction and Transformation
2
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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