GSM8752635: I0508-2; synthetic construct; OTHER
Plasmid DNA was extracted from bacteria using GeneJET Plasmid Miniprep Kit (Thermo Fisher Scientific, K0503). Genomic DNA was extracted from thawed cell pellets using DNeasy Blood and Tissue Kit (Qiagen, 69506) Plasmid DNA was used as template material for PCR amplification of a 360bp segment corresponding to the cEFhand of STIM1. The extracted genomic DNA was used as the template material for PCR amplification of a 568bp segment corresponding to the cEFhand of STIM1. To avoid sequencing any lingering unintegrated plasmid DNA, a forward primer hybridizing to the Bxb1 integrase coding region encoded in the landing pad was directly 5-prime of the region of interest, so that only integrated plasmids would be amplified. Each sample was amplified with a single 40 μL reaction containing 2 μg of genomic DNA (as assessed by spectrophotometry at A260), 0.25uM forward primer containing an i7 index and p7 cluster generator sequence, 0.25uM reverse primer containing i5 index barcode and p5 cluster generator sequence, and 20 μL of Phusion plus high-fidelity PCR mix (Thermo Scientific, F631S). The amplicons were created with an initial 95°C incubation for 3 mins, and 28 total cycles of 95°C for 15 secs, 60°C for 15 secs, 72°C for 30 secs, followed by a final extension step of 72°C for 1 min. The PCR reaction contents were mixed with 6x DNA loading dye (New England Biolabs, B7024S) and separated on 1% agarose gel prepared in Tris-acetate-EDTA buffer, and electrophoresed. Double stranded DNA bands migrating at 568 nt for genomic and 360nt for plasmid were observed with SYBR Safe DNA Gel Stain (Thermo Scientific, S33102) were excised and extracted with GeneJET Gel Extraction and DNA Cleanup Micro Kit (Thermo Fisher Scientific, K0832). The concentrations of the final DNA were quantified with a Qubit dsDNA BR assay kit (Life technologies, Q32853). Amplicons from all the experimental conditions were pooled together in equimolar ratios, subject to a final quality control step for homogeneity with an Agilent Fragment Analyzer and cluster estimation with quantitative PCR.