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Rewiring of endogenous signaling pathways to genomic targets for therapeutic cell reprogramming

Data underlying the figures in the publication “Rewiring of endogenous signaling pathways to genomic targets for therapeutic cell reprogramming”, published in Nat Commun, 2020, 11, 608. <a href="https://www.nature.com/articles/s41467-020-14397-8">https://www.nature.com/articles/s41467-020-14397-8</a> Table of contents: 1. Source Data; Excel file with the data for Figures 2, 3b, 3d, 3c, 4a, 4b, 4c, 5 as well as S2, S3, S4, S5, S6, S7a, S7b, S8, S9, S10, S11, S12, S13a and S13b. Data 1 – Transgene expression by SEAP Data for Figures 2, S2, S4, S5, S7a, S8, S9, S12 and S13b. SEAP (human placental secreted alkaline phosphatase) levels were profiled in cell culture supernatants using a colorimetric assay. 100 µL 2x SEAP assay buffer (20 mM homoarginine, 1 mM MgCl2, 21% diethanolamine, pH 9.8) was mixed with 80 µL heat-inactivated (30 min at 65 °C) cell culture supernatant. After the addition of 20 µL substrate solution (120 mM p-nitrophenyl phosphate; cat. no. AC128860100, Thermo Fisher Scientific, Waltham, MA, USA), the absorbance time course was recorded at 405 nm and 37 °C using a Tecan Genios PRO plate reader (cat. no. P97084; Tecan Group AG, Maennedorf, Switzerland) and the SEAP levels were determined as follows: first, absorbance change over time (slope) was calculated. According to the Beer–Lambert’s law, absorbance is proportional to the concentration of a colored compound and depends on the light path length (d) and extinction coefficient (ε) (ε for p-nitrophenyl (εpNP) = 18.600 M−1 cm−1). Enzymatic activity EA [U/L] was calculated from the equation: EA = slope × dilution factor × εpNP−1 × d−1 Values in the file present determined SEAP levels. Data 2 – Endogenous gene expression by qPCR Data for Figures 3b, 3d, 4a, 4b, 5, S3, S6, S10, S11 and S13a. Total RNA of HEK293T cells was isolated using the Quick-RNA kit (Zymo Research, Irvine, CA, USA). Reverse transcription was performed using a High-Capacity cDNA Reverse Transcription Kit (cat. no. 4368814, Thermo Fisher Scientific, Waltham, MA, USA). Quantitative PCR was performed with the SsoAdvanced Universal SYBR® Green Supermix (cat. no. 1725270, Bio-Rad, Hercules, CA, USA). The Eppendorf Realplex Mastercycler (Eppendorf GmbH) was set to the following amplification parameters: 30 s at 95 °C and 40 cycles of 15 s at 95 °C followed by 30 s at X °C (X = 59 for insulin, 64 for IL-12, 59 or 64 for GAPDH). The relative threshold cycle (Ct) was determined and normalized to the endogenous glyceraldehyde 3-phosphate dehydrogenase (GAPDH) transcript. The fold change for each transcript relative to the control was calculated using the comparative Ct method. Values in the file present determined mRNA levels relative to GAPDH. Data 3 – Secreted protein level by ELISA Data for Figures 3c and 4c. Human Insulin was quantified with the Mercodia Insulin ELISA (cat. no. 10-1113-01, Mercodia, Uppsala, Sweden). IL-12 was quantified with the human IL-12 (p40) ELISA Kit (cat. no. KAC1561, Thermo Fisher Scientific, Waltham, MA, USA). Values in the file present protein levels as determined by ELISA kit. Data 4 – Nanoluc luciferase Data for Figure S7b. NanoLuc® luciferase was quantified in cell culture supernatants using the Nano-Glo® Luciferase Assay System (cat. no. N1110; Promega, Duebendorf, Switzerland). In brief, 7.5 µL of cell culture supernatant was added per well of a black 384-well plate and mixed with 7.5 µL substrate-containing assay buffer. Total luminescence was quantified using a Tecan Genios PRO plate reader (Tecan Group AG). Values in the file present luminescence as measured.

ShareScore

32/100

Overall dataset sharing score

Score breakdown

These five areas show where the dataset supports — or may limit — practical reuse.

Stewardship: 8
Harmonization: 4
Access: 16
Reuse readiness: 4
Engagement: 0

Topics

GEARs, Therapeutic Cell Engineering, Synthetic Biology