This README.txt file was generated on April 28 2026 by Tien G. Pham ------------------- GENERAL INFORMATION ------------------- 1. Title of Dataset: Source Data for "Orthogonal RNA-regulated destabilization domains for three-color RNA imaging with minimal RNA perturbation" 2. Author Information First Author Contact Information Name: Tien G. Pham Institution: University of Massachusetts, Amherst Address: Department of Chemistry, University of Massachusetts, Amherst, Massachusetts 01003, USA Email: tgpham@umass.edu Corresponding Author Contact Information Name: Jiahui Wu Institution: University of Massachusetts, Amherst Address: Department of Chemistry, University of Massachusetts, Amherst, Massachusetts 01003, USA Email: jiahuiwu@umass.edu Author Contact Information Name: Omoyemi Ajayi Institution: University of Massachusetts, Amherst Address: Department of Chemistry, University of Massachusetts, Amherst, Massachusetts 01003, USA Email: ooajayi@umass.edu Author Contact Information Name: Jiaze He Institution: University of Massachusetts, Amherst Address: Department of Chemistry, University of Massachusetts, Amherst, Massachusetts 01003, USA Email: jiazehe@umass.edu Author Contact Information Name: Irina Sagarbarria Institution: University of Massachusetts, Amherst Address: Department of Chemistry, University of Massachusetts, Amherst, Massachusetts 01003, USA Email: msagarbarria@umass.edu Author Contact Information Name: Jeanne Hardy Institution: University of Massachusetts, Amherst Address: Department of Chemistry, University of Massachusetts, Amherst, Massachusetts 01003, USA Email: jhardy@umass.edu --------------------- DATA & FILE OVERVIEW --------------------- Directory of Files A. Filename: Source Data Fig.1.xlsx Short description: Excel file containing normalized yellow cytosolic fluorescence intensity of different mDeg variants with and without MS2 RNA aptamer in HEK293T cells. B. Filename: Source Data Fig.2.xlsx Short description: Excel file containing normalized yellow cytosolic fluorescence intensity of pDeg variant2 with and without PP7 RNA aptamer in HEK293T cells. C. Filename: Source Data Fig.3.xlsx Short description: Excel file containing normalized cytosolic fluorescence intensity of three different fluorescent protein-fused mDeg with and without MS2 RNA aptamer in HEK293T cells. D. Filename: Source Data Fig.4.xlsx Short description: Excel file containing diffusion coefficient values for endoplasmic reticulum–tethered green fluorescent RNA puncta from mDeg-MS2, pDeg-PP7, and tDeg-Pepper pairs using endoplasmic reticulum tethering assay in U2OS cells. E. Filename: Source Data Fig.5.xlsx Short description: Excel file containing normalized yellow cytosolic fluorescence intensity of three destabilization domains, mDeg, pDeg, and tDeg, to test their orthogonality in HEK293T cells. F. Filename: Source Data Fig.6.xlsx Short description: Excel file containing relative mRNA levels of MS2 RNA tags with different lengths using real-time quantitative PCR. G. Filename: Source Data Extended Data Fig.3.xlsx Short description: Excel file containing normalized yellow cytosolic fluorescence intensity of different pDeg variants with and without PP7 RNA aptamer in HEK293T cells. H. Filename: Source Data Extended Data Fig.4.xlsx Short description: Excel file containing normalized cytosolic fluorescence intensity of three different fluorescent protein-fused pDeg with and without PP7 RNA aptamer in HEK293T cells. I. Filename: Source Data Extended Data Fig.6.xlsx Short description: Excel file containing diffusion coefficient values for plasma membrane–tethered red fluorescent RNA puncta from both mDeg-MS2 and pDeg-PP7 pairs using the Stargazin tethering assay in U2OS cells. J. Filename: Source Data Extended Data Fig.7.xlsx Short description: Excel file containing relative mRNA levels of MS2 and PP7 RNA tags with different lengths using real-time quantitative PCR. K. Filename: Source Data Extended Data Fig.8.xlsx Short description: Excel file containing relative mRNA levels of three different 3' untranslated regions of the minimal repeats of MS2 and PP7 tag lengths in U2OS cells. --------------------- EXPERIMENTAL & COMPUTATIONAL DATA INFORMATION --------------------- 1. Sample-specific information: Cell culture & live-cell imaging All mammalian cell lines, HEK293T/17 (ATCC, CRL-11268) and U2OS (ATCC, HTB-96), were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 100 U per ml of penicillin and 100 μg per ml of streptomycin under 37 °C with 5% CO2. For cell passage, TrypLE Express was used for dissociating the adherent mammalian cells from culture flasks. For RNA live-cell imaging, cells were seeded into 35-mm glass bottom dishes precoated with poly-D-lysine, respectively, and cultured overnight. After 24 hours, cells were transfected using FuGENE HD reagent followed by the manufacturer's protocol. Cells were changed with new media on the day after transfection. Then, cells were imaged 2 days after transfection and cell culture media was changed to FluoroBrite DMEM before live-cell imaging. RT-qPCR Total cellular RNAs were extracted using GeneJET RNA Purification Kit according to the manufacturer’s protocol. The purified RNAs were then reverse-transcribed to the complementary DNAs (cDNAs) using SuperScript IV First-Strand Synthesis kit with an oligo-dT and random hexamer primers according to the manufacturer’s instructions. qPCR was performed to measure the relative mRNA level by mixing the cDNA with SYBR Green Supermix. 2. Equipment-specific information: Nikon Ti2-E epifluorescence inverted microscope with a Prime BSI Express sCMOs monochrome camera, Lumencor SOLA V-NIR Light Engine for Epifluorescence light source and temperature and CO2 stage top incubator (Tokai Hit) was used for live-cell imaging. NIS-Elements Advanced Research software was used for data acquisition. A ×20/0.80-NA (numerical aperture) or a ×60/1.42-NA oil immersion objective (Nikon) with TYPE 37 immersion oil was used for HEK293T cells and U2OS cells, respectively. A GFP filter cube (excitation: 470 ± 20 nm; dichroic mirror: 495 nm long-pass; emission: 525 ± 25 nm) was used to detect fluorogenic proteins emitting green fluorescence. A YFP filter cube (excitation: 500 ± 10 nm; dichroic mirror: 515 nm long-pass; emission: 535 ± 15 nm) was used for detecting proteins with yellow fluorescence emission. Red fluorescent proteins were detected using a dsRed filter cube (excitation: 545 ± 15 nm; dichroic mirror: 570 nm long-pass; emission: 620 ± 30 nm), while far-red fluorescent proteins were imaged using a Cy5 filter cube (excitation: 620 ± 30 nm; dichroic mirror: 660 nm long-pass; emission: 700 ± 37.5 nm). A DAPI filter cube (excitation: 395 ± 12.5 nm; dichroic mirror: 425 nm long-pass; emission: 460 ± 25 nm) was used for imaging Hoechst-stained nuclei and mTagBFP2. Biorad CFX96 Real-time system instrument was used for obtaining RT-qPCR data. 3. Date of data collection: 2024-2025