NicheXplorer Bridges Genetic Screening and Niche Engineering for Functional Microenvironment Discovery
NicheXplorer Bridges Genetic Screening and Niche Engineering for Functional Microenvironment Discovery
For C3H10T1/2 cells, lentiviral constructs expressing GFP, Foxp1, EP300, or combinations including EP300 + Foxp1, GFP + GFP (dual control), GFP + Foxp1, and EP300 + GFP were transduced at MOI=5 with 8 μg/ml polybrene. A total of 1×10⁵ transduced cells per well were seeded in 6-well plates. After 4 days, cells were washed twice with DPBS, digested with 0.25% trypsin/EDTA at 37°C for 2–3 min, and neutralized with complete DMEM. The suspension was centrifuged at 300 ×g for 5 min. For BA/F3 cells, co-culture with EP300-/Foxp1-overexpressing C3H10T1/2 was performed in IL-3-depleted complete DMEM (ratio 1:2) for 48 h. Suspended BA/F3 cells were isolated by gentle pipetting to avoid dislodging adherent cells, followed by centrifugation at 300 ×g for 5 min. Supernatants from both cell types were discarded, and pellets were resuspended in 1 mL TRIzol™ reagent (Invitrogen) with vigorous pipetting (10×) to ensure complete lysis. Total RNA was isolated from C3H10T1/2 and BA/F3 using the Direct-zol RNA Miniprep (Catalog R2050) according to the manufacturer's instructions. RNA integrity was confirmed by Bioanalyzer analysis (RIN > 8.0) and quantified via Qubit fluorometry. RNA library for RNA-seq was prepared as fllows: mRNA was purified from total RNA using polyT and then fragmented into 300~350 bp fragments, the first strand cDNA was reverse-transcribed using fragmented RNA and dNTPs (dATP, dTTP, dCTP and dGTP) and second strand cDNA synthesis was subsequently performed. Remaining overhangs of double-strand cDNA were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3' ends of DNA fragments, sequencing adaptors were ligated to the cDNA and the library fragments were purified. The template was enriched by PCR, and the PCR product was purified to obtain the final library.