NicheXplorer Bridges Genetic Screening and Niche Engineering for Functional Microenvironment Discovery
NicheXplorer Bridges Genetic Screening and Niche Engineering for Functional Microenvironment Discovery
For B16F10-OVA cells, lentiviral constructs expressing mCherry, HEY1, ETS1, or a combination of HEY1 and ETS1 were transduced. A total of 1 × 10⁵ cells were seeded per well in a 6-well plate. After 4 days, wash the cells with DPBS, then digest them with 0.25% trypsin/EDTA for 2-3 minutes at 37°C. To terminate the digestion, add complete DMEM medium, then centrifuge the cells at 300g for 5 minutes. Discard the supernatant and resuspend the pellet in 1 mL of TRIzol reagent, pipetting up and down to ensure complete lysis. Finally, store the lysed samples at -80°C and ship on dry ice to the sequencing company. B16-OVA (Cat# SCSP-5096, Cell Bank of Chinese Academy of Sciences) were cultured in the high-glucose DMEM medium (Cat# C11995500BT, Gibco). Doxycycline (1 μg/mL) was added to the culture medium every 24 hours to induce gene expression Total RNA was extracted using the RNAprep Pure Tissue Kit (DP431) following the manufacturer's protocol. 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.