GSM9431931: Nucleo-seq of MCF10A in 5k nucleoli; Homo sapiens; OTHER
Nucleoli Extraction and Purification: Nuclei were isolated from crosslinked cells (1% formaldehyde) using lysis buffers and SDS/Triton X-100 treatment. Purified nuclei were sonicated to release nucleoli, which were then separated from nuclear debris by filtration and purified through a S2/S3 density gradient centrifugation;Nucleoli Staining and Fluorescence-Activated Nucleoli Sorting:Purified nucleoli were stained with a PE-conjugated anti-NPM1 antibody. Using a flow cytometer calibrated with size-standardized beads, nucleoli were identified as PE-positive particles measuring 500-1300 nm in diameter and sorted for subsequent analysis. Intact nuclei or sorted nucleoli were re-crosskined for WGS preparation, or digested in situ with DpnII restriction enzyme and re-ligated for Nucleo-C Library Construction. For WGS and Nucleo-seq, libraries were conducted based on Tn5 tagmentation and adapter addition via TruePrep DNA Library Prep Kit V2 for Illumina (Vazyme, TD502/503), according to total input DNA amount (TD502 for 4–6 ng DNA, TD503 for 1–2 ng DNA), respectively acoording to the manufacturer's instruction.The final library was size-selected (300-500bp) for paired-end sequencing on an Illumina platform. For minimized Hi-C and Nucleo-C, proteins were degraded with proteinase K, crosslinks were reversed, and DNA was purified via ethanol precipitation.The digested DNA was fragmented using dsDNA Fragmentase. Biotinylated fragments were captured on streptavidin beads, followed by end repair, adapter ligation, and PCR amplification (11-13 cycles). The final library was size-selected (300-500bp) for paired-end sequencing on an Illumina platform. For RNA-seq, cells were collected using TRNzol reagent and subsequently constructed for RNA extraction and sequencing. The RNA library preparation was performed by using NEBNext UltraTM RNA Library Prep Kit for Illumina, followed by size selection with AMPure XP Beads. Sequencing was performed using Illumina's sequencing-by-synthesis technology.