Effects of long-term depletion of Pu.1 in adult zebrafish microglia in mosaic condition

In order to uncover the key downstream targets that mediate the out-competition of pu.1-deficient DsRed+ microglia in mosaic condition, we manually picked DsRed+ (pu.1-deficient) and eGFP+ (control) microglia from adult pu.1KI/Δ839;Tg(coro1a:CreER) zebrafish at 3 months post 4-OHT injection and performed RNA-seq. We found the tumor suppressor gene tp53 was one of the top targeted genes robustly upregulated in pu.1-deficient DsRed+ microglia which may contribute to the cell competition-mediated chronic elimination of pu.1-deficient microglia. 3~5 DsRed+ and eGFP+ microglia from adult pu.1KI/Δ839;Tg(coro1a:CreER) zebrafish at 3 months post 4-OHT injection were manually picked under the fluorescent microscope (Nikon Ti-S) equipped with the micro-manipulator (NT-88-V3, Nikon). Subsequently, picked cells were transferred by the glass needle to lysis buffer (mainly including Triton X-100 and RNase inhibitor) for thorough vertex. Then the cell lysate was used for reverse-transcription and amplification with the Smart-Seq2 method to generate the cDNA library. cDNA was sent to Novogene Company for Illumina sequencing with an average depth of 6×10^6 raw reads per sample. Sequence data were aligned to zebrafish genome by STAR (Spliced Transcripts Alignment to a Reference). Samples with strong expression of DC-related genes were discarded.