Project description:A mass spectrometry-based proteomics analysis was performed to study the protein binders of GTSF1L in germ cell culture (BmN4; ovary-derived) of Bombyx mori. Anti-HA pull-downs were performed in BmN4 cells transfected with the HA-BmGtsf1L plasmid or the HA-eGFP control plasmid. Proteins were digested in-gel by trypsin. The resultant peptides were then dimethyl labelled and combined. Peptides were measured on a Q Exactive Plus Orbitrap mass spectrometer.
Project description:BmN4 cells are cultured cells derived from Bombyx mori ovaries and widely used to study transposon silencing by PIWI-interacting RNAs (piRNAs). A high-accurate genome sequence of BmN4 cells is required to analyze the piRNA pathway using RNA-seq. The genome sequence of BmN4 cells was assembled using Pacific Biosciences (PacBio) HiFi and Oxford Nanopore technology Ultralong (ONT-UL) reads. Microscopic observation and image analysis showed that BmN4 cells were octoploid on average, and the number of chromosomes per cell was highly variable. We concluded the haplotype-resolved assembly of such a complex genome would be difficult; therefore, we assembled a consensus genome sequence. RNA-seq analysis of Siwi knockdown cells also revealed that Siwi-piRISC may target Countdown (Cd), an LTR retrotransposon. By comparing the consensus genome sequence with the reads, we identified differences between haplotypes, particulary structural variants, suggesting that some transposons, including Countdown, increased their copy number in BmN4 cells.