Total RNA of conjugating E. vannus: 8 hours post-mixing, WT
Ribosomal RNA was depleted fromtotal RNA using the Ribo-off rRNA Depletion Kit (Cat. N406-01, Vazyme, China). The resulting RNAwas ligated at the 3 end with an RNA adapter using T4 RNA Ligase 1 (Cat. M0204S, NEB). First-strand cDNA synthesis was performed using Maxima H Minus Reverse Transcriptase (Cat. EP0752,Thermo Scientific), followed by second-strand synthesis and amplification using LongAmp Taq DNAPolymerase (Cat. M0287S, NEB). The resulting double-stranded cDNA was purified using AMPureXP beads and used for library preparation. The sequencing was carried on a PromethION platform(Oxford Nanopore Technologies, Oxford, UK). Raw ONT data was basecalled by dorado (v0.9.0,https://github.com/nanoporetech/dorado), filterd with Nanofilt (2.8.0, -q 7 -l 10) (112), and trimmedusing Pychopper (2.7.10, -m edlib, https://github.com/epi2me-labs/pychopper).