title: scRNA-seq of danio rerio: juvenile PCV
design: Liibrary preparation, sequencing and pre-processing of single-cell RNA-Seq dataPlate-based single-cell RNA sequencing libraries were processed using the MARS-seq protocol, as previously described. Sequence ready library was sequenced using Illumina NextSeq 500 to565 reach 50,000 reads per cell. We sequenced 669 cells in total. GRCz10 was used for genomealignment. Data pre-processing was carried out following cross-platform single-cell data pre-processing (https://doi.org/10.1038/s41467-022-34681-z). Practically, the protocol is padding theR2 reads with AAAAAAAAA (awk NR%4==0 {print IIIIIIIII substr($0,0,15) II};NR%4==2 {print AAAAAAAAA substr($0,0,15) AA}; NR%2==1 {print}). Swapping R1 and R2 filenames and then executing standard Cell Ranger with --r1-length=26 --chemistry=SC3Pv2Cells. We used the Seurat R package (Seurat ver. 5.0.0) for downstream analysis: cells with more than 1800 nFeatures (genes) were removed as suspected doublets; cellswith less than 100 nFeatures (genes) were removed for low coverage or empty wells. Cells with more than 8% mitochondrial gene content were excluded for low quality. Overall, 643 cells passed quality filters for downstream analysis. Mitochondrial (^mt-) and ribosomal (^rp[sl]|^RP[SL],zgc:158463) genes were removed for downstream analysis.