Twenty female larvae were collected at the wandering L3 stage (108-117 h AEL at 25oC) and bled in Schneider medium complemented with N-phenylthiourea on ice. Both circulating and resident pools of hemocytes were collected by first puncturing the cuticle to release the circulating hemocytes, then by scrapping the cuticle with fine forceps to release the resident hemocytes (FBrf0230406). After bleeding, the hemolymph was filtered on a 100um-mesh to remove cell aggregates and wasp eggs.

For each condition, single-cell RNA-seq libraries were prepared from 10,000 cells using the Chromium Single Cell 3' Reagent Kits v2 (10x Genomics).

RNA-seq libraries were sequenced on the Illumina HiSeq 4000 platform with paired-end 100 bp reads.

Reads were processed using Cell Ranger v3.0.1 (10x Genomics). They were mapped to the Drosophila melanogaster reference genome BDGP6_ens95. For quality filtering, cells with less than 200 UMI were removed. Further analysis was performed using Seurat v3 (https://satijalab.org/seurat/). Dimensionality reduction and clustering were performed by computing 20 principal components and with a clustering resolution of 0.55.