title: Drosophila Sociality Influences Immune Peptide Load and Apoptosis-induced Tumor Suppression independently of the Antitumor Peptide Defensin
design: Drosophila Sociality Influences Immune Peptide Load and Apoptosis-induced Tumor Suppression independently of the Antitumor Peptide Defensin
library_name: Sample 7_s
library_construction_protocol: For each replicate, groups of 100 tumorous flies were beheaded on dry ice; heads and bodies were store separately at -80°C. These flies were selected 10 days post tumor induction, as we assumed that the social-induced effect occur along the entire process of tumor growth. 24 days past heat-shock, midguts from individual flies were dissected and dissociated into single cells as previously described (AKIKI et al. 2024). Samples containing the intestinal cells of single (Fig. 4 and Table S6) or group of two to three (Fig. 5 and Table S7) flies  were processed on a CytoFLEX S (Beckman Coulter) cytometer and analyzed with CytExpert 2.4. Results are presented as mean percentages of tumorous cells (labelled by GFP) over the total intestinal cell number (inclusing tumorous cells). Statistics were conducted on GraphPad Prism 8.0.1. using a Mann-Whitney test.  The yw,HS-flp;esg-gal4,UAS-GFP; FRT82B,Tub-Gal80 (line 1)  and w,HS-flp;UAS-RasV12;FRT82B,Apc2N175K,ApcQ8 (line 2)  stocks (MARTORELL et al. 2014) were maintained with co-segregating SM5-TM6B balancers. Prior to experiments, these lines were isogenized on chromosomes I, II and III from individual males crossed to a FM7; SM5-TM6B stock. Next, the UAS-RasV12 chromosome II from line 2 was recombined with either UAS-p35 (BDSC#5072), UAS-JNKK-RNAi (VDRC#109277), UAS-Defensin or the defsk3 mutant allele (PARVY et al. 2019), the latter being also recombined with the esg-gal4,UAS-GFP chromosome II from line 1; these recombined chromosomes were re-introduced into the parental lines. Control w1118 flies were used as healthy females in H-groups.  To generate tumors, females from line 1 or its derivative recombined to the defsk3 mutation were crossed to males from line 2 or its recombined derivatives. In their progeny non-balanced virgin females were selected and heat-shocked at 37°C for 1h15mins, two to three days after adult eclosion as previously detailed (DELAMOTTE et al. 2025). Fly groups were established immediately after heat-shock and maintained in a 25°C incubator on our standard media (GARRIDO et al. 2015), changed twice a week into new vials.  DNA reads were converted into raw data (FASTQ files) and preprocessed to remove adapters, low-quality sequences and artefacts. Cleaned sequences were aligned to the reference genome \"dmel-all-chromosome-r6.42.fasta\" using HISAT2. Differential analyses between replicates were performed using the DESeq2 tool to identify genes whose expression significantly differs between experimental conditions. RNA extraction from bodies was performed as described for heads (AKIKI et al. 2024). Briefly, RNAs were extracted using Trizol-chloroform and libraries for Illumina sequencing were prepared using the Illumina TruSeq total RNA stranded kit (GARRIDO et al. 2015) and sequenced using a NextSeq 550 instrument. Analysis type: HISAT2 Reference genome: dmel-all-chromosome-r6.42.fasta (from FlyBase)