Supplementary Files Supplementary Section 1. General Feature Format File of AgamP4. 61Embryo transcriptome annotation and InterproScan output of potential novel genes. File/s: -AgamP4. 61Embryo. gtf -NovelGenesᵢnterproScan. xlsx Supplementary Section 2. DESeq2 counts generated from the short-read dataset across all conditions and timepoints and PCA output across all conditions and timepoints. File/s: -PCAdata. xlsx Supplementary Section 3. Differential transcript expression (DESeq2) across timepoints for each experimental condition. Data are not filtered for significance. File/s: -DTEₐcrossTimeMales. xlsx -DTEₐcrossTimeWT. xlsx -DTEₐcrossTimeFemales. xlsx Supplementary Section 4. Z-score values across all conditions and timepoints used to perform k-mean analysis and list of gene IDs used for GO enrichment analysis performed using ShinyGo (v0. 8). File/s: -Clusterₐnalysisᵦₛcores. xlsx Supplementary Section 5. Conservation scores value obtained using An. gambiae conservation score tool. Pkl files containing per-base Cs values (3kb upstream, TSS, TED, 3kb downstream) for each transcript category (potential novel genes, potential novel transcript, 5000 random annotated transcripts) and for the embryonic clusters. -Plottingcsclusters (Python script to plot conservation score values across different embryonic clusters) -Plottingcsₜranscriptomecategories (Python script to plot conservation score values of transcript belonging to different annotation categories). Pkl files containing per-base Cs values: transcriptomecscluster₀. pkltranscriptomecscluster₁. pkltranscriptomecscluster₂. pkltranscriptomecscluster₃. pkltranscriptomecscluster₄. pkltranscriptomecscluster₅. pkltranscriptomecscluster₆. pkltranscriptomecsₚotenialₙovelgenes. pkltranscriptomecsₚotenialₙovelᵢsoforms. pkltranscriptomecsᵣealₘatching₅000. pkl Supplementary Section 6. DenoiSex pipeline script and output. The folder contain the scripts to perform the sex bias correction on estimates counts generated using Kallisto (01Biascorrectionₐnalysis), the generation of reduced transcriptome (02Generateᵣeducedₜranscriptomes) and the estimation of Noise frool for expression analysis (03Noisefloorₑxpressionₐnalysis). The file 'DenoiSexcorrectedcountsbooleans. xlsx' contains sex-corrected estimated transcript counts (generated using Kallisto) alongside with the original counts. The file also contains boolean values that indicating transcript above/below quantification noise treshold. File/s: -01Biascorrectionₐnalysis -02Generateᵣeducedₜranscriptomes -03Noisefloorₑxpressionₐnalysis -DenoiSexcorrectedcountsbooleans. xlsx Supplementary Section 7. Differential transcripts expression across timepoints and between Male and Female embryo pools (DESeq2). Data are annotated based on AgamP4. 61Embryo transcriptome and are not filtered for significance. The file also incorporate information from the DenoiSex pipeline (Boolean value for quantificaiton noise tresholds). File/s: -DTEₐcrossTimeFemale. xlsx -DTEₐcrossTimeMale. xlsx -DTEₐcrossTimeWT. xlsx -DTEMaleᵥersusFemale. xlsx Supplementary Section 8. Differential splicing analysis output (splicing events) across timepoints for all the experimental conditions and between Male and Female embryo pools (SUPPA2). Transcript are annotated based on AgamP4. 61Embryo transcriptome and are filtered for significance as explained in the main text. File/s: -Differentialₛplicingₑventsₜimeₛex. xlsx Supplementary Section 9. The folder contains the output of a Motif enrichment analysis on splicing events during An. gambiae embryogenesis. Transcripts were selected based on SUPPA2 results used to identify differential splicing between male and female embryogenesis and across developmental stages. This file contains the output of SEA (https: //doi. org/10. 1101/2021. 08. 23. 457422) performed on transcript regions involved in splicing across time and between sexes selected using MoSEA (https: //github. com/comprna/MoSEA). The folder also contains the input sequences (fasta) for each splicing event category used as input for the SEA analysis. Control fasta sequences are also provided. File/s: -PlottingSEAₒutput (script to plot merged SEA output files) -allₑventsₘergedₛequencesAnophelesₛex (merged SEA sequences output files. differential splicing between sexes) -allₑventsₘergedₛequencesAnophelesₜime (merged SEA sequences output files, differential splicing across time) Sex: -SEAₒutput (contains folders for each SEA analysis on each splicing event category). -MaleᵥersusFemaleₐ3ₜot. fasta-MaleᵥersusFemaleₐ5ₜot. fasta-MaleᵥersusFemaleₐfₜot. fasta-MaleᵥersusFemaleₐlₜot. fasta-MaleᵥersusFemalecontrol. fasta-MaleᵥersusFemaleₘxₜot. fasta-MaleᵥersusFemaleᵣiₜot. fasta-MaleᵥersusFemaleₛeₜot. fasta Time: -SEAₒutput (contains folders for each SEA analysis on each splicing event category). -splicingWTₜimeₐ3ₜot. fasta-splicingWTₜimeₐ5ₜot. fasta-splicingWTₜimeₐfₜot. fasta-splicingWTₜimeₐlₜot. fasta-splicingWTₜimecontrol. fasta-splicingWTₜimeₘxₜot. fasta-splicingWTₜimeᵣiₜot. fasta-splicingWTₜimeₛeₜot. fasta Supplementary Section 10. Fle binding motif scan using FIMO. The folder contains FIMO output on differentially expressed transcripts and differential spliced transcripts between Male and Female embryos across development. -FIMODifferentialₛplicingFleₘotif (folder containing FIMO output) -FIMODifferentialₜranscriptFleₘotif (folder containing FIMO output) Supplementary Section 11. List of Primers used for experimental validation of novel genes, sex-specific splicing and qPCR. -Listₒfₚrimers
Matteo Vitale (Tue,) studied this question.