DOI: 10.5061/dryad.0k6djhbd8
title: Data from: Counting cytoplasmic incompatibility factor mRNA using digital droplet PCR

description: Wolbachia bacteria inhabit over half of all insect species and often
 spread through host populations via efficient maternal transmission and
 cytoplasmic incompatibility (CI), killing aposymbiotic embryos when
 fertilized by symbiotic males. Wolbachia's cifB gene triggers CI in
 males, while cifA, expressed in females, rescues embryos from CI-induced
 lethality. In some systems, cifA also contributes to CI induction. CI
 strength—the percentage of embryos that die from CI—is a key determinant
 of Wolbachia's prevalence in host populations, and cifB mRNA levels
 in testes generally correlate with CI strength. Yet, cifB's rarity
 can hamper precise quantification, necessitating tissue pooling for
 reverse transcription quantitative PCR (RT-qPCR) to achieve reliable
 measurements, obscuring variation at the level of individual insect
 tissues. Here, we present four RT digital droplet PCR (RT-ddPCR) assays to
 count rare cifA and cifB mRNA from wMel Wolbachia in Drosophila
 melanogaster. These assays count cif transcripts alongside a synthetic
 spike-in RNA or a D. melanogaster housekeeping gene to normalize for
 technical or biological variation. These assays have a limit of detection
 of about 1 cifA and 3 cifB copies per reaction. We expect these methods to
 be useful for mosquito-control programs that use wMel to block the spread
 of pathogens from Aedes aegypti to humans. Moreover, the oligos were
 designed with homology to cifA and cifB sequences from at least 33
 Wolbachia strains, suggesting utility beyond wMel. These methods will
 allow researchers to measure cif mRNA levels from individual insect
 tissues, enabling efforts to pair molecular and phenotypic data at
 unprecedented resolutions.
description: # Data from: Counting cytoplasmic incompatibility factor mRNA using
 digital droplet PCR Dataset DOI:
 [10.5061/dryad.0k6djhbd8](10.5061/dryad.0k6djhbd8) ## Description of the
 data and file structure All data associated with Van Vlaenderen L, Conner
 WR, Shropshire JD. (2025) Counting cytoplasmic incompatibility factor mRNA
 using digital droplet PCR. ### Files and variables #### File: Data-S1.csv
 **Description:** RNA extraction and purification measurements. #####
 Variables * Sample ID: A unique sample identifier * Testes: Number of
 testes in the sample * pre-Qubit concentration (ng/L): RNA concentration
 measured through Qubit before reprecipitation * pre-Nanodrop concentration
 (ng/L): RNA concentration measured through Nanodrop before reprecipitation
 * pre-A260/A280: Nanodrop quality ratio before reprecipitation *
 pre-A260/A230: Nanodrop quality ratio before reprecipitation *
 pre-A260: Nanodrop measurement before reprecipitation * pre-A280: Nanodrop
 measurement before reprecipitation * pre-A230: Nanodrop measurement before
 reprecipitation * post-Qubit concentration (ng/L): RNA concentration
 measured through Qubit after reprecipitation * post-Nanodrop concentration
 (ng/L): RNA concentration measured through Nanodrop after reprecipitation
 * post-A260/A280: Nanodrop quality ratio after reprecipitation *
 post-A260/A230: Nanodrop quality ratio after reprecipitation *
 post-A260: Nanodrop measurement after reprecipitation *
 post-A280: Nanodrop measurement after reprecipitation *
 post-A230: Nanodrop measurement after reprecipitation #### File:
 Data-S2.fa **Description:** FASTA file containing cifA sequences
 homologous to the cifA RT-ddPCR oligos. #### File: Data-S3.fa
 **Description:** FASTA file containing cifB sequences homologous to the
 cifB RT-ddPCR oligos. #### File: Data-S4.csv **Description:** cifA/spike
 RT-ddPCR annealing temperature measurements. ##### Variables *
 Well: Reaction well * Assay: Assay performed * Sample type: * Annealing
 temperature: * Target: The target amplified * Conc(copies/L): Target
 copies detected per uL * Supermix: Supermix used in the reaction *
 DyeName(s): Dye used to measure targets * Accepted Droplets: Droplets
 measured * Positives: Droplets with the target * Negatives: Droplets
 without the target #### File: Data-S5.csv **Description:** cifB/spike
 RT-ddPCR annealing temperature measurements. ##### Variables *
 Well: Reaction well * Assay: Assay performed * Sample type: The type of
 sample added to the reaction * Annealing temperature: * Target: The target
 amplified * Conc(copies/L): Target copies detected per uL *
 Supermix: Supermix used in the reaction * DyeName(s): Dye used to measure
 targets * Accepted Droplets: Droplets measured * Positives: Droplets with
 the target * Negatives: Droplets without the target #### File: Data-S6.csv
 **Description:** cifA/spike RT-ddPCR dilution series measurements. #####
 Variables * Well: Reaction well * Sample type: The type of sample added to
 the reaction * Sample ID: A unique sample identifier * Assay: Assay
 performed * Target: The target amplified * Conc(copies/L): Target copies
 detected per uL * Supermix: Supermix used in the reaction *
 DyeName(s): Dye used to measure targets * Accepted Droplets: Droplets
 measured * Positives: Droplets with the target * Negatives: Droplets
 without the target #### File: Data-S7.csv **Description:** cifB/spike
 RT-ddPCR dilution series measurements. ##### Variables * Well: Reaction
 well * Sample description 1: The type of sample added to the reaction *
 Sample ID: A unique sample identifier * Assay: Assay performed *
 Target: The target amplified * Conc(copies/L): Target copies detected per
 uL * Supermix: Supermix used in the reaction * DyeName(s): Dye used to
 measure targets * Accepted Droplets: Droplets measured *
 Positives: Droplets with the target * Negatives: Droplets without the
 target #### File: Data-S8.csv **Description:** cifA/β-Spec RT-ddPCR
 dilution series measurements. ##### Variables * Well: Reaction well *
 Sample type: The type of sample added to the reaction * Sample ID: A
 unique sample identifier * Assay: Assay performed * Target: The target
 amplified * Conc(copies/L): Target copies detected per uL *
 Supermix: Supermix used in the reaction * DyeName(s): Dye used to measure
 targets * Accepted Droplets: Droplets measured * Positives: Droplets with
 the target * Negatives: Droplets without the target #### File: Data-S9.csv
 **Description:** cifB/β-Spec RT-ddPCR dilution series measurements. #####
 Variables * Well: Reaction well * Sample type: The type of sample added to
 the reaction * Sample ID: A unique sample identifier * Assay: Assay
 performed * Target: The target amplified * Conc(copies/L): Target copies
 detected per uL * Supermix: Supermix used in the reaction *
 DyeName(s): Dye used to measure targets * Accepted Droplets: Droplets
 measured * Positives: Droplets with the target * Negatives: Droplets
 without the target #### File: Data-S10.png **Description:** DNase
 treatment raw gel images. #### File: Data-S11.csv **Description:** DNase
 treatment RT-ddPCR results. NA = Not applicable. ##### Variables * Well:
 Reaction well * Assay: Assay performed * DNase conditions: DNase protocol
 used * RT : Whether the RNA was reverse transcribed or not * Target: The
 target amplified * Conc(copies/L): Target copies detected per uL *
 Supermix: Supermix used in the reaction * DyeName(s): Dye used to measure
 targets * Accepted Droplets: Droplets measured * Positives: Droplets with
 the target * Negatives: Droplets without the target #### File:
 Table_S1.csv **Description:** List of *Wolbachia* genome accession numbers
 used in this study. ##### Variables * Host (Wolbachia): The relevant
 *Wolbachia* strain's host. The *Wolbachia* strain is listed in
 parentheses if available. * Wolbachia accession: The accession number
 associated with the *Wolbachia* genome.