A two-step enrichment process facilitates robust transcriptional profiling of Bb during the tick bloodmeal.
(A) Schematic of Bb during tick feeding. Bb in the nymphal tick midgut respond to the bloodmeal by multiplying and changing their transcriptional state. At the same time, the tick gut undergoes numerous changes to digest the bloodmeal. After two to three days of feeding, a small number of Bb leave the midgut and enter the salivary glands (blue), while the majority are left behind in the gut after engorgement. (B) Schematic of Bb enrichment process from feeding ticks. Whole ticks are dissociated, anti-Bb antibodies are added to lysates, and antibodies and Bb are captured magnetically. RNA is extracted and RNA-seq libraries are prepared. DASH is then used to remove rRNA before sequencing. This process increases Bb reads in the resulting sequencing. (C) RT-qPCR results showing the percentage of Bb flaB and tick gapdh RNA in the enriched versus depleted fractions after the enrichment process. Data come from 4 replicates each from day 2, day 3, and day 4, mean +/− SE. Nearly all Bb flaB RNA was found in the enriched fraction, while the majority of tick gapdh was found in the depleted fraction. (D) The percentage of reads mapping to rRNA before and after DASH. Data are shown as mean +/− SD. rRNA reads are drastically reduced after DASH. (E) The percentage of reads in RNA-seq libraries mapping to Bb. Bb mRNA reads make up a small but sufficient proportion of libraries for transcriptomics. Data are shown as mean +/− SD. (F) The number of reads in millions (M) mapped to Bb for each day. Data are shown as mean +/− SD. An average of 4.3 million reads per sample mapped to Bb genes, covering 92% of genes with at least 10 reads.