Methylation sequencing
Blood of 34 adults of 17 breeding pairs was sampled in the years 2013 (N=16), 2014 (N=32) and 2017 (N=26) using larval stages of the bloodsucking bug Dipetalogaster maximus. Bugs were placed into dummy eggs with holes through which they could stick out their mouth pieces and placed in the nests of incubating focal birds (Beckeret al. 2006; Arnold et al. 2008; Bichet et al.2019). After 20-30 minutes, “bug eggs” were collected and the focal birds’ blood, sucked by the bug, was removed from the bugs’ abdomen using a syringe. Upon collection, whole blood was stored in EDTA buffer (2%) in a fridge (3-7°C) for up to 3 weeks, before red blood cells were transferred to glycerol buffer (40%) and frozen at -80° C. Each bug was used only once to prevent contamination of blood samples.
Genomic DNA was extracted and libraries for RRBS were generated as described in Klughammer et al. (2015), following standard steps such as MspI digestion, end-fill-in, A-tailing and size selection. This included the enrichment of the libraries with Pfu-Turbo Cx Hotstart DNA polymerase to allow the assessment of the bisulfite conversion rate. After clean-up and quality control, libraries were sequenced with an Illumina HiSeq 4000 (50bp SE).