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).