RNA extraction and gene expression analyses:
We extracted RNA from liver, gut, and spleen tissue samples of each
sparrow using a standard phenol:chloroform protocol (Sambrook and
Russell, 2012). Reverse transcription was carried out using the iScript
cDNA Synthesis kit (Bio-Rad 1708891) according to the manufacturer’s
instructions. We then quantified the absolute copy numbers of DNMT1,
DNMT3, TET2 using droplet digital PCR (ddPCR). Each ddPCR reaction
contained 5 µl ddPCR Multiplex Supermix (12005909, Bio-Rad), 2.25 µl of
forward and reverse primers (10 µM), 0.63 µl of probe FAM, 0.63 µl of
probe HEX, and 0.63 µl of FAM + HEX probe mixture (for 50% FAM + HEX,
0.31 µl of each), and 1 µl of cDNA sample (3500 ng/µl; see Supplemental
Table for details). The reactions were run on a C1000 Touch™ Thermal
Cycler with a 96–Deep Well Reaction Module (1851197, Bio-Rad). After
amplification, droplets were separated and analyzed as positive
(containing the target sequence) or negative (without the target
sequence) using the QXDx Droplet Reader (12008020, Bio-Rad). Expression
data were analyzed using QuantaSoft™ Analysis Pro software (version
1.05).