Embryonic heat conditioning in chicks induces transgenerational heat/immunological resilience via methylation on regulatory elements

We used in ovo embryonic heat conditioning (EHC) of first-generation chicks and assessed the effect on their untreated offspring by measuring genome-wide alterations in DNA-methylation patterns. DNA was extracted from the APH and reduced representation bisulfite sequencing (RRBS) was performed Overall design: DNA was isolated by TRI Reagent (Molecular Research Center). RRBS libraries were prepared using Zymo-Seq RRBS Library Kit (Zymo) and prepared from 70–140 ng of DNA. Sequencing was done on a NovaSeq 6000 instrument (Illumina) using an SP 100 cycle kit (paired end sequencing). Trimming of Illumina adapters, as well as quality trimming, were performed with Trim Galore, using the following options: --nextseq --non_directional –rrbs. Reads were mapped to the chick genome (galGal5.0) using Bismark v. 0-22-3, bowtie mode. Methylation calls were extracted with Bismark, methylation extractor mode. DMS was extracted and analyzed by edgeR (39), with the recommended cutoff of at least 8 reads in every sample and FDR < 0.05. DMRs were extracted and analyzed by metilene v. 0.2-8. Motif enrichment analysis and DMS/DMR annotation were performed by HOMER tools. Segregating Hyper/Hypo-TADs according to TAD methylation was achieved considering only TADs with more than three DMSs. ChromHMM was used to establish a chromatin state model from an independent study that utilized bulk hypothalamic tissue, and to determine fold enrichment of DMS/DMR over state model. APH mRNA profile of 10 day old noncoditioned F1 chicks, nonchallenged (t0)/6 hours into heat challenge/6 hours into LPS challenge, by 3'-mRNA- sequencing (RNA-seq).