Embryonic heat conditioning induces paternal heredity of immunological cross- tolerance: coordinative role of CpG DNA methylation and miR-200a regulation [BiSulfite-seq]

The first-generation embryos underwent inovo heat conditioning (EHC) and the offspring were subjected to an intracranial lipopolysaccharide (LPS) challenge, demonstrating immune resilience in second generation which is mediated through the paternal DNA methylation pattern. DNA was extracted from the sperm and reduced representation bisulfite sequencing (RRBS) was performed DNA was isolated from the sperm sample using Genomic Mini AX Swab & Semen Spin (A&A Biotechnology, Gdansk, Poland). 70-140ng of DNA was used for RRBS library preparation using Zymo-Seq RRBS Library Kit (Zymo Research, Irvine, CA). The paired end sequencing was done using a SP 100 cycle kit on Nova seq 6000 instrument (Illumina). The quality trimming and trimming of Illumina adapters was performed with Trim galore and the reads were mapped to the chicken genome (galGal 5.0) using Bismark v. 0-22-3 bowtie mode. The methylation calls were extracted with Bismark, methylation extractor mode. Edge R was used for the extraction and analysis of differential methylation on single CpGs (DMS) with the cutoff of 8 reads in every sample and FDR <0.05. DMRs are extracted and analysed by metilene v. 0.2-8. PO/AH mRNA profile of 3 day old Naive (t0) and 6h hours into miR-200a challenge, by bulk MARS-seq method