Embryonic heat conditioning induces paternal heredity of immunological cross- tolerance: coordinative role of CpG DNA methylation and miR-200a regulation [RNA-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 Overall design: RNA was isolated from PO/AH and mRNA profile of 3 day old Naive (t0) and 6h hours into miR-200a challenge, by bulk MARS-seq method. 15ng of purified RNA was used for library preparation with the bulk MARS-seq method, involving barcoding of samples by reverse transcription using an oligo dT primer, pooling of samples, and subsequent molecular reactions for linear amplification and preparation for Illumina sequencing. 100bp single reads were sequenced on a Novaseq 6000 sequencing system. The output was ~16 million reads per sample. Poly-A/T stretches and Illumina adapters were trimmed from the reads using Cutadapt (28). The resulting reads shorter than 30bp were discarded. The remaining reads were mapped onto 3' UTR regions (1000 bases) of the Gallus genome (galGal5.0, UCSD) according to Refseq annotations, using STAR with EndToEnd option and out Filter Mismatch Nover Lmax was set to 0.05. Deduplication was carried out by flagging all reads that were mapped to the same gene and had the same UMI. Read counts per gene were quantified using HTSeq-count (30). UMI counts were corrected for saturation by considering the expected number of unique elements when sampling without replacement. Differentially expressed genes were identified using DESeq2 (31) with the betaPrior, cooksCutoff and independent Filtering parameters set to False. Raw P values were adjusted for multiple testing using the procedure of Benjamini and Hochberg. Pipeline was run using snakemake.