The homologous genomic sequences of LNCR rasiRNAs from cluster 1 are present at two distinct locations, whereas homologous genomic sequences of LNCR rasiRNAs from cluster 2 are present at three distinct locations. The precise positions of LNCR rasiRNAs on BAC 83A24 are indicated in Table S4. To monitor the chromatin dynamics that occurs in this genomic region Evofosfamide CYP17 inhibitor during development, ChIP-qPCR analysis was performed. Quantification of immunoprecipitated DNA was performed as described in Material and Methods, and the results of ChIP-qPCR analysis are presented in Figure 5B. Interestingly, this region showed a dynamic epigenetic landscape. The heterochromatic marker H3K9me2 is almost absent at this genomic region during early stages of egg development. In contrast, at L2 larval stage, the H3K9me2 marker is abundant and is distributed almost uniformly all over the analysed genomic region. A considerable loss of this heterochromatin marker was detected at the pupal stage, whereas in adults, heterochromatin organization of this genomic region is established again. This analysis revealed the dynamic aspect of heterochromatin marker accumulation related to the development of S. frugiperda. The Dabrafenib presence of the euchromatic marker H3K4me2 is also shown, indicating that there is little variation of this euchromatic marker in the analysed genomic region during development. The overall quantity of histone H3 is presented as a control, representing overall variation of histone H3 in the chromatin establishment during different developmental stages. The presence of both euchromatic and heterochromatic markers was detected in a number of loci, which may be caused by the fact that chromatin was prepared from a mixture of different cell types coming from whole insect bodies. Apparently, the rDNA locus in eggs does not contain H3K9me2 heterochromatic marker but there is another locus at the position of primer 7 with statistically significant enrichment of the H3K9me2 marker, confirming that the ChIP analysis was technically appropriate. We next investigated whether the increased H3K9me2 is indeed occurring on the all genomic copies of TE-LNCR at the same time as it was detected in the analysed genomic region of BAC 83A24. To do this, we inspected the epigenetic profile of TE-LNCR copies at the adult developmental stage where formation of heterochromatin was already detected. To evaluate the organisation of chromatin at the TE-LNCR genomic copies, two pairs of primers were designed within the LNCR sequence. Chromatin immunoprecipitation was performed with the above mentioned antibodies for modifications of histone H3. The quantity of immunoprecipitated DNA in each sample was evaluated by qPCR and relative enrichment factor was calculated as described in Materials and Methods. Ribosomal gene L37 was used as a positive control for euchromatin and rDNA was a positive control for heterochromatin marker.