RNA-Seq and ChIP-Seq gene profiling of retinal ganglion cells in Math5Cre-driven Knockout of Ezh2 and G9a in murine retinal ganglion cells at P0

Epigenetic mark deposition during embryonic development contribute to postnatal homeostasis and tissue stability. Previously, we found out that Ezh2 contributes critically to the function and postnatal cell survival in bipolar cells but not in retinal ganglion cells in the retina. (Yan et al. Postnatal onset of retinal degeneration by loss of embryonic Ezh2 repression of Six1. Sci Report. doi:10.1038/srep33887; (Cheng L, Wong LJ, Yan N, Han RC et al. Ezh2 does not mediate retinal ganglion cell homeostasis or their susceptibility to injury. PLoS One 2018;13(2):e0191853.). In this study, we used RNA-seq to define up- and down regulated genes in both Ezh2 and G9a deficient (Math5Cre; Ezh2f/fG9af/+; dKO) retinal ganglion cells (RGC) to evaluate the hypothesis of Ezh2 and G9a interaction that has been discussed in other tissues but in the retina. ChIP-Seq was applied to evaluate H3K27me3 histone marks in retinal ganglion cells in wild type (WT), Ezh2 (Math5Cre; Ezh2-/-, sKO) and combined G9a-Ezh2 (Math5Cre; G9a+/-Ezh2-/-, dKO) knockout mutant mice at P1. mice pups (Math5Cre+;Ezh2f/fG9a+/- [dKO] or wild type [WT]) were sacrificed at P0 and the retina was harvested immediately. After enzymatic digestion, retinal ganglion cells (RGC) were purified from other retinal cells using magnetic labelling with anti-CD90-antibody which binds specifically to RGC. After elution, samples were immediately proceed to RNA extraction. We collected triplicates for each cell samples. Library was prepared with NEBNext Poly A, Sequencing was done with Mid-Output single read 75 cycles flow cell.胚胎发育过程中的表观遗传标记沉积有助于出生后机体稳态与组织稳定性维持。此前本团队研究发现，Ezh2对视网膜双极细胞的功能及出生后细胞存活具有关键调控作用，但对视网膜神经节细胞（retinal ganglion cells, RGC）并无此类作用（Yan等. 胚胎期Ezh2解除对Six1的抑制引发视网膜退行性变. *Scientific Reports*. doi:10.1038/srep33887; Cheng L, Wong LJ, Yan N, Han RC等. Ezh2不介导视网膜神经节细胞稳态及其损伤易感性. *PLOS ONE* 2018;13(2):e0191853.）。本研究通过RNA测序（RNA-seq）分析Ezh2与G9a双缺陷（Math5Cre; Ezh2^f/fG9a^+/-; dKO）视网膜神经节细胞中的差异表达基因，以验证Ezh2与G9a的相互作用假说——该假说已在其他组织中被提出，但尚未在视网膜领域开展相关研究。本研究同时采用染色质免疫沉淀测序（ChIP-Seq）检测出生后1天（P1）龄野生型（wild type, WT）、Ezh2单敲除（Math5Cre; Ezh2^-/-, sKO）及G9a-Ezh2双敲除（Math5Cre; G9a^+/-Ezh2^-/-, dKO）小鼠视网膜神经节细胞中的H3K27me3组蛋白修饰标记。实验所用幼鼠分为两组：Math5Cre⁺;Ezh2^f/fG9a^+/-（dKO）组与野生型（WT）组，于出生后0天（P0）龄处死后立即采集视网膜组织。经酶消化后，利用特异性结合RGC的抗CD90抗体磁珠标记法，从其他视网膜细胞中纯化得到视网膜神经节细胞。洗脱后立即对样本进行RNA提取，每组细胞样本均设置3次生物学重复。采用NEBNext Poly A试剂盒构建文库，使用Mid-Output单端测序75循环流动槽完成测序。