RNA-seq profiling of RPE reprogramming

The plasticity of the retinal pigment epithelium (RPE) has been observed during proliferative vitreoretinopathy (PVR), a defective repair process in humans. In contrast, in the embryonic chick, the RPE can be efficiently reprogrammed to regenerate a complete neural retina after surgical removal and when supplied an exogenous source of FGF2. Here, we analyzed discrete RPE cell populations during early times of transiently reprogrammed (RPE 6 hours post-retinectomy) and reprogrammed (RPE 6 hours post-retinectomy and FGF2 treatment) cells, using laser capture microdissection followed by RNA sequencing (LCM-seq) and computational analysis. RPE from chicken embryos were isolated via the Veritas Laser Capture Microdissection system in triplicate across three conditions: 1) intact embryonic day 4 (E4) RPE, 2) E4 RPE 6 hours post retinectomy (transiently reprogrammed RPE; t-rRPE), and 3) E4 RPE 6 hours post retinectomy and FGF2 treatment (reprogrammed RPE; rRPE). Total RNA extraction was performed using PicoPure RNA Isolation Kit (Arcturus, Applied Biosystems, Foster city, CA), including a treatment with DNase I. Intact, total RNA was reverse-transcribed using an Oligo(dT) primer, and limited cDNA amplification was performed using the SMARTer® Ultra® Low Input RNA Kit for Sequencing – v4 (Takara Bio USA, Inc., Mountain View, CA). The resulting full-length cDNA was fragmented and tagged, followed by limited PCR enrichment to generate the final cDNA sequencing library (Nextera® XT DNA Library Prep, Illumina, San Diego, CA). Libraries were sequenced as single-end 75 base pair reads on an Illumina NextSeq500 to generate 44-68 million single-end 75 bp reads for each sample. Reads were quality trimmed and adapter sequences removed using Trim Galore v0.6.1 and Cutadapt v1.18. Trimmed reads were aligned to Gallus gallus genome GRCg6a with Ensembl annotation 98 using STAR v2.7.3. Assembly and quantification of transcripts was performed with Stringtie v2.0.4 and normalization of gene counts was performed with DESeq2 v1.22.2.

视网膜色素上皮（retinal pigment epithelium, RPE）的可塑性已在人类增殖性玻璃体视网膜病变（proliferative vitreoretinopathy, PVR）——一种存在缺陷的修复过程中被观测到。与之相对，在胚胎鸡中，手术移除视网膜后，若补充外源性成纤维细胞生长因子2（fibroblast growth factor 2, FGF2），RPE可被高效重编程以再生完整的神经视网膜。本研究针对瞬时重编程（视网膜切除术后6小时的RPE）与重编程（视网膜切除术后6小时并经FGF2处理的RPE）两类细胞的早期阶段的离散RPE细胞群，采用激光捕获显微切割结合RNA测序（laser capture microdissection followed by RNA sequencing, LCM-seq）及计算分析开展研究。我们通过Veritas激光捕获显微切割系统，从三个实验组中分离鸡胚RPE，每组设置3次生物学重复：1）完整的胚胎第4天（embryonic day 4, E4）RPE；2）视网膜切除术后6小时的E4 RPE（瞬时重编程RPE；t-rRPE）；3）视网膜切除术后6小时并经FGF2处理的E4 RPE（重编程RPE；rRPE）。总RNA提取采用PicoPure RNA分离试剂盒（Arcturus、Applied Biosystems，美国加利福尼亚州福斯特城），并辅以DNase I处理。完整的总RNA使用寡聚(dT)引物进行反转录，针对微量cDNA扩增采用SMARTer® Ultra® 超低起始量RNA测序试剂盒v4（Takara Bio USA, Inc.，美国加利福尼亚州芒廷维尤）。所得全长cDNA经片段化并添加标签，随后通过有限轮PCR富集以构建最终cDNA测序文库（Nextera® XT DNA文库制备试剂盒，Illumina，美国加利福尼亚州圣迭戈）。采用Illumina NextSeq500平台进行单端75碱基对测序，每个样本可获得4400万至6800万条单端75bp读段。使用Trim Galore v0.6.1与Cutadapt v1.18对读段进行质量修剪并移除接头序列。将修剪后的读段比对至带有Ensembl注释版本98的家鸡（Gallus gallus）基因组GRCg6a，比对工具为STAR v2.7.3。采用Stringtie v2.0.4完成转录本组装与定量，并通过DESeq2 v1.22.2对基因计数进行标准化处理。