Gene Manipulation Laboratory, Department of Biotechnology and Medical Engineering, National Institute of Technology Rourkela, Odisha, 769 008, India.
Gene Manipulation Laboratory, Department of Biotechnology and Medical Engineering, National Institute of Technology Rourkela, Odisha, 769 008, India.
Anal Biochem. 2021 Feb 1;614:114059. doi: 10.1016/j.ab.2020.114059. Epub 2020 Dec 4.
RNA sequencing (RNAseq) has divulged newer role of spermatozoal RNA in male fertility. This study aimed to evaluate different sperm purification and RNA extraction methods for long-read RNA sequencing of poly(A) transcriptome in goat spermatozoa. Sperm samples were purified by swim-up separation using different purification medium. Spermatozoal RNA was extracted by seven different methods with additional supplementation of reducing agents in lysis buffer. poly(A) selected RNA was used for cDNA library preparation and long-read RNAseq in Nanopore sequencer. Sperm purification by 1 h swim-up resulted in higher recovery (89.20 ± 1.15%), motility (82.33 ± 1.53%), viability (88.10 ± 5.03%) and plasma membrane integrity (71.33 ± 4.51%) in sperm TALP (sp-TL) medium. A monophasic solution of GITC with phenol and DTT resulted in the highest yield of large sized RNAs (3.89 ± 0.46 ng/million cells) suitable for long-read RNAseq of poly(A) transcripts. RNAseq resulted in reads of length, ranging from 500bp to 2 Kb. A total of 123 transcripts were identified in goat spermatozoa by de novo assembly and included sperm-specific transcripts such as CATSPERG, PRM2, CYLC2, SPATA6, PLCZ1 etc. This study is the first report of long-read RNAseq of poly(A) transcriptome in goat spermatozoa.
RNA 测序（RNAseq）揭示了精子 RNA 在男性生育力中的新作用。本研究旨在评估不同的精子纯化和 RNA 提取方法，以用于山羊精子多聚 A 转录组的长读 RNAseq。使用不同的纯化介质通过游泳分离法对精子样本进行纯化。通过七种不同的方法提取精子 RNA，并在裂解缓冲液中额外添加还原剂。使用 poly(A) 选择的 RNA 进行 cDNA 文库制备和 Nanopore 测序仪的长读 RNAseq。在 sp-TL 培养基中进行 1 小时游泳分离可使精子回收率（89.20±1.15%）、活力（82.33±1.53%）、活力（88.10±5.03%）和质膜完整性（71.33±4.51%）更高。含 GITC、苯酚和 DTT 的单相溶液可获得最高产量的大尺寸 RNA（3.89±0.46ng/百万个细胞），适合多聚 A 转录物的长读 RNAseq。RNAseq 产生的读长范围从 500bp 到 2kb。通过从头组装在山羊精子中鉴定了 123 个转录本，包括精子特异性转录本，如 CATSPERG、PRM2、CYLC2、SPATA6、PLCZ1 等。这是山羊精子多聚 A 转录组长读 RNAseq 的首次报道。
