期刊
ANALYTICAL BIOCHEMISTRY
卷 614, 期 -, 页码 -出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2020.114059
关键词
MinION; Sperm RNA; DTT; RNA Xpress (TM) reagent; Nanopore sequencing
资金
- Council of Scientific and Industrial Research (CSIR), India
This study evaluated different sperm purification and RNA extraction methods for long-read RNA sequencing of poly(A) transcriptome in goat spermatozoa. Results showed that sperm purification by 1 h swim-up in sp-TL medium and using a monophasic solution of GITC with phenol and DTT resulted in the highest yield of large sized RNAs suitable for long-read RNAseq of poly(A) transcripts.
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.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据