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.
Keywords: DTT; MinION; Nanopore sequencing; RNA Xpress(TM) reagent; Sperm RNA.
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