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Collected oocytes were lysed in TRIzol reagent (Takara), and ERCC Spike-In RNA (Thermo Fisher Scientific) was also added to the TRIzol reagent before RNA extraction. Total RNA was isolated by chloroform extraction, followed by isopropanol precipitation. To facilitate RNA precipitation, 1/10 volume of 3 M NaAc and 1 uL glycogen were added to the aqueous phase. The RNA pellet was washed twice with 75% ethanol and resuspended in nuclease-free water. Purified RNA was used for library preparation using the SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian (Takara) according to the manufacturer's instructions. Briefly, fragmented total RNA was first reverse-transcribed into cDNA, followed by limited-cycle PCR to add Illumina adapters. After purification, ribosomal cDNA was depleted, and the remaining fragments were further amplified using universal primers. Final libraries were purified and sequenced on an Illumina NovaSeq platform (173 bp paired-end reads) at Nanjing Jiangbei New District Biomedicine Public Service Platform Co., Ltd. |
RNA-Seq |
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PCR |
PAIRED
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