GSM9443446: NC_wk23_A_R4; Mus musculus; RNA-Seq
Liver tissues were homogenized into RNA STAT-60 (Tel-Test Inc., Friendswood, TX) using a polytron homogenizer, followed by total RNA isolation using the MagMAX mirVana Total RNA isolation kit (Thermo Fisher Scientific Inc., Foster City, CA) according to the manufacturer's instructions. Total RNA samples were qualified and quantified on the Fragment Analyzer (Agilent Technologies, Santa Clara, CA) per manufacturer's instructions. For the TruSeq Stranded mRNA protocol, total RNA was utilized as the input for Illumina's TruSeq Stranded mRNA library preparation kit (Platform: Illumina NovaSeq6000). This method purified polyA-containing mRNA molecules using oligo-dT attached magnetic beads and involved two rounds of purification. During the second elution of the polyA RNA, the RNA was fragmented and primed for complementary DNA (cDNA) synthesis. Subsequently, the cleaved RNA fragments, primed with random hexamers, were reverse transcribed into first-strand cDNA. The RNA template was then removed, and a replacement strand was synthesized, incorporating dUTP in place of dTTP to generate double-stranded cDNA (ds cDNA). An adenine (A) nucleotide was added to the 3ʹ ends of the blunt fragments to prevent them from ligating to one another during the adapter ligation reaction. Multiple indexing adapters were ligated to the ends of the ds cDNA fragments in preparation for hybridization. Polymerase chain reaction (PCR) was employed to selectively enrich those DNA fragments that possessed adapter molecules on both ends and to amplify the quantity of DNA in the library. After amplification, the targeted libraries were quantified using quantitative PCR (qPCR) and pooled into a single lane. The pooled libraries were subsequently prepared for cluster generation and sequencing on high-throughput Illumina NovaSeq instruments. At least 4 gigabases of 50 base pairs of paired-end data were generated by the sequencing machine, in accordance with the project's raw data requirements.