GSM8586268: CC4:2-3; Rattus norvegicus; RNA-Seq
Total RNA was extracted from the tissue using TRIzol® Reagent (Qiagen) according to the manufacturer's instructions. RNA quality was determined using the 5300 Bioanalyser (Agilent) and quantified with the ND-2000 (NanoDrop Technologies). Only RNA samples meeting specific criteria (OD260/280 = 1.8 ~ 2.2, OD260/230 ≥ 2.0, RQN ≥ 7.0, 28S: 18S ≥ 1.0, > 1μg) were used to construct the sequencing library. RNA purification, reverse transcription, library creation, and sequencing were conducted by Shanghai Majorbio Bio-pharm Biotechnology Co., Ltd. (Shanghai, China). The mRNA-seq transcriptome library was generated according to the Illumina® Stranded mRNA Prep, Ligation protocol (San Diego, USA) using 1 μg of total RNA. Initially, messenger RNA was isolated using the polyA selection method with oligo (dT) beads and subsequently fragmented using a fragmentation buffer. Following this, double-stranded cDNA was produced using a SuperScript double-stranded cDNA synthesis kit (Invitrogen, USA) with random hexamer primers. The resulting cDNA underwent end-repair, phosphorylation, and adapter ligation according to the library preparation protocol. cDNA target fragments of 300 bp were size-selected on 2% Low Range Ultra Agarose and amplified by PCR using Phusion DNA polymerase (NEB) for 15 cycles. After post-quantification using Qubit 4.0, the sequencing libraries were sequenced on either a NovaSeq X Plus platform (PE150) with the NovaSeq Reagent Kit or a DNBSEQ-T7 platform (PE150) with the DNBSEQ-T7RS Reagent Kit (FCL PE150) version 3.0.