GSM9335825: D5_1; Mus musculus; RNA-Seq
In this study, 6-8-week-old female mice bearing orthotopic renal carcinoma models received tail vein injection of 4-thiouridine (4sU) for 1.5-hour metabolic labeling before kidney tissue collection. Tissue dissociation was performed using the Multi Tissue Dissociation Kit 2 with gentleMACS Octo Dissociator, followed by filtration through 100 μm and 40 μm strainers to obtain single-cell suspensions. The Well-paired-seq microfluidic chip was employed to pair cells with barcoded beads in dual-chamber structures, with cell lysis achieved using 5% sodium lauroyl sarcosinate to facilitate mRNA capture on beads, establishing the foundation for subsequent transcriptomic analysis of 4sU-labeled newly synthesized RNA versus total RNA. Following cell lysis and mRNA capture, barcoded beads underwent 4sU-to-C chemical conversion using iodoacetamide treatment. Reverse transcription with template switching was performed, followed by Exonuclease I digestion to remove excess primers. Second-strand synthesis utilized Klenow exo- enzyme and cDNA was amplified via PCR. The resulting products were purified twice with DNA Clean Beads, and sequencing libraries were prepared using a Vazyme Kit with 5 ng input. Final libraries underwent additional bead purification and were sequenced on a HiSeq X Ten platform with 150 bp paired-end reads.