GSM9796482: Day_1_Kidney_tamoxifen_ILY_treated; Mus musculus; RNA-Seq
Kidneys were harvested post-perfusion and processed for single-cell suspension preparation. Tissue dissociation was initiated by mechanical disruption of the kidney, followed by enzymatic digestion. The dissected kidney was incubated in 10 mL of Hank's Balanced Salt Solution (HBSS), containing 0.75 mg/mL collagenase IV (Worthington Biochemical) and 20 µg/mL DNase I (Sigma-Aldrich), for 40 minutes at 37°C. After digestion, the cell suspension was passed through a 40 µm cell strainer to remove debris and larger tissue fragments, and the resulting single-cell suspension was maintained on ice. The cell suspension was then washed twice with PBS by centrifugation at 500 x g for 10 minutes. To enrich hematopoietic cells, the cells were subjected to density gradient centrifugation using Percoll. The pellet obtained from the previous wash was resuspended in 10 mL of 30% Percoll solution and carefully layered over 3 mL of 70% Percoll solution. The gradient was centrifuged at 500 x g for 30 minutes without the application of a brake. Following centrifugation, the interphase layer between the 30% and 70% Percoll solutions, containing enriched single cells, was carefully collected. The collected cells were washed twice with 10 mL of PBS (500 x g, 10 minutes). The final cell pellet was resuspended in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) for downstream single-cell RNA sequencing (scRNA-seq) analysis. Cell number and viability were assessed using an automated cell counter, yielding a viability rate of approximately 84%, suitable for scRNA-seq. scRNA-seq was performed using the 10× Genomics Chromium platform to analyze transcriptomic profiles of kidney cells. A target of 5000 viable cells per sample was set based on prior optimization using 10× Genomics Single Cell 3′ RNAseq technology (10x Genomics). Live cells were quantified and sorted before processing, ensuring the cell viability exceeded 80% for downstream analysis. Following cell capture, full-length barcoded cDNA libraries were generated from each sample according to the manufacturer's protocol. Briefly, individual cells were partitioned into droplets containing reagents for reverse transcription, generating unique cDNA barcodes for each cell. The cDNAs were then amplified via polymerase chain reaction (PCR) to obtain sufficient material for library preparation. The cDNA libraries were pooled and diluted to a final concentration of 1.8 pM for sequencing. The pooled libraries were sequenced using an Illumina NextSeq 2000 platform, with paired-end single index reads. Sequencing output was analyzed to ensure adequate coverage and depth for each sample.