title: Illumina HiSeq 2500 paired-end Sequencing of KAS22012272 (KAE22117610)
design: Reference sequence : mouse mm10
library_name: kidney_1year-001_TRANSCRIPTOMIC SINGLE CELL
library_construction_protocol: For generation of single-cell libraries, TAM-injected Lrig1-tdTomato mice were sacrificed at 1 and 365 days after injection. The kidneys were collected and digested, and renal epithelial cells were isolated. Cells were passed through a 40 micrometer strainer to obtain single tubule epithelial cells. Prior to library preparation, single tubule epithelial cells were enriched for live cells using a dead cell removal magnetic cell sorting kit (The Dead Cell Removal Kit, Miltenyi Biotec Inc.) according to the manufacturer's instruction. Isolated live renal epithelial cells were captured for library preparation using the Chromium controller according to the 10x Single Cell 3' v3 protocol (10x Genomics). In brief, the cell suspensions were diluted in nuclease-free water to achieve a targeted cell count of 10,000. The cell suspension was mixed with master mix and loaded with Single Cell 3' Gel Beads and Partitioning Oil into a Single Cell 3' Chip. RNA transcripts from single cells were uniquely barcoded and reverse transcribed within droplets. cDNA molecules were pooled and subjected to an end-repair process, the addition of single 'A' bases, and ligation of the adapters. The products were purified and enriched with PCR to create the final cDNA library. The purified libraries were quantified using qPCR according to the qPCR Quantification Protocol Guide (KAPA) and qualified using the Agilent Technologies 4200 TapeStation. The libraries were sequenced using HiSeqX (Illumina) with a read length of 28 bp for read 1 [with cell barcodes and unique molecular identifiers (UMIs)], 8 bp for the index read (sample barcode), and 91 bp for read 2 (RNA read).