GSM8763804: Lgr5 Old Saline B; Mus musculus; RNA-Seq
Sytox blue-negative, live cells were sorted for scRNA-seq using the Chromium Next GEM Single Cell 3ʹ Reagent Kit v3.1 (Dual Index, 10x Genomics, 1000269) with Feature Barcode technology for Cell Multiplexing (10x Genomics, 1000262). Libraries were prepared according to the manufacturer's instructions, with quality control and cell counting performed prior to loading cells onto the microfluidic chip. All cell suspensions were >75% viable and within the recommended concentration range. Following GEM generation and barcoding via reverse transcription (RT), post GEM-RT cleanup and cDNA amplification were conducted. Gene expression and cell multiplexing library construction followed, with quality control performed on generated libraries before sequencing. Gene expression libraries were pooled with cell multiplexing libraries and sequenced on the NovaSeq 6000 (Illumina, San Diego, CA) to yield a minimum of 2,000 reads per cell for cell multiplexing libraries and 20,000 reads per cell for gene expression libraries, following the 28/10/10/90 (R1/i7/i5/R2) bp configuration as per 10X Genomics guidelines. 10x 3' scRNA-seq kit instructions were followed as standard