GSM4709004: Cell_9 (A9); Mus musculus; RNA-Seq
Sorted mammary epithelial cells were washed and resuspended at a concentration of ~500 cells/µl. For Fluidigm microfluidics scRNAseq, primary cell suspensions were mixed with Fluidigm C1 Suspension Reagents (Fluidigm 100-5315) at a ratio of 8:2 before loading mix onto C1 chip (Fluidigm 100-5760). Captured cells were visualized and counted using a Keyence BZ-X710 microscope (Keyence Corporation, Itasca, Illinois, USA). Single-cell RNA isolation and amplification were performed using the Fluidigm C1 Single Cell Auto Prep IFC following the Fluidigm Protocol: 100-7168 I1. cDNA library preparation was performed following the Fluidigm C1 Protocol: 100-7168 I1 (1771x/1772x/1773x). RNA spike-in controls were omitted. Next, reverse transcription and cDNA pre-amplification were performed using the SMARTer Ultra Low RNA Kit (Clontech) and processed in the C1 system by running script mRNA Seq: RT and Amp (1771x/1772x/1773x). After completion of RT and cDNA amplification, the samples were harvested and quantified through the PicoGreen assay (Thermo-Fisher) using the Agilent Bioanalyzer (Agilent Genomics). Equal amounts of cDNA from each sample were tagmented and prepared for library synthesis using the using Nextera XT DNA Sample preparation kit with 96 indices (Illumina), per the protocol supplied by Fluidigm. Libraries were multiplexed and sequenced as paired-end on a Next-Seq 4000 Illumina sequencing platform. cDNA amplification, library preparation, quality control, and sequencing were performed at the Genomics High Throughput Sequencing facility at the University of California, Irvine.
GEO Accession
GSM4709004