GSM9407563: Lymph node cells, remitter-rep2; Mus musculus; RNA-Seq
The isolated mouse islets were incubated in 2ml Accutase™ Cell Dissociation reagent at room temperature for 5 mins on a tube rotator. After incubation, islet cells were resuspened thoroughly by pipet up and down. Five-time volume of culture medium with 10% FBS was added to stop excessive dissociation reactions. Then dissociated islet cells were washed and resuspened in PBS with 0.04% BSA. Pancreatic draining lymph node cells were dispersed into single cells by grinding the samples using the blunt end of a syringe over a cell strainer. Alive cells were counted using trypan blue and hemacytometer. Library was constructed according to the manufacturer's protocols (Chromium Next GEM Single Cell 5' v2, Dual Index, 10X Genomics). Briefly, cells were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). Gel bead primers contain an Illumina TruSeq Read 1 primer, a 10x Barcode, a unique molecular identifier (UMI), and template switch oligo (TSO). Then the poly-A mRNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which was amplified. The amplifiedd cDNA from poly-A mRNA and the amplified DNA from cell surface protein Feature Barcode are separated by size selection for generating V(D)J and/or 5' Gene Expression libraries and Cell Surface Protein (hashtag) libraries, respectively.