title: GSM8162244: Mouse small intestine immune cells WT/Bmal1/Apc/Dko scRNA-seq 20220504; Mus musculus; RNA-Seq
library_construction_protocol: FACS-isolated CD45+ cells from the small intestine of WT, Bmal1-/-, Apc+/- and Apc+/-;Bmal1-/- mice (n=3 mice/genotype) were washed once in PBS with 0.04% BSA, and a total of 0.2-2x106  cells per genotype were used for cell multiplexing barcoding using the CellPlex Kit (10X Genomics), according to manufacturer's instructions. Briefly, cells were pelleted after washing by centrifugating at 300 rcf, for 5 minutes at room temperature, and resuspended in 100 µl Cell Multiplexing Oligos (one oligo/barcode per genotype). Oligos were allowed to bind cell membranes by incubating at room temperature for 5 min, and unbound multiplexing oligos were washed 3 times with 2 ml ice-cold ml PBS + 1% BSA. After washing, cells were resuspended in chilled PBS + 1% BSA to a concentration of approximately 1,000 cell/μL, and loaded onto the 10X Genomics Chromium Controller for droplet-enabled scRNAseq according to the manufacturer's instructions. Library generation was performed following the Chromium NextGEM Single Cell v3.1 Cell Multiplexing Reagents Kits User Guide: CG000388Rev B. A total of 3 libraries were generated, each of them consisting of a pool of equal cell numbers from CD45+ cells from the small intestine of each genotype.