GSM5579377: P14-stage Whole Mesenchyme Single Cell RNA seq; Mus musculus; RNA-Seq
Cells were isolated from entire small intestinal tissues, epithelium denuded via 20 minutes in warm 10mM EDTA/HBSS (at 37 degress C), and tissue further minced by fine scalpels. Digestion of tissue was performed using 3 mg/ml collagenase II (Worthington) in 5%FBS/HBSS at 37 degrees C. Supernatants were collected 40 minutes after digestion, washed to remove debris, treated with ACK lysis buffer to remove RCBs, and strained through a 40 uM filter. Lysates were washed in PBS/0.1%BSA and antibody-labelling was performed prior to FACS. FACS-sorted cells were spun at 300 rcf for 10 minutes, and loaded on a 10X Chromium Controller (10X Genomics) for targeting 5-10,000 cells using the single cell 3' V3.1 assay. Reverse transcription, cDNA amplification and library prep were completed according to manufacturer's protocol. Sequencing of library was performed using a HiSeq4000 Illumina System. Chromium Next GEM Single Cell 3' Reagent Kits v3.1 (10X Genomics)