Illumina NovaSeq 6000 paired end sequencing; Intestinal fibroblastic reticular cell niches control innate lymphoid cell homeostasis and function - Intestinal fibroblasts
Intestinal fibroblastic reticular cell niches control innate lymphoid cell homeostasis and function - Intestinal fibroblasts
Total lamina propria fibroblasts and EYFP+ cells from the small intestine of Ccl19-EYFP and Ccl19-EYFP Ltbrfl/fl  mice were isolated from small intestine. Tissue was harvested and incubated three times for 15 min at room temperature under constant agitation with PBS containing 5% FCS , 5 mM EDTA , 10 mM HEPES  and 1 mM DTT  in order to dissociate the epithelial layer.  The tissue was subsequently washed with BSS containing 10 mM HEPES and digested three times for 20 min at 37°C under constant agitation with 120 µg /ml collagenase P , 25 µg/ml DNase I  and 5 µg/ml Dispase I in medium. To enrich for lamina propria fibroblasts, hematopoietic and erythrocytes were depleted by incubating the cell suspension with MACS anti-CD45 and anti-Ter119 microbeads and passing through a MACS LS column . Unbound single cell suspensions were stained with different markers and sorted with a BD FACS Melody cell sorter.  Sorted single cell suspensions were run on the 10x Chromium analyzer (10X Genomics) cDNA library generation was performed following the established commercial protocol for Chromium Single Cell 3' Reagent Kit (v3 Chemistry).