title: Illumina NovaSeq 6000 paired end sequencing; Intestinal fibroblastic reticular cell niches control innate lymphoid cell homeostasis and function - Innate lymphoid cells
library_construction_protocol: For the isolation of lymphocytes and ILCs from the lamina propria, 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 and 25 µg/ml DNase I in medium. single cell suspension was resuspended with 5 ml 30% Percoll diluted with medium and was underlayed with 3 ml 70% Percoll diluted with medium . After centrifugation, the lymphocyte/ILC layer was retrieved and washed with MACS buffer (PBS buffer with 2% FCS and 2 mM EDTA. The cell suspension from the small intestine of Ccl19-EYFP Ltbrfl/fl  mice and the co-housed littermates were enriched for ILCs via the Easysep Mouse Pan ILC enrichment kit  and staining with the lineage cell markers and CD127  for sorting. Sorted single cell suspensions were run on the 10x Chromium analyzer. cDNA library generation was performed following the established commercial protocol for Chromium Single Cell 3' Reagent Kit (v3 Chemistry).