title: NextSeq 500 paired end sequencing; Single-cell RNAseq of murine Bcl6-deficient ILC against wild-type controls
design: Single-cell RNAseq of murine Bcl6-deficient ILC against wild-type controls
library_name: KO_p
library_construction_protocol: We performed single-cell RNA sequencing from small intestine ILC as previously described (Fiancette et al., 2021). Briefly, Id2-CreERT2 ROSA26-tdRFP Bcl6-fl/fl and Id2-CreERT2 ROSA26-tdRFP control mice were treated with 5mg of tamoxifen in corn oil 5 times over the course of 2 weeks via oral gavage. Mice were sacrificed through cervical dislocation 3 weeks after the last dose. Fat and Peyer's patches were removed from the small intestine, which was opened longitudinally and washed vigorously in PBS. Tissue was incubated for two concurrent rounds in PBS containing 1 mM EDTA, 1 mM dithiothreitol and 5 % FBS for 25 min at 37 °C with constant mixing. Then, tissues were washed in PBS and digested in complete RPMI with in 1 mg/ml collagenase VIII (Sigma-Aldrich) and 20 μg/ml DNase (Sigma-Aldrich) for 25 min at 37 °C. The digested tissues were filtered through a 70 μm nylon strainer and stained for fluorescence-activated cell sorting. Live CD45+ B220- CD11c- CD11b- CD3- CD5- TCRβ- TCRγδ- CD90.2+ CD127+ ROSA26-tdRFP+ ILC were sorted into PBS 10% FBS using a BD FACS Aria II. Sorted cells were passed through the 10x Chromium (10x Genomics) system as indicated on the manufacturer's protocol (CG000315 Rev B), at a target recovery rate of 10,000 cells. cDNA library generation was performed as indicated in the protocol for Chromium Single Cell 3' Reagent kits v3.1 (10x Genomics). Resulting libraries were prepared in parallel in a single batch.