Single-Cell RNA-Seq of Lgr5+ Intestinal Cells

Lgr5-eGFP-IRES-CreER mice were treated with adenovirus in vivo and then 26h post-treatment, the proximal jejunum was harvested to generate a single cell suspension and FACS isolated using the endogenous GFP signal, as above. The sorted cellular suspensions were loaded on a GemCode Single Cell Instrument (10x Genomics, Pleasanton, CA) to generate single-cell GEMs. Approximately ~1200–2800 cells were loaded per channel. Two technical replicates were generated per sorted cell suspension. Single-cell RNA-Seq libraries were prepared using GemCode Single Cell 3′ Gel Bead and Library Kit (now sold as P/N 120230, 120231, 120232, 10x Genomics) as per Zheng et. al^{29 (link)}. Sequencing libraries were loaded at 2.1pM on an Illumina NextSeq500 with 2 × 75 paired-end kits using the following read length: 98bp Read1, 14bp I7 Index, 8bp I5 Index and 5bp Read2. Note that these libraries were generated before the official launch of GemCode Single Cell 3′ Gel Bead and Library Kit. Thus 5bp UMI was used (the official GemCode Single Cell 3′ Gel Bead contains 10bp UMI.).