title: Illumina HiSeq 4000 sequencing; Ablation of Lgr5-positive cells in intestinal polyps from ApcMin;Lgr5DTR mice
design: Ablation of Lgr5-positive cells in intestinal polyps from ApcMin;Lgr5DTR mice
library_construction_protocol: Lgr5+ cells in the ApcMin;Lgr5DTR mice were ablated with a single intraperitoneal dose of Diphtheria Toxin (DT) (Cat # 322326-1mg, Merck) in saline (50 μg kg−1). The ApcMin;Lgr5DTR mice were culled 24 hours and 5 days post DT. Untreated ApcMin control mice were aged until symptomatic, then culled. Mice were approximately 4 months of age. The entire intestinal tract was removed and divided into small intestine (proximal, middle and distal) and colon. The intestines were flushed with PBS, opened longitudinally using a gut preparation apparatus (Rudling et al 2006). For fresh frozen samples, individual polyps were cut out with a scalpel and snap frozen in liquid nitrogen for subsequent RNA extraction.  The remaining tissue was fixed overnight in 10% neutral buffered formalin (Merck) and processed into FFPE blocks for subsequent histological analysis. For FFPE samples, the tissue was fixed overnight in 10% neutral buffered formalin (Merck) and processed into FFPE blocks.  4um sections were cut and H and E stained to identify polyps.  Tissue from identified polyps were removed from deparaffinised slides with a needle and collected. For fresh frozen samples, RNA was extracted using the RNeasy Micro Kit from Qiagen (74004) and DNase treatment was performed using the DNA-free kit from Life Technologies (AM1906). For FFPE samples, RNA was extracted using the High Pure FFPET RNA Isolation Kit (Roche). 3'RNAseq libraries were prepared using the QuantSeq 3' mRNA-Seq FWD Library Prep Kit (Lexogen, Austria). RNA input varying from 20-1000ng and 14-20 PCR cycles were used. Library quality and quantity were assessed using the HS 1000 DNA TapeStation (Agilent) and DNA High Sensitivity Qubit Assay (ThermoFisher) respectively.