description: Protocols: 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 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.
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2022-06-30
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2022-06-30
attributes.tag: External Id
attributes.value: SAMEA14392621
attributes.tag: INSDC center alias
attributes.value: OXFORD
attributes.tag: INSDC center name
attributes.value: University of Oxford
attributes.tag: INSDC first public
attributes.value: 2022-06-30T00:19:17Z
attributes.tag: INSDC last update
attributes.value: 2022-06-30T00:19:17Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-104701652713385:MA11
attributes.tag: age
attributes.value: 16
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: common name
attributes.value: house mouse
attributes.tag: developmental stage
attributes.value: adult
attributes.tag: disease
attributes.value: intestinal polyp
attributes.tag: genotype
attributes.value: ApcMin;Lgr5DTR
attributes.tag: individual
attributes.value: 17
attributes.tag: isolate
attributes.value: not applicable
attributes.tag: organism part
attributes.value: ileum
attributes.tag: sample name
attributes.value: E-MTAB-104701652713385:MA11
attributes.tag: sampling site
attributes.value: neoplasm
attributes.tag: sex
attributes.value: male
attributes.tag: specimen with known storage state
attributes.value: frozen specimen
attributes.tag: strain
attributes.value: C57BL/6J