GSM2416067: Lgr5_high_751P_04_SCBC-20; Mus musculus; RNA-Seq
Intestinal crypt cells were purified as previously described (Merlos-Suárez et al., 2011). Proximal small intestines (duodenum and jejunum) were dissected from age and sex-matched mice and cut open longitudinally. They were later incubated with HBSS EDTA (8 mM) for 5 min at room temperature. After this incubation, intestines were shaken vigorously to remove villi and the first supernatant was discarded. The intestines were then incubated with HBSS EDTA on ice for 15 min, after which shaking was repeated. This yielded the first crypt fraction. This process was repeated twice and after microscopic inspection of the samples the fractions enriched in crypts were kept (usually fractions 2 and 3). These fractions were then pooled together for each mouse, centrifuged at 1200 rpm for 5 min (4 ºC) and filtered through a 70 μm mesh (BD Biosciences) to further remove villi. Filtered fractions were enzymatically disaggregated by adding dispase (0.4 mg/mL, Gibco) and incubated for 20 min at 37ºC. The media was then supplemented with 5% FBS (Gibco) to neutralize dispase and the samples were filtered through a 40 μm mesh. For intestinal LRC analysis in compound mice, single cells were stained with an APC-conjugated antibody raised against EphB2 (Clone mAb 2H9, 6 µg/mL, BD). Cells were stained with DAPI (1 µg/mL, Sigma Aldrich) to exclude dead cells. The single-cell preparations of small intestinal crypts were analyzed by flow cytometry in a FACS Aria 2.0 (BD). Data was analyzed with FACSDiva software (BD) or FlowJo for EdU retention assays. Isolation of single cells, cDNA synthesis and amplification were performed on the C1 mRNA Seq HT IFCs (Fluidigm) with the SMARTer Ultra Low RNA Kit (Clonetech). 400 capture sites on a C1 HT IFC medium chip (10-17μm). Spike-in artificial transcripts (ERCC RNA Spike-In Mix, Ambion) from The External RNA Controls Consortium were added during sample preparation as internal control to supported subsequent quality control steps (Baker et al., 2005). Quantity and quality of cDNA were assessed with a High Sensitivity DNA Chip (Agilent Technologies). Libraries were generated by Nextera XT DNA Sample Prep Kit (Illumina) and sequenced on a HiSeq 2500 (Illumina) using 100-bp mode of Truseq Rapid SBS kit v2. Sequencing was carried out as paired-end reads. Read one (12 bp) contained cell and unique molecular barcodes (Islam et al., 2014; Kivioja et al., 2012); read two (85 bp) contained the transcript sequence. Primary data analysis was carried out with the standard Illumina pipeline. Fluidigm C1 HT-Seq technology includes a two-level indexing system, which allows the multiplexed sequencing of multiple single cells and library pools. Here, we processed 10 libraries (up to 40 cells per library) of Lgr5-high cells.
GEO Accession
GSM2416067