Instrument: Illumina NovaSeq 6000
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: PAIRED
Construction protocol: The small intestine was dissected, flushed with cold PBS, opened longitudinally and cut into small fragments roughly 2-4 mm in length. Intestine fragments were washed twice with cold PBS and then incubated with 20 mM EDTA-PBS on ice for 30 min. The tissue was then shaken vigorously, and the supernatant was collected as fraction1 in a new conical tube. The tissue was incubated in fresh EDTA-PBS and a new fraction was collected every 30 min. Fractions were collected until the supernatant consisted almost entirely of crypts. The final fraction was filtered through a 70-μm cell strainer (enriched for crypts), washed twice in PBS, centrifuged at 300 g for 3 min, and dissociated with TrypLE Express (Invitrogen) at 37 ºC for 5 min. The single-cell suspension was then passed through a 40-μm cell strainer. Cells were stained with antibodies and gated on 7AAD- CD45- CD31- TER119- EpCAM+, GFP high (stem cells) and GFP low (TA cells), and sorted by a BD AriaIII cell sorter. Sorted samples were processed by extracting RNA with RNeasy Micro Kit (Qiagen) RNA libraries were prepared for sequencing using standard Illumina protocols