title: GSM5939684: ISC 22 month (old) SM6 WT rep3; Mus musculus; RNA-Seq
library_construction_protocol: The isolated small intestine was flushed with PBS and opened longitudinally. The tissue was then scraped with a glass coverslip to remove villi, cut into 5-mm pieces and washed with PBS five times (20 inversions of the tube per wash). Following incubation for 30 min at 4°C in 4mM EDTA-PBS, intestinal crypts were released from small intestinal tissue fragments by mechanically pipetting with a 10ml pipette in PBS and repeating this step twice. After centrifugation (430xg for 5 min at 4°C), the pellet was resuspended in PBS and strained using a 70-μm cell strainer (BD Biosciences). Following centrifugation (430xg for 5 min at 4°C), the collected crypts were incubated for 30 minutes at 4°C in DMEM/F12 – 10% serum (Gibco), pelleted again by centrifugation (430xg for 5 min at 4°C) and then dissociated in TrypLE Express (Invitrogen) supplemented with 10 μM Rock inhibitor (Y-27632, Abcam) and 2.5µg/ml DNAse 1 (Sigma-Aldrich) for 4 minutes at 37°C. The dissociated cells were strained using a 70-μm cell strainer and were washed twice with PBS and collected by centrifugation at 4°C at 430xg for 5 minutes. Cellularized intestinal epithelial were labelled with antibodies and ISCs isolated by FACS (SM6 strategy, CD31-/CD45-/CD24med/CD166+/CD44high/GRP78low/EPHB2high/EPCAM+). A 3-prime biased approach was used to generate sequencing libraries (see methods in publication for further details). An 8 bp sample index and a 10 bp unique molecular identifier (UMI) were added during initial poly(A) priming and pooled samples were amplified using a template switching oligonucleotide. The Illumina P5 (5' AAT GAT ACG GCG ACC ACC GA 3') and P7 (5' CAA GCA GAA GAC GGC ATA CGA GAT 3') sequences were added by PCR and Nextera transposase, respectively. The library was designed so that the forward read (R1) utilizes a custom primer (5' GCC TGT CCG CGG AAG CAG TGG TAT CAA CGC AGA GTA C 3') to sequence directly into the index and then the 10 bp UMI. The reverse read (R2) uses the standard R2 primer to sequence the cDNA in the sense direction for transcript identification. Sequencing was performed on the NextSeq550 (Illumina), using the V2 High output kit (Illumina, #TG-160-2005) in accordance with the Illumina Protocol 15046563 v02, generating 2 reads per cluster composed of a 19 bp R1 and a 72 bp R2.