title: GSM8454788: Young_1_ProxSI; Mus musculus; RNA-Seq
library_construction_protocol: Crypts from different sections of intestine were isolated as described previously(Asad A Ahmad et al., 2014). After centrifugation, the crypts were re-suspended in 3ml of Single-Cell Isolation Solution (TrypLE supplemented with: 1mg/ml DNaseI, 5mM MgCl2, 80μM Y27) and incubated at 37°C for 20min, vortexing for 5 seconds after first 10min. Reaction was quenched by addition of 27ml ice-cold PBS and cells were centrifuged at 1000g for 5minutes at 4°C. Cell pellet was re-suspended in FACS staining medium (FSM - PBS supplemented with 2% Fetal Bovine Serum, 80μM Y27, 3mM EDTA) and treated with CD326 (EpCAM) (G8.8), PE-Cyanine7 coupled rat monoclonal antibody and a TotalSeq Hashtag antibody for 30min on ice in the dark. The cells were then centrifuged at 450g for 5min at 4°C and re-suspended in 500μl fresh FSM. An input of 10000 EpCAM positive FACS sorted single cells per compartment was added to each 10X Chromium Controller channel. The 10X Single Cell 3' v2 or v3.1 reagent kit (10X Genomics) was used for cell partitioning, cDNA amplification and library construction.