GSM7730184: Sample2_RNA; Mus musculus; RNA-Seq
Mouse distal small intestine (2x 3cm) was collected, flushed with ice cold PBS and opened, while villi were scraped with a coverglass. After an additional wash, tissue was incubated in HBSS Ca+Mg+ Free (14175095, ThermoFischer Scientific) containing 5mM EDTA (15575-020, Invitrogen) 10mM HEPES (15630-056, Gibco/Life Technologies ) for 25 min at 37C, vortexing every 5 min. Crypts were shaken out, washed with PBS and then incubated in TrypLE  (12605036, ThermoFisher Scientific) supplemented with 10uM y27632 (Y0503, Sigma-Aldrich), 1mM NAC (A9165, Sigma-Aldrich), 50ug/ml DNaseI (10104159001, Roche) for 21min at 37C and pipetted 50x every 5min to dissociate into single cells. Cells were then pelleted in aDMEM/F12 (12634-028, Gibco/Life Technologies) containing 5% FBS, washed in 0.1% BSA/PBS, strained through 100um cell strainer, washed again in 0.1% BSA/PBS and filtered through blue-capped FACS tubes. Subsequently, cells were stained in 0.1% BSA/PBS, containing following antibodies: EpCAM-APC (AB_1659714, eBioscience, 1:500) CD31 PeCy7 (AB_10612003, BD biosciences, 1:200), CD45 PeCy7 (AB_394489, BD biosciences, 1:200), DAPI 1ug/mL, Hashing Ig (1:100) and transferred to low retention tubes, incubated 15min in the dark on room temperature, washed with 0.1%BSA and resuspended in ultra clean 0.1% BSA/PBS for flow cytometry. Samples were sorted with BD FACS Aria III sorter, 70u nozzle, 1.0 ND filter into an Eppendorf tube with 2uL ultra clean 0.1% BSA/PBS.  Hashing was performed so that each sample contained cells from one mouse and was labelled by a distinct hashing Ig before the sort. This means we had 8 hashed samples from 4 mice that were traced 48h (# 1-4) and 4 mice traced 72h (# 5-8). We sorted GFPhigh clones and GFP- cells from the epithelium as control into 2 eppendorfs so that each contained cells from 48h and 72h clones, as well as the control epithelium, and each contained all 8 hashes. Thus, we had 2 samples for library preparation: Sample 1 contained control epithelium from mice number 1,2,5,6, 48h clones from mice 3,4 and 72h clones from mice 7,8; Sample 2 contained control epithelium from mice number 3,4,7,8, 48h clones from 1,2 and 72h clones from 5,6. The libraries were prepared using the Chromium Next GEM Single Cell 3' Reagent Kits v3.1 according to the 10X Genomics protocols v3.1. Maximum 10,000 cells were loaded into 36uL of ultra clean 0.1%BSA/PBS for the formation of Gel Bead-in-Emulsions and followed by reverse transcriptase. cDNA was amplified with 12 polymerase chain reaction (PCR) cycles, while sample-indexing PCR was performed with 14 cycles. The libraries were sequenced on an Illumina NextSeq 2000 platform with a High Output 150 cycles kit.