title: GSM6925088: scRNA-seq_IR_56h_Ki67-RFPcells_rep1; Mus musculus; RNA-Seq
library_construction_protocol: Crypts were isolated from proximal half of small intestine of Mki67tm1.1Cle/J mice after 56 hours post-irradiation and their non-IR littermate control. Briefly, freshly harvested intestine was flushed with cold PBS, opened longitudinally, cut into 1 cm pieces, and then rotated in 3 mM EDTA in PBS at 4 °C for 5 minutes, 10 minutes and 40 minutes (refreshing EDTA/PBS every time). The tissue was then vigorously shaken to release the epithelium, and non-IR crypts passed through a 70-μm cell strainer. 100-μm cell strainer was used in IR samples, as crypts were expanded during regeneration after IR. Crypts were pelleted by centrifugation at 300 g for 3 minutes at 4 °C and then washed with cold PBS. To dissociate single cells for FACS, isolated crypts were further rotated with 5U/ml dispase (Stem Cell 07913) and 200 U/ml of DNase I (Sigma D4513) at 37 °C for 30 minutes, and were then washed twice with 1% BSA/PBS, and filtered with a 40-μm cell strainer. Cells were prepared in 1% BSA/PBS with 200 U/ml of DNase I for sorting. Ki67-RFP+ DAPI- cells from mice 56 hours post-IR and non-IR condition were detected and sorted with Beckman Coulter Astrios EQ High Speed Cell Sorter, respectively. Dead cells were eliminated using 0.5 μg/ml DAPI. Kaluza analysis 2.1.3 software was used for FACS data analysis. Total 5500 cells were sorted and concentrated into 5 μL for initiating the PIPseq pipeline (PIPseq™ T2 3' Single Cell Capture and Lysis Kit v2.1, Fluent BioSciences). Cells were captured and lysed according to the manufacturer's instructions. PIPseq™ T2 Single Cell Sequencing Kit (v2.1, Fluent BioSciences).