title: GSM9342217: sham_ileum; Rattus norvegicus; RNA-Seq
library_construction_protocol: Small intenstine tissue from rat was stored in the Tissue Storage Solution (miltenyi biotech, cat no. 130-100-008) at 4°C. Prior to dissociation, tissue was washed with phosphate buffered saline (PBS), and 0.2g of tissue was minced into 1–2 mm pieces.The minced tissue was then transferred into a gentleMACS C tube (miltenyi biotech, cat no. 130-093-237) containing 2.3mL of enzyme mix from the Human Tumor Dissociation Kit (miltenyi biotech, cat no. 130-095-929). Using the gentleMACS Dissociator with Heaters (miltenyi biotech, cat no. 130-096-427), the C tube was mounted upside down and processed with the 37C_h_TDK_1 program.  After termination of the program, the C tube was detached and cell suspension was sequentially passed through 70 and 30-μm strainer to separate cells from debris and undigested tissue chunks. The cell suspension was pelleted by centrifuging at 300g, 4°C, for 5 min. The supernatant was discarded, and pellet was gently resuspended in cold Ca2+ and Mg2+ free 0.04% BSA/PBS. And then, the cell suspension was counted with a LUNA-FX7™ Automated Fluorescence Cell Counter (logos biosystems) using a acridine orange (AO) and propidium iodide (PI) staining (logos biosystems, cat no. F23001).  Dead cells were removed using the Dead Cells Removal Kit (miltenyi Biotech, Cat no 130-090-101). The cell suspension was centrifuged at 300g, 4°C, for 5 min and resuspended in 100ul of Dead Cell Removal MicroBeads. The sample was mixed well and incubated for 15 min at room temperature. The labeled cell suspension was then applied to an MS columns (miltenyi biotech, cat no. 130-042-201), and the flow-through containing unlabeled live cells were collected. The MS column was washed 4 times with 500ul of 1x Binding Buffer, and all unlabeled cells were pooled and pelleted by centrifuging at 300g, 4°C, for 5 min.The resulting pellets were washed 3 times and gently resuspended in cold Ca2+ and Mg2+ free 0.04% BSA/PBS.  Finally, as described above, cell counts and viability were assessed using a LUNA-FX7™ Automated Fluorescence Cell Counter. According to the 10x Chromium Single Cell 3' v3.1 protocol (10x Genomics, document no. CG000315) Single Cell RNA-seq libraries were prepared using the 10x Chromium controller and Next Gem Single cell 3' Reagent v3.1 kits (10x genomics, PN-1000123). Briefly, the cell suspension (target recovery 10,000 cells) was mixed with reverse transcription master mix and loaded with Single Cell 3′ Gel Beads and Partitioning Oil into a Single Cell G Chip (10x genomics, PN-1000120) to generate single-cell Gel Bead-in-emulsion (GEM). Poly-adenylated mRNA from single cells were uniquely barcoded and reverse-transcribed within GEM. After barcoded full-length cDNA was generated from mRNA through GEM-RT incubation, the barcoded cDNA molecules were amplified with PCR. For 3' Gene Expression Library construction, the enriched cDNA was sequentially subjected to the enzymatic fragmentation, end-repair, A-tailing, adaptor ligation and index PCR. The purified libraries were quantified using qPCR according to the qPCR Quantification Protocol Guide (KAPA) and qualified using the Agilent Technologies 4200 TapeStation (Agilent technologies). The library was sequenced using HiSeq platform (Illumina), and 150 bp paired-end reads were generated.