GSM5354715: intestinal CD45+ cells - D6 - S6; Mus musculus; RNA-Seq
The cells living in the epithelial layer and laminar propria of the small intestine were isolated via a Lamina Propria Dissociation Kit (130-097-410, Miltenyi Biotec) and the procedures were according to the manufacturer's instructions. Three male D6KO mice and three littermate male WT mice at 11 weeks of age were euthanized by CO2 inhalation, and perfused with 4°C Hank's Balanced Salt Solution without calcium and magnesium salts (HBSS w/o) containing 5% fetal bovine serum (FBS) to remove the blood leukocytes. Approximately 20 cm of intestine was harvested, and the luminal content was removed by flushing with HBSS w/o. Peyer's patches were also removed to exclude the leukocytes of secondary gut-associated lymphoid tissue. The intestine was longitudinally slit, washed by HBSS w/o, and laterally cut into pieces of around 0.5 cm in length. The intestinal pieces were incubated with predigestion solution (HBSS w/o containing 5 mM EDTA, 5% FBS, and 1 mM DTT) for 20 mins at 37°C on a shaker at 220 rpm, and the solution was passed through the 100 μm strainer. The flow-through fraction was placed on ice for 10 mins, and the supernatant containing the suspended intraepithelial leukocytes was collected. The intestinal pieces were conducted with the above procedures thrice to obtain the cells from the epithelial layer and to remove the entire epithelial layer. For the isolation of cells from laminar propria, the remaining pieces were further incubated with enzymes for 20 mins at 37°C on a shaker at 220 rpm, churned by gentleMACS Dissociator, and passed through the 100 μm strainer. All fractions were centrifugated at 300 rcf for 5 mins at 4°C, and the pellet was resuspended in 4°C PB buffer. The cells isolated from the epithelial layer and laminar propria were pooled together, passed through the 70 μm strainer, and washed with 4°C PB buffer once. The live cells were isolated using a Dead Cell Removal Kit (130-090-101, Miltenyi Biotec). The CD45+ cells were selected through a MACS column with CD45 MicroBeads (130-052-301, Miltenyi Biotec). To increase the purity of live CD45+ intestinal cells, the cells were further stained with Live/Dead fluorescent reactive dye (L34957, Invitrogen) and PE anti-mouse CD45 antibody (103105, BioLegend) and sorted by BD Influx™ Cell Sorter (BD Biosciences) in 100% FBS. The sorted cells were centrifugated at 300 rcf for 5 mins at 4°C, and finally resuspended in 4°C PBS. Due to low sorting efficacy, one D6KO sample was abandoned. Ultimately, three WT samples and two D6KO samples were chosen for following single-cell RNA sequencing. The isolated intestinal CD45+ cells were counted by Cellometer Auto T4 (Nexcelom Bioscience). Three WT samples and two D6KO samples were pooled separately. Subsequently, WT and D6KO samples were processed by BIOTOOLS Co., Ltd through the GemCode Single Cell Platform using the GemCode Gel Bead, Chip and 3'-Library v2 Kits (10X Genomics, Pleasanton) according to the manufacturer's instructions. In brief, cell suspensions were loaded onto a Chromium Single-Cell Chip along with the reverse transcription master mix and single cell 3′ gel beads, aiming for 5,000 cells per channel. Following generation of single-cell gel bead-in-emulsions, reverse transcription was carried out in a Mastercycler ep gradient S thermocycler (Eppendorf). The amplified cDNA was subsequently purified using SPRIselect beads and sequenced on an Illumina HiSeq X Ten PE150 platform. The raw data contained the following read regions: 26 bp Read 1 (16 bp single cell barcode, 10x barcode; 10 bp Unique molecular identifier, UMI), 98 bp Read 2 (transcript insert), 8 bp i7 Index (sample index). The Cell Ranger software pipeline (version 2.0.1, 10X Genomics) was used to demultiplex cellular barcodes, map reads to the genome and transcriptome using the STAR aligner, and down-sample reads as required to generate normalized aggregate data across samples, producing a matrix of gene counts (27,998 genes) versus cells (3,996 cells in WT and 3,908 cells in D6KO). Single cell RNA-seq