GSM8684657: CD4+ cells from IRI-6h mice; Mus musculus; RNA-Seq
Small intestinal lamina propria cells were collected utilizing a Lamina Propria Dissociation Kit (Miltenyi Biotec) following the manufacturer's protocol, followed by density gradient separation using Percoll (Cytiva). Single-cell suspensions were obtained from gradient layers between 30 % and 70 % Percoll. Live CD4+ cells were enriched using Dead Cell Removal Kit and CD11b or CD4 Microbeads (all from Miltenyi Biotec), and fixed for 19 hours at 4 °C with Chromium Next GEM Single Cell Fixed RNA Sample Preparation Kit (10x Genomics). For each fixed sample, the detection probe of the Chromium Fixed RNA Kit was hybridized to RNA in the cells, and a ligation reaction performed to bind adjacent hybridized probes. After mixing each sample, barcode primer beads and the cells were encapsulated, annealing the probes and the primers, and an extension reaction performed to add a cell identification adapter sequence to the detection probe using ChromiumX. To construct the library, samples were amplified and purified by polymerase chain reaction using index primers.