GSM6730789: female infected with Citrobacter rodentium; Mus musculus; RNA-Seq
After perfusion, large intestines were harvested and minced before transferring to conical tubes. The minced pieces were resuspended in 10ml of complete RPMI-1640 containing 1% P/S, 20mM HEPES, 0.05 mg/ml Liberase TL (Roche), 0.05% DNaseI (Roche) and shaken at 200 rpm for 30 min at 37°C in 50-ml Falcon tubes. The tissue suspension was collected and passed through a 70 mm cell strainer and the cells were pelleted by centrifugation at 1500 rpm. The cells were then resuspended and purified by 47% Percoll and centrifuged at 1500 rpm for 10 min. The pellet were collected, washed and resuspended in complete RPMI media. CD45+ immune cells were then enriched for by Miltenyi Beads. Library was performed according to the manufacter's instructions (SC RNA3' V3.1, 10X Genomics) GEMS were generated, poly-A RNA in every GEM was retrotranscripted to cDNA, cleaned up with Dynabeads, amplified by PCR and the appropriate sized fragments were selected with SPRIselect for library constrution. A sample index (i7) was added to each of the four samples during library construction.