GSM6837106: Chow_LaminaPropria_1; Mus musculus; RNA-Seq
Mice were euthanized using isoflurane (VETONE) and secondary cervical dislocation. The intestines were maintained in a moistened state, and the Peyer's patches and the remaining mesentery/fat were excised. The small intestine from ileum to jejunum was collected and gently flushed with a syringe with ice cold 1 × Hanks' Balanced Salt Solution (HBSS) media (without Ca2+ and Mg2+) (SIGMA, Cat# H4641) to remove the feces. The intestine was cut open and dissected into 1 cm segments, which were then transferred into a 50 mL tube and gently shaken to let the tissue stretch. They were then washed twice with ice cold 1 × HBSS and the extra HBSS removed using a pipet. 20 ml pre-digestion solution (1 × HBSS with 5% FBS, 5 mM EDTA and 1 mM DTT) was added and incubated for 20 min at 37 °C with 250 rpm rotation in an incubator. The 1 × HBSS containing the epithelial cell layer was collected on ice after vortex and passed through a 100 μm cell strainer. For intraepithelial immune cell isolation, the 1 × HBSS containing the epithelial cell layer was centrifuged at 2000 rpm for 5 mins. The cell pellet was resuspended in 20 ml 40% percoll (Sigma-Aldrich, Cat# P4937) and 20 ml 70% percoll added to the bottom of each tube, spun at 2000 rpm for 20 mins (accelerate at 6 and brake at 2) at room temperature. The middle layer of cells was collected, washed, spun down with 1 × PBS, and resuspended in RPMI 1640 culture media (Corning Cellgro, Cat# 10-040-CV) (supplemented with 10% FBS (Corning, Cat# MT35015CV), 1% Penicillin-Streptomycin-Glutamine (Gibco, Cat# 10378016), 1% MEM Non-Essential Amino Acids Solution (Gibco, Cat# 11140050), 1% HEPES (Gibco, Cat# 15630056), and 1% Sodium Pyruvate (Gibco, Cat# 11360070)) for further FACS or flow cytometry analysis. For intraepithelial immune cell scRNA-seq, single cell suspensions were sorted using a FACSAria III flow cytometer to purify live hematopoietic cells (gated as DAPI-CD45.2+ cells). For IEC isolation, the epithelial cell layer in the 1 × HBSS was spun at 2000 rpm for 5 mins. The cell pellet was resuspended in RPMI 1640 culture media for further FACS. For epithelial structural cell scRNA-seq, single cell suspensions were sorted using a FACSAria III flow cytometer to purify live non- hematopoietic cells (gated as DAPI-CD45.2- cells). For lamina propria immune cell isolation, the epithelial cell layer was removed and the remaining intestine tissues washed twice with RPMI 1640 culture media. The extra media was removed and 10 ml RPMI 1640 culture media with 1mg/ml Collagenase type I (Thermo Fisher Scientific, Cat# 17100017) was added. The sample was incubated for 20 min at 37 °C with 250 rpm rotation and vigorously vortexed to allow the tissue to fully dissolve before passing through 100 μm cell strainer. Media containing the lamina propria cells was centrifuged at 2000 rpm for 5 mins and the cell pellet resuspended in 20 ml 40% percoll. 20 ml 70% percoll was added to the bottom of each tube and spun at 2000 rpm for 20 mins (accelerate at 6 and brake at 2) at room temperature. The middle layer of cells was collected, washed, spun down with 1 × PBS, and resuspended in RPMI 1640 culture media for further FACS or flow cytometry analysis. For lamina propria immune cell scRNA-seq, single cell suspensions were sorted using a FACSAria III flow cytometer to purify live hematopoietic cells (gated as DAPI-CD45.2+ cells). Library was performed according to the manufacter's instructions (single cell 3' v2 protocol, 10x Genomics). Briefly, GCs were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the apropiated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.