GSM7290763: C1T; Homo sapiens; RNA-Seq
Freshly collected DC and Si8 from RedMUC298trTg mice were flushed with ice-cold HBSSwo (Mg2+/Ca2+ free supplemented with 10mM HEPES, pH 7.2) to remove luminal content. The tissue was inverted and inflated by injection of HBSSwo. Epithelial isolation was performed by incubation of tissue with pre-digestion buffer (HBSSwo, 5 mM EDTA, 5% v/v FCS, 1 mM DTT) in a shaking incubator. The isolated epithelial cells were resuspended in digestion buffer (HBSS with Mg2+/Ca2+ supplemented with 2 mg/ml collagenase type I and 40 U/ml DNase I) and incubated at 37 °C for 30 min. Cells were washed and re-suspended in PBS and stained with a Viability Dye, washed and resuspended in FACS buffer (HBSSwo, 2% v/v FCS, 5mM EDTA). Alive GCs were sorted using a FACS Jazz (Becton Dickinson) according to the presence or absence of mCherry signal using a C57Bl6 mouse as negative control. Goblet cells were washed in HBSS-/- and resuspended in HBSS-/- supplemented with 0.04% w/v BSA. Library was performed according to the manufacter's instructions (single cell 3' v3 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.