title: GSM6720644: Gp130F/F stomach and tumour (antrum); Mus musculus; RNA-Seq
library_construction_protocol: Freshly collected gastric tissue from Gp130F/F and wildtype Gp130+/+ mice was cut into 1 mm pieces and digested in Collagenase/Dispase (Roche) and DNase I (Roche) in Ca2+- and Mg2+-free Hanks medium plus 5% FCS for 30 minutes at 37°C with gentle shaking. Samples were then vortexed for 15 seconds, filtered and washed in PBS plus 5% FCS. Single cell suspensions were blocked with FC block (Invitrogen) for 20 minutes at 4°C, before staining with fluorophore-conjugated primary antibodies for EpCAM and CD45.2 for 20 minutes at 4°C in the dark. Cells were washed twice and re-suspended in PBS supplemented with 5% FCS prior to staining with Sytox blue for viability and sorted with an Aria III cell sorter. Library was performed according to the manufacturer'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 Illumina 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 appropriately sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Illumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.