GSM7157726: Uninfected wound from dorsal skin replicate 1; Mus musculus; RNA-Seq
6-mm biopsy punch was used to isolate the wounds that were washed with 0.5% BSA/HBSS on ice. Any underlying fat tissue were removed and the tissues were cut into smaller pieces using scissors and blades. The minced tissue were incubated in Dispase/Liberase/Collagenase I cocktail at 37°C for two hours, followed by smashing with a piston. Partly digested tissue was sifted through a 70µm cell strainer, and rinsed three times with 0.5% BSA/HBSS. The flow-through was kept on ice. Undigested remaining tissue in the cell strainer was incubated in 0.05% trypsin/EDTA/HBSS for 15 minutes at 37°C. The trypsin-digested tissue was smashed once more, and sifted through a 70µm cell strainer. The flow-throughs from both incubations were pooled per sample and filtered through a 40µm cell strainer. Cell suspensions were centrifuged 300 xg for 10 min at 4°C twice, where the supernatant from each run were carefully discarded and the pellets were resuspended in 500µL of 0.04% BSA/PBS. Cell viability was determined by mixing 10µL of cell suspension with 10µL of trypan blue, and at least four separate counts were averaged for one sample. Cell concentrations were adjusted to 1200 cells/µl with 0.04% BSA/PBS. A more detailed protocol can be found at dx.doi.org/10.17504/protocols.io.yxmvmn8m9g3p/v1. Library was performed according to the manufacture's instructions (10X Genomics, Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 Protocol [CG000315]). Briefly, cells isolated from the infected and uninfected skin wounds 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 transcribed 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 appropriated 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.