title: GSM8571922: Albino BrafCA/+; PtenΔ/+ tumor-adjacent skin, replicate 3, scRNAseq; Mus musculus; RNA-Seq
library_name: GSM8571922
library_construction_protocol: The mice were euthanized, shaved, and depilated. For non-tumor bearing samples, a 2 by 3 cm section of the back skin was retrieved, and the fat scraped off from the underside. For melanoma-bearing samples, the skin sample on top of the tumor piece was also collected, with the mouse-matched adjacent skin next to the tumor (termed “tumor-adjacent skin”) as sample-matched control. The tumor or skin sample was then physically minced, and suspended in a gentle-macs C-tube dissociation buffer (5mL of RPMI, 50 µL  of liberase 0.25 mg/mL, 116 µL of Hepes 23.2 mM, 116 µL of Sodium Pyruvate 2.32 mM, 500 µL of Collagenase :Dispase 1 mg/mL) for 50 min at 37°C incubation with gentle agitation at a speed of 85-90rpm. After initial 50 minute digestion i, 10 µL of DNaseI (23 µL was added for another 10 min of 37°C agitated incubation and then inactivated with 400 µL of fetal bovine serum and 10 µL of EDTA (1 mM). The tissue suspension was further dissociated mechanically with GentleMACS by running the setting m_imptumor_04.01 twice. The digested suspensions were filtered twice through a 70 mm strainer and dead cells removed by centrifugation at 300 x g for 15 min. The live cells were washed with 0.04% UltraPure BSA:PBS buffer, gently re-suspended in the same buffer, and counted using trypan blue and cell counter. Libraries were prepared using the Chromium Single Cell 3' v2 protocol (10X Genomics). Briefly, individual cells and gel beads were encapsulated in oil droplets where cells were lysed and mRNA was reverse transcribed to 10X barcoded cDNA. Adapters were ligated to the cDNA followed by the addition of the sample index. Prepared libraries were sequenced using paired end 100 cycles chemistry for the Illumina HiSeq 4000.