title: Melanoma B16 transcriptome sequencing in C57BL16 mice under dacarbazine treatment
design: Melanoma B16 transcriptome sequencing in C57BL16 mice under dacarbazine treatment
library_construction_protocol: Before the experiment, the mice were adapted to the conditions of the vivarium, the period of adaptation was at least 2 weeks. The average weight of mice at the beginning of the experiment was 16.0 g.B16 melanoma cells were kindly provided by the Research Institute of Fundamental and Clinical Immunology (Novosibirsk, Russia).A melanoma model in the form of a subcutaneously growing node was created in 2 stages. Briefly, the first stage involved subcutaneous implantation of B16 melanoma cells into two mice. Then, having obtained tumor growth in the form of nodes in these mice, tumor nodes were isolated and crushed into cells, and then these melanoma cells were transplanted into all other animals. For subcutaneous implantation, thawed melanoma cells B16 in a concentration of 1x107 in a volume of 0.5 ml were injected subcutaneously in the form of a papule into the right side of the abdomen. After 2-3 weeks, the animals were sacrificed, and tumor nodes, the diameter of which was close to 2 cm, were removed under sterile conditions. Primary melanoma nodes were freed from stroma and other tissues, and then crushed with scalpels to a homogeneous suspension, adding Hanks' medium (PanEko, Russia) to the tumor tissue in the final concentration 1:10. Cell transplantation consisted of subcutaneous injection of 0.5 ml of the resulting homogenate from the tumor in Hanks' solution into the right lateral surface of the abdomen of each mouse. Poly-A RNA Control Stock diluted with Poly-A RNA Control Dil Buffer from the GeneChip™ Poly-A RNA Control Kit (Applied Biosystems by Termo Fisher Scientific, Vilnius, Latvia) was used as an exogenous positive control. The required concentration of RNA, 250 ng/µl, was prepared on ice. A diluted Poly-A RNA Control Stock solution and nuclease-free water were added to the RNA. Next, the synthesis of the first strand of cDNA was performed, for which a mix for the synthesis of the first strand of DNA was added to the samples, consisting of First-Strand Buffer (Applied Biosystems by Termo Fisher Scientific, Vilnius, Latvia) and First-Strand Enzyme (Applied Biosystems by Termo Fisher Scientific, Vilnius, Latvia). The resulting solution was incubated in a thermal cycler for 1 hour at 25°C, then for 1 hour at 42°C, then 2 minutes at 4°C. The resulting single-stranded cDNA was converted to double-stranded cDNA by adding a second-strand synthesis mix consisting of Second-Strand Buffer (Applied Biosystems by Termo Fisher Scientific, Vilnius, Latvia) and Second-Strand Enzyme (Applied Biosystems by Termo Fisher Scientific, Vilnius) on ice. , Latvia). The resulting solution was incubated in a thermal cycler for 1 hour at 16°C, then for 10 minutes at 65°C, then 2 minutes at 4°C. After that, synthesis and amplification of cRNA were performed on a double-stranded DNA template at room temperature using IVT Buffer (Applied Biosystems by Termo Fisher Scientific, Vilnius, Latvia) and IVT Enzyme (Applied Biosystems by Termo Fisher Scientific, Vilnius, Latvia). The resulting solution was incubated in a thermal cycler for 16 hours at 40°C. The amplified cRNA was purified using magnetic beads (Purification Beads) (Applied Biosystems by Termo Fisher Scientific, Vilnius, Latvia) and a three-step wash with 80% ethanol. Magnetic particles were added to the test samples, which bind RNA and settle when placed on a magnetic plate. The settled magnetic particles were washed three times with 80% alcohol, after which the cRNA was eluted with nuclease-free water heated to 65°C. The concentration of isolated RNA was measured on a NanoDrop spectrophotometer. The cRNA samples were diluted to the required concentration (625 ng/µl) with nuclease-free water. 2nd-Cycle Primers (Applied Biosystems by Termo Fisher Scientific, Vilnius, Latvia) were added to the test samples, with which they were incubated in a thermal cycler for 5 minutes at 70°C, then for 5 minutes at 25°C, then 2 minutes at 4 °C. using reverse transcription reaction, cRNA was converted into cDNA using a mix for the synthesis of single-stranded cDNA of the 2nd cycle, which includes 2nd-Cycle ss-cDNA Buffer (Applied Biosystems by Termo Fisher Scientific, Vilnius, Latvia) and 2nd-Cycle ss-cDNA Enzyme (Applied Biosystems by Termo Fisher Scientific, Vilnius, Latvia). The resulting solution was incubated in a thermal cycler for 10 minutes at 25°C, then for 90 minutes at 42°C, then for 10 minutes at 70°C, then 2 minutes at 4°C. For hydrolysis of the cRNA, RNase H (Applied Biosystems by Termo Fisher Scientific, Vilnius, Latvia) was added to the resulting cDNA, with which the samples were incubated in a thermal cycler for 45 minutes at 37°C, then 5 minutes at 95°C, then 2 minutes at 4 °C After digestion, cycle 2 single strand cDNA was purified using magnetic beads and a three-step wash with 80% ethanol. Purified cDNA was fragmented with a fragmentation mix containing Nuclease Free Water, 10x cDNA Fragmentation Buffer, UDG, 10 U/μL, APE 1, 1,000 U/μL from the GeneChip™ WT Terminal Labeling Kit (Applied Biosystems by Termo Fisher Scientific, Vilnius , Latvia). The resulting solution was incubated in a thermal cycler for 1 hour at 37°C, then for 2 minutes at 93°C, then 2 minutes at 4°C. For transcriptome sequencing, library construction was carried out in several stages. First, to enrich the mRNA fraction according to the poly-A principle, particles with poly-T tails were used, to which 200 ng of total RNA was added. Next, the mRNA molecules were fragmented to small sizes. Reverse transcription was performed on these fragments using random primers. The second strand cDNA was synthesized with dUTP instead of dTTP. The resulting double-stranded cDNA was subjected to end repair and 3'-end polyadenylation. Adapter ligation was then performed. The next step was cleavage of the uracil-labeled second strand using the enzyme uracil DNA glycosylase (UDG), followed by PCR amplification.