Genomic dna extraction kit

For the rats at 7–10 days after birth, the toes and tail tips were collected and numbered. Then, rats' genomic DNA was extracted by using TransGen's genomic DNA extraction kit (ee101-12), and PCR detection was performed with the amplification primer: mouse ATP5D KO-F: 5′-CTGATGTGCTCAGGGTGTTGACC-3′; mouse ATP5D KO-R: 5′-GCTGCTGATGTTAGGGAGGAGGC-3′. Mouse ATP5D KI-F: 5′-CTGATGTGCTCAGGGTGTTGACC-3′; mouse ATP5D KI-R: 5′-GC TGCTGATGTTAGGGAGGAGGC-3′.

Vof16 knockout and miR‐185‐5p knockout rats were constructed in Cyagen Biosciences Inc. The tail tips were collected and then the rats' genomic DNA was extracted using Transgen's genomic DNA extraction kit (ee101‐12), and the DNA sequencing primers for vof16 and miR‐185‐5p were as following:

Rat vof16‐F: 5′‐ AGTTTGTCCGAGTGATGGGAATACAC ‐3′

Rat vof16‐R: 5′‐ GACTCCGTGAGCCATTTATCATTCTG ‐3′

Rat miR‐185‐5p‐F: 5′‐ CTGATGTGCTCAGGGTGTTGACC ‐3′

Rat miR‐185‐5p‐R: 5′‐ GCTGCTGATGTTAGGGAGGAGGC ‐3′.

Additionally, the reaction was performed in mixtures system included 10 μL PCR master mix 0.6 μL upstream primers, 0.6 μL downstream, 3 μL DNA template, and 5.8 μL water. The thermal cycling conditions were performed as: initial denaturation at 94°C for 5 min, and 35 cycles of denaturation at 94°C for 30 s, annealing at 60°C for 30 s, with elongation at 72°C for 30 s, followed by elongation at 72°C for 5 min and the storage at 12°C. Afterward, genotype was detected by agarose gel electrophoresis system. After genotype identification, the knockout rats were subjected to SCT operation, and the corresponding experiments were performed on vof16 or miR‐185‐5p knockout rats and their wild‐type mates.

Tail tips were collected and numbered for neonatal rats at 7–10 days. Then, Transgen’s genomic DNA extraction kit (ee101-12) was used to extract rats’ genomic DNA and were further detected by PCR with the amplification primer:
Rat DPYSL2 forward: 5′-ACTGAGCAGGTTCAGTCCGTGC-3′.
Rat DPYSL2 reverse: 5′-ACTTGTGGTGGAAGCTCTGACTCCC-3′.
The PCR amplification reaction system was conducted with 10 μl PCR master mix: 0.6 μl upstream primers, 0.6 μl downstream, 3 μl DNA template, and 5.8 μl water. The thermal cycling conditions were performed as initial denaturation at 94 °C for 5 min; 35 cycles of denaturation at 94 °C for 30 s; annealing at 60 °C for 30s, with 1 min elongation at 72 °C; and followed by final elongation at 72 °C for 10 min. Subsequently, the agarose gel electrophoresis system was applied to visualize the final genotype detection under U.V after 55 min electrophoresis at 150 V.

DMEM medium, fetal bovine serum, T4 ligase, T7 endonuclease, Escherichia coli DH5α, plasmid extraction kit, genomic DNA extraction kit, reverse transcription kit, and SYBR^® Green RT-PCR Master Mix were purchased from TransGen Biotech Co (Beijing). ACCUTASE™ cell digestion solution was purchased from eBioScience Inc. Western blot antibodies, STAT1 (14994), p-STAT1 (Tyr701, 7649P). STAT3 (9132), p-STAT3 (Tyr705, 9145), STAT5 (9363p), p-STAT5 (Y694, 4322). AURKA (14475), p-AURKA (Thr288, 3079), PD-L1 antibody (13684), p-AKT (Ser473, 4060), AKT (pan) (4691), p-ERK/ERK antibody (4370), GAPDH (5174), β-tubulin (2128) were purchased from Cell Signaling Technology, CD3 histochemical antibody (100219), CD8 histochemical antibody (104705) were purchased from Biolegend, and 4% paraformaldehyde were purchased from Wuhan Seville Biological LTD. DAB histochemical staining kit was purchased from Zhongshan Jinqiao Biological Technology LTD (Beijing). ECL luminescence color reagent was purchased from Healthcare. Trizol RNA extraction reagent was purchased from Sigma-Aldrich (T9424). Anti-mouse PD-L1 Antibody was purchased from BioXcell.