GSM8723649: HIR_40Gy; Sus scrofa; RNA-Seq
The liver tissue samples were subjected to nuclear isolation, sequencing, and library preparation following the 10X Genomics protocol. Approximately 500 mg of hepatic tissue was dissociated into a singular nuclear suspension via tissue homogenization in a chilled lysis buffer (0.25 M sucrose, 5 mM CaCl2, 3 mM MgAc2, 10 mM Tris-HCl pH 8.0, 1 mM DTT, 0.1 mM EDTA, 1 × Protease Inhibitor and 1U/µL RiboLock RNase Inhibitor (Thermo Scientifc, cat no. O0381) with pestle strokes. The resulting homogenate was subsequently filtered through a 70 µm cell strainer, yielding a nuclear fraction collected in a 15ml centrifuge tube, with a 1 ml volume. This nuclear fraction was mixed with an equal volume of 50% iodixanol solution (0.16 M sucrose, 10 mM NaCl2, 3 mM MgCl2, 10 mM Tris-HCl pH 7.4, 1 U/µL RiboLock RNase Inhibitor, 1 mM DTT and 0.1 mM PMSF Protease Inhibito (Thermo Scientifc, cat no. 36978), yielding a final concentration of 25%, and supplemented with 1 mL of 33% iodixanol solution at the tube's base and 30% iodixanol solution at the top. The solution centrifugation at 500 × g for 8 min at 4℃, subsequent to myelin layer removal from the gradient's apex. Nuclei were harvested from the 30% iodixanol interface, resuspended in nuclear wash and resuspension buffers, and centrifuge (0.04% bovine serum albumin, 0.2 U/µL RiboLock RNase inhibitor, 500 mM mannitol and 0.1 mM PMSF protease inhibitor in PBS) at 500 × g and 4℃ for 5 min. Filtration through a 40 µm cell filter eliminated cell debris and sizable aggregates. The nuclei's total count, concentration, and integrity ratio were ascertained via hemocytometer-assisted microscopic examination of trypan-stained samples. Ultimately, the nuclear concentration was regulated to 700-1,200 nuclei/µL, with nuclei inspected using the 10X Chromium platform. Cellular suspensions were loaded on a 10X Genomics GemCode Single-cell instrument that generates single-cell Gel Bead-In-EMlusion (GEMs). Using Chromium Next GEM Single Cell 3'Reagent Kit v3.1 for Library Generation and Sequencing of cDNA. Upon dissolution of the Gel Bead in a GEM, primers containing (i) an Illumina® R1 sequence (read 1 sequencing primer), (ii) a 16 nt 10 × Barcode, (iii) a 10 nt Unique Molecular Identifier (UMI), and (iv) a poly-dT primer sequence were released and mixed with cell lysate and Master Mix. Silane magnetic beads were used to remove leftover biochemical reagents and primers from the post GEM reaction mixture. Full-length, barcoded cDNAs were then amplified by PCR to generate sufficient mass for library construction. R1 (read 1 primer sequence) were added to the molecules during GEM incubation. P5, P7, a sample index, and R2 (read 2 primer sequence) were added during library construction via End Repair, A-tailing, Adaptor Ligation, and PCR. The final libraries contained the P5 and P7 primers used in Illumina bridge amplification. A Single Cell 5' Library comprised standard Illumina paired-end constructs which begin and end with P5 and P7. The Single Cell 5′ 16 bp 10X Barcode and 10 bp UMI were encoded in Read 1, while Read 2 was used to sequence the cDNA fragment. Sample index sequences were incorporated as the i7 index read. Read 1 and Read 2 were standard Illumina® sequencing primer sites used in paired-end sequencing.