GSM9683421: Liver, 2months, sample3; Mus musculus; OTHER
Fresh mouse liver tissue specimens were adhered to BGI-manufactured Stereo-seq spatial transcriptomics chips through brief thermal conditioning at 37 °C for 3 min. Subsequent fixation involved immersion in chilled absolute methanol (-20 °C) for 40 min prior to initiating library construction workflows. For morphological validation, select sections underwent nuclear counterstaining with nucleic acid dye (Thermo Fisher Scientific, Q10212), with fluorescent micrographs captured using a Motic Custom PA53 FS6 microscope system. These quality-controlled specimens were subsequently processed through FITC-channel spatial barcoding workflows for transcriptome capture. The tissue sections were initially treated with a buffer solution containing 0.1× SSC (Thermo, AM9770) and 0.05 U/mL RNase inhibitor (NEB, M0314L) to prepare them for subsequent steps. Permeabilization was achieved by immersing the sections in a solution containing 0.1% pepsin (Sigma, P7000) in 0.01 M HCl, followed by incubation at 37 °C for 5 min. After this step, the sections were again washed with the same buffer containing RNase inhibitor. The RNA released from the permeabilized tissue was captured by DNA nanoballs (DNBs) and subjected to reverse transcription. This process utilized SuperScript II Reverse Transcriptase (Invitrogen, 18064-014) in a reaction mixture containing 10 U/mL reverse transcriptase, 1 mM dNTPs, 1 M betaine, 7.5 mM MgCl₂, 5 mM DTT, 2 U/mL RNase inhibitor, 2.5 mM Stereo-seq TSO, and 1× First-Strand buffer. The reverse transcription was carried out overnight at 42 °C. Following this, the tissue sections underwent two washes with 0.1× SSC buffer and were then treated with Tissue Removal buffer (comprising 10 mM Tris-HCl, 25 mM EDTA, 100 mM NaCl, and 0.5% SDS) at 55 °C for 10 min to facilitate tissue digestion. The cDNA-containing chips were subsequently treated with a cDNA Release Mix (comprising cDNA Release Enzyme and buffer) overnight at 55 °C. Finally, the cDNA was purified using VAHTSTM DNA Clean Beads at a 0.8× ratio to ensure high-quality cDNA recovery. cDNA amplification was performed using KAPA HiFi HotStart DNA Polymerase Master Mix (Roche, KK2602) supplemented with 0.8 μM cDNA-specific amplification primers. Thermal cycling parameters were configured with the following profile: initial denaturation at 95 °C for 5 min; 15 amplification cycles consisting of denaturation at 98 °C for 20 sec, primer annealing at 58 °C for 20 sec, and strand extension at 72 °C for 3 min per cycle; concluding with a terminal extension phase at 72 °C for 5 min to ensure complete product elongation. DNA quantification of PCR products was performed using the QubitTM dsDNA Assay Kit (Thermo, Q32854). For library preparation, 20 ng of purified DNA underwent fragmentation via incubation with custom-formulated Tn5 transposase at 55 °C for 10 min, with enzymatic activity terminated through the addition of 0.02% SDS solution followed by gentle mixing at 37 °C for 5 min. Amplification reactions were conducted in 100 μL volumes containing 25 μL of fragmented DNA, 1× concentration of KAPA HiFi HotStart Ready Mix, 0.3 μM Stereo-seq-Library-F primer, 0.3 μM Stereo-seq-Library-R primer, and nuclease-free water to reach the final volume. Thermal cycling conditions comprised an initial denaturation at 95 °C for 5 min, followed by 13 cycles of 98 °C for 20 sec, 58 °C for 20 sec and 72 °C for 30 sec, concluding with a final extension at 72 °C for 5 min. Amplified libraries underwent sequential purification using AMPure XP magnetic beads with size selection ratios of 0.63 and 0.153, followed by DNB generation through standard protocols. Final sequencing was executed on the MGI DNBSEQ-Tx platform utilizing a paired-end 150 bp configuration.