GSM9653148: snRNA-Seq, Liver, 18months, sample4; Mus musculus; RNA-Seq
The mouse liver was placed in 1 mL lysis buffer (LB) and homogenized using a tissue homogenizer. The homogenate was then lysed on ice for 1-10 min. A 40 μm cell strainer was used to filter out debris, and the filtrate was centrifuged at 4 °C and 500 g for 5 min. After removing the supernatant, 300 μL of LB solution was added to the sediment, and the nucleus was resuspended by pipetting. The suspension was transferred to a new centrifuge tube, and 300 μL of resuspension buffer (RB) was added, followed by thorough mixing. Next, 600 μL of PB1 solution was slowly added to the bottom of the tube to form a layer. Similarly, 600 μL of PB2 solution was slowly added to the bottom of the tube to form another layer. The mixture was then centrifuged at 4 °C and 4,000 g for 20 min, with the nucleus located at the interface between PB1 and PB2. A volume of 150-300 μL of the nucleus layer was transferred to 1 mL of RB solution, mixed thoroughly, and filtered through a 40 μm cell strainer. The filtrate was centrifuged at 4 °C and 500 g for 5 min. After removing the supernatant, the nucleus sediment was resuspended in 100 μL of EB solution. Cellular lysis and subsequent RNA barcoding were achieved through GEM-based reverse transcription. cDNA synthesis was conducted in a S1000TM Touch Thermal Cycler (Bio-Rad) using the following thermal profile: 45 min at 53 °C, 5 min at 85 °C, followed by 4 °C hold. Amplified cDNA products underwent quality control verification using the Agilent 4200 (service provided by CapitalBio Technology, Beijing). The constructed libraries were subsequently sequenced on an Illumina NovaSeq 6000 platform, employing a paired-end 150 bp (PE150) strategy, with a sequencing depth of at least 100,000 reads per cell. This sequencing process was carried out by CapitalBio Technology in Beijing.
GEO
GSM9653148_r1