GSM5703385: IR_2; Homo sapiens; RNA-Seq
Liver tissues were cut into pieces and placed into a pre-prepared gentleMACSTM C tubes with dissociation enzyme mix. Then, perform an appropriate gentleMACSTM program (37C_h_TDK_1) to dissociate tissues for I h using the gentleMACSTM Octo dissociator with heaters. Subsequently, the cell suspension was filtered by 40 μm nylon cell strainer (Falcon, Cat#352340) and transferred to a 50 ml centrifuge tube. The erythrocytes were lysed by using 5 mL ACK lysing buffer (Gibco, Cat#A1049201). Dead cells were removed by the Dead Cell Removal Kit (Miltenyi Biotec, Cat#130090101) according to the manufacturer's recommendations. Finally, the cell pellet was washed twice and resuspended in PBS, and the cell viability was calculated by trypan blue (Gibco, Cat#15250-061), and then placed on ice for later use. The Chromium Single Cell 3′ Library & Gel Bead Kit v3 (10x Genomics; Pleasanton, CA, USA) was used for the construction of sequencing libraries. Liver single cell suspensions were loaded onto the 10x Genomics Chromium chip (10x Genomics; Pleasanton, CA, USA) to generate droplets. The obtained Gel Beads-in-emulsion (GEMs) were followed by reverse transcription using ProFlex PCR System (Thermo Fisher, MA, USA). Then the resulting cDNA was purified, amplified. According to the cDNA concentration quantified by Qubit (Thermo Fisher, MA, USA), libraries were constructed by the Chromium Single Cell 3′ Library & Gel Bead Kit v3 (10x Genomics; Pleasanton, CA, USA) according to the manufacturer's instructions.