GSM8075326: Mod, replicate3, scRNAseq; Homo sapiens; RNA-Seq
A total of 5ml peripheral blood was collected from each sample, and peripheral blood mononuclear cells (PBMCs) were isolated using density gradient centrifugation with Ficoll-Paque plus (17144003, Cytiva). After washing twice in PBS, PBMCs were preserved in serum-free cell lyophilization solution (abs9417, absin) and were stored in liquid nitrogen at −196°C until thawing. After PBMC resuscitation,  incubation, centrifuge, wash, and resuspend with an appropriate volume of resuspended buffer and filter through 40 µm cell sieve. The single-cell suspension, oil, beads are added into C4 scRNA slide and instrument in order. Cell lysis and magnetic bead capture mRNA are performed in droplets. Prepare reagents for emulsion breakage in advance, vacuum pump is used for emulsion breakage. The reaction system is configured. After reacting at the suitable temperature for a fixed period of time, reverse transcription is conducted. The reaction system is configured. After reacting at the suitable temperature for a fixed period of time, cDNA second strand is synthesised. The reaction system is configured. After reacting at the suitable temperature for a fixed period of time, cDNA and oligo products were amplified. The purification was conducted separately. The cDNA and oligo products are subjected to quality detection of their concentration and the size distribution of the fragments. The reaction system is configured. After reacting at the suitable temperature for a fixed period of time, Oligo products are subjected to amplification, index ligation and purification for further circularization. The reaction system is configured. After reacting at the suitable temperature for a fixed period of time, the cDNAs are subjected to fragmentation, end repair and addition of “A” base at the 3'-end of each strand. Finally, the products are purified. The reaction system is configured. After reacting at the suitable temperature for a fixed period of time, the cDNAs are subjected to adaptor ligation. Finally, the products are purified. The PCR reaction system is configured. After reacting at the suitable temperature for a fixed period of time, amplification is processed via PCR. The products are then purified. The corresponding library quality control protocol will be selected depending upon product requirements.  cDNA and Oligo products are respectively denatured into single stranded. The reaction system and program for circularization are respectively configured and set up. Single-stranded cyclized products are produced, while uncyclized linear DNA molecules are digested.