GSM8322610: ctrl,replicate2,scRNAseq; Rattus norvegicus; RNA-Seq
Tissue samples are collected at day 4 and immediately cut into 2-3 mm tissue block using a scalpel in tissue preservation solution. After centrifuging, tissue preservation solution is removed. The tissue block is transferred into a centrifuge tube and enzymed under 37 ℃ for digestion. Repetitive pipetting is done every 15-20 mins. After 30 mins, tissue digestion is examined via trypan blue staining until no obvious tissue block exists and the digestion stops. 40 µm cell strainer is used to filter the cell suspension. The residual cells are cleaned using the 2-3mL precooled complete DMEM medium in which the cleaning solution is filtered and collected into a centrifuge tube. Centrifuge at 300g and 4 ℃ for 5 mins and discard the supernatant. Then, red blood cell lysis buffer at room temperature is added to resuspend cells and lyse the red blood cells for 5 mins. Equal amounts of PBS-BSA (0.04 %) solution are added to stop the lysis of the red blood cells. Solutions are centrifuged at 300 g and 4 ℃ for 5 mins and discard the supernatant. Cell status is observed via trypan blue staining in which then the Dead Cell Removal Kit can be used to remove dead cells and debris. PBS-BSA (0.04%) is used to wash cells. A right amount of PBS-BSA (0.04%) is used to resuspend the precipitation followed by counting the cells using a hemacytometer. The cellular suspension is stained with 0.4% trypan blue to assess cell viability under microscopic observation. Prepared single-cell suspension is then partitioned into GEMs (Gel Beads in Emulsions) in the automated Chromium Controller, and then mRNAs are reverse transcribed into cDNAs. The reaction system is configured for breaking GEMs. After reacting at the suitable temperature for a fixed period, the products are purified. The reaction system is configured for PCR. After reacting at the suitable temperature for a fixed period, the products are purified. The reaction system is configured. A certain amount of cDNA amplified products is taken. 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. Then, the cDNAs are subjected to adaptor ligation. Finally, the products are purified. The PCR reaction system is configured. Amplification is processed via PCR. The products are then purified. The corresponding library quality control protocol will be selected depending upon product requirements. Single-stranded PCR products are produced via denaturation. The reaction system and program for circularization are subsequently configured and set up. Single-stranded cyclized products are produced, while uncyclized linear DNA molecules are digested. Single-stranded circle DNA molecules are replicated via rolling cycle amplification, and a DNA nanoball (DNB) which contain multiple copies of DNA is generated. Sufficient quality DNBs are then loaded into patterned nanoarrays using high-intensity DNA nanochip technique and sequenced through combinatorial Probe-Anchor Synthesis (cPAS).