GSM8209058: Lung, control, scRNA-seq; Mus musculus; RNA-Seq
The correct enzymes are selected according to different tissue types. The enzyme solution is then prepared using centrifuge tubes and preheated into 37℃ water bath. The tissue sample is taken from a 4℃ refrigerator. After centrifuging, tissue preservation solution is removed. Tissue is then cleaned using DMEM and immediately cut into 2-3mm tissue block using a scalpel. The tissue block is transferred into a centrifuge tube filling with enzymatic hydrolysate and placed in a 37 ℃ water bath 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. 40µm cell strainer is used to filter the cell suspension. The residual cells are cleaned using the 2-3mL precooled complete 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. According to different tissue types and colours of cell precipitations, red blood cell lysis buffer at room temperature is added to resuspend cells and lyse the red blood cells for 2-5 mins. Equal amounts of PBS-BSA (0.04%) solution are added to stop the lysis of the red blood cells. Centrifuge at 300g 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. Centrifuge at 300g and 4℃ for 5 mins and discard the supernatant. A right amount of PBS-BSA (0.04%) is used to resuspend the precipitation followed by counting the cells using a hemacytometer. 1 Microscope Assessment of the Cell or Nuclei The cellular or nuclei suspension is stained with 0.4% trypan blue to assess cell viability under microscopic observation. Cells with greater than 80% viability are qualified for library construction process. 2 GEM Generation and Reverse Transcription 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. 3 Breaking GEMs The reaction system is configured for breaking GEMs. After reacting at the suitable temperature for a fixed period of time, the products are purified. 4 cDNA Amplification The reaction system is configured for PCR. After reacting at the suitable temperature for a fixed period of time, the products are purified. 5 Fragmentation, end Repair & A-tailing The reaction system is configured. A certain amount of cDNA amplified products is taken. 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. 6 Adaptor Ligation 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. 7 PCR 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. 8 Library QC The corresponding library quality control protocol will be selected depending upon product requirements. 9 Circularization 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. 10 Sequencing 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).