GSM8833635: treat3D; Rattus; RNA-Seq
Bile duct samples were rinsed twice with Dulbecco's modified Eagle's medium (DMEM) (Gibco, Grand Island, NY, USA) and cut into 1 mm3 pieces. Next, 5 mL of a mixed digestion enzyme solution (0.03% collagenase type I (Gibco), 0.05% collagenase type IV (Gibco), 0.05% dispase, 0.01% DNase I) was added, and the tissues were incubated at 37°C for 50-70 minutes. The reaction was stopped with DMEM containing 10% fetal bovine serum (FBS) (Gibco) and the sample was filtered through a 40 µm nylon mesh cell strainer. The precipitate was centrifuged at 350 x g for 5 minutes, and washed twice with DMEM containing 10% FBS. The cells were lysed with red Puregene RBC Lysis Solution (Qiagen, Duesseldorf, Germany) for 5 minutes at room temperature, and then dead cells and debris were removed with a Miltenyi Biotec Dead Cell Removal Kit (Miltenyi Biotec, Bergisch Gladbach, Germany). The live cell rate was assessed using trypan blue staining, with a minimum requirement of 85%. The 10x Genomics Chromium is based on GemCode technology and allows high-throughput single-cell 3' mRNA quantitative analysis in which describes intercellular heterogeneity. The cell 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. 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 GEMs is broken and purified for a fixed period of time. A certain amount of cDNA amplified products is taken and subjected to fragmentation, end repair and addition of “A” base at the 3'-end of each strand. After that, the products are purified. The cDNAs are subjected to adaptor ligation and purified. The cDNA with adaptor is amplified via PCR and the products are subjected to library quality control. 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).
GEO
GSM8833635_r1