GSM7918436: Middle-Aged (12-month-old male); Mus musculus; RNA-Seq
The collected lymphatic vessels were transferred into 1.5 mL Eppendorf tubes containing 1 mL of digestion buffer [10 mL Dulbecco's Modified Eagle Medium (DMEM) with 20 mg Collagenase I, 5 mg Elastase, 8 mg Dispase, and 1 mg DNaseI]. The vessels were incubated in a 37 oC swirling water bath for approximately 40 minutes, with the digestion content being resuspended every 5 minutes. After digestion, the samples were filtered through a 70 μM nylon mesh and quenched by adding Fetal Bovine Serum (FBS) to a final concentration of 10%. The samples were then centrifuged at 300 rcf for 5 minutes at 4 oC. The pelleted cells were resuspended in 1 mL of ACK lysis buffer and kept at room temperature for 2 minutes to remove the red blood cells. The ACK lysis reaction was quenched by adding 9 mL of FACS buffer (2% FBS + 1X DPBS). The resulting 10 mL sample was centrifuged at 300 rcf for 5 minutes at 4 oC. The pelleted cells were resuspended in 150-200 μL of FACS buffer and filtered through a 35 μM nylon cell strainer. Cell viability was confirmed by Trypan Blue (Gibco) staining through manual counting as well as TC20 automated cell counting (Bio-Rad). Only samples with > 74% live cells were processed with 10X Genomics Single Cell 3' Reagent Kit-based Gel Bead-In-Emulsions (GEM) reactions, followed by Illumina sequencing. 10x genomics Chromium Single Cell 3' Reagent (v2 Chemistry) was used for library preparation. 10x genomics single-cell mRNA sequencing.