GSM9176942: Mature AVF 3; Homo sapiens; RNA-Seq
Upper arm veins or venous AVF segments 0.5-1 cm in length were cut longitudinally with a sterile scalpel and then into 1-2 mm square sections. Samples were enzymatically digested for 90 minutes at 37°C with shaking using a combination of collagenase type II, soybean trypsin inhibitor, elastase, Dispase, DNAse I, collagenase type XI, hyaluronidase type I, and HEPES in HBSS with Ca2+ and Mg2+. After 90 minutes, an equal volume of Accumax was added to the dissociation mix and incubated for an additional 5 minutes. Single cells were filtered through a 40 μM strainer, washed with HBSS twice, and incubated with red blood cell lysis buffer for 5-10 min. Cells were washed once again with HBSS and resuspended in 0.1% BSA in PBS for counting and RNA sequencing. Samples were run using the Chromium Single Cell 3′ Library & Gel Bead Kit v3 (10X Genomics) with a target capture of 10,000 cells. Each sample was processed on an independent Chromium Single Cell A Chip (10X Genomics) and subsequently run on a thermocycler (Eppendorf). Sequencing libraries were evaluated for quality on the Agilent Tape Station (Agilent Technologies, Palo Alto, CA) and quantified using a Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA) and qPCR before sequencing on the Illumina NovaSeq 6000.