GSM8147636: Digested control cells, macrophages alone, replicate 3; Homo sapiens; RNA-Seq
Biowires were digested in collagenase type I (1370U/mL), hyaluronidase (122U/mL), DNase I (146U/mL), Dispase (1U/mL) , ROCK inhibitor (1uM) and 1uM of Flavopiridol for 40 minutes at 37°C with gentle shaking. After 40 minutes, the digestion mix was pipetted 10 times to physically dissociate cells and was washed in 1mL media. Cells were pelleted (300g for 5 minutes at 4°C) and stained with cell surface antibodies. Cells were stained in FACS buffer (2% heat-inactivated bovine serum in PBS containing 1mM EDTA) with mouse anti-human CD14 (clone M5E2; 1:100) and mouse anti-human CD45 (clone 2D1; 1:100) for 20 minutes at 4°C. Cells were washed once in FACS buffer (2% heat-inactivated bovine serum in PBS containing 1mM EDTA) and spun down 300xg for 5 minutes at 4°C. Finally, cells were resuspended in BD FACS Pre-Sort Buffer with 1:100 DAPI and sorted on Aria Fusion (BD Biosciences) under low pressure (100um nozzle) directly into 350uL of RLT lysis buffer in DNA lobind tubes. Total RNA from sorted hESC-macrophages or hESC-cardiomyocytes was prepared using the Qiagen RNeasy Micro kit with on-column treatment with RNAase-free DNAase. Sample library preparation using the Takara SMARTer Stranded Total RNA-seq Kit v3