Illumina HiSeq 2000 paired end sequencing; RNA-seq of Ins+ cells in human differentiation protocol across lines and isolated from human islets
RNA-seq of Ins+ cells in human differentiation protocol across lines and isolated from human islets
hESCs were differentiated to Stage 6 cells, INS+ cells comprised approximately 4% of all Stage 6 cells. A large proportion of INS+ cells also co-expressed the pancreatic hormone somatostatin, consistent with previous reports. Also see Hrvatin et al. 10.1073/pnas.1400709111 SI Experimental Procedures. Directed Differentiation. Islets were dispersed to a single cell suspension using TrypLE Express (Invitrogen). Cells were passed through a 40 um filter (BD Falcon 352340) and washed with PBS at least twice. Cells were fixed and permeabilized with 4% PFA (Electron Microscopy Sciences), 0.1% saponin (Sigma-Aldrich 47036) in molecular grade PBS (Ambion) supplemented with 1:100 RNasin Plus RNase Inhibitor (Promega, N2615) for 30min at 4C. All the subsequent steps were carried out at 4C. Cells were pelleted by centrifugation at 3000g for 3min at 4C and washed in Wash Buffer: PBS containing 0.2% BSA (Gemini Bio-Products), 0.1% saponin (Sigma-Aldrich), 1:100 RNasin Plus RNase Inihibitor. Primary antibody staining was carried out while 3D rocking for 30min at 4C in Staining buffer containing PBS with 1% BSA, 0.1% saponin and 1:25 RNasin Plus RNase Inhibitor (note that certain antibodies may require longer incubation times). Cells were washed twice in Wash Buffer followed by secondary antibody staining in Staining buffer. Following secondary antibody staining cells were washed twice in Wash buffer and resuspended in Sort buffer containing PBS, 0.5% BSA, and 1:25 RNasin Plus RNase Inhibitor. Fixation, washing, staining and sorting were performed at a concentration of 5-10 M cells/ml. After sorting, cells were pelleted by centrifugation at 3000 g for 5' at 4 degrees C. The supernatant was discarded. Total RNA was isolated from the pellet using the RecoverAll Total Nucleic Acid Isolation kit (Ambion), starting at the protease digestion stage of manufacturer-recommended protocol. The following modification to the isolation procedure was made: instead of incubating cells in digestion buffer for 15 minutes at 50 degrees C and 15 minutes at 80 degrees C, we carried out the incubation for 3 hours at 50 degrees C. Cell lysates were frozen at -80 degrees C overnight before continuing the RNA isolation by the manufacturer's instructions. NA
Experimental Factor: cell or organism part
H1
Experimental Factor: antibody target
insulin