Illumina HiSeq 2000 paired end sequencing; RNA-seq of definitive endoderm and pancreatic progenitor cells derived in vitro from human pluripotent stem cells in order to study the role of transcription factor GATA6 in human pancreas development
RNA-seq of definitive endoderm and pancreatic progenitor cells derived in vitro from human pluripotent stem cells in order to study the role of transcription factor GATA6 in human pancreas development
hPSCs were routinely cultured in feeder-free conditions on vitronectin-coated (Stemcell Technologies #07180) sterile plates (Corning, Costar) with Essential 8 (E8) media (Life Technologies #A1517001). Subculture was performed every 4-6 days by aspirating the cell culture media, washing the cells once with D-PBS, incubating them in 0.5 mM EDTA (Invitrogen, #15575020) for 5 min at room temperature, aspirating the EDTA and adding appropriate volumes of fresh E8 media (Table 2) to the cells. hPSCs were further physically detached from the plates by gentle pipetting, collected in a 15 ml Falcon tube (BD Falcon) and allowed to settle for 10 min. The supernatant was aspirated and hPSCs were re-suspended in fresh E8 media, and then dissociated into smaller clumps by gentle pipetting. The cells were split at a split ratio of 1:6 and plated onto fresh vitronectin-coated plates containing appropriate volumes of media. Cells were maintained by refreshing E8 media on a daily basis. Cells were grown in 12 well plates for total RNA isolation. 3 wells were harvested per sample to obtain technical triplicates. RNeasy Mini Kit together with the Qiacube (Qiagen) was used for total RNA extraction. Cell culture media was aspirated and the cells were washed once with D-PBS. The D-PBS was completely aspirated and cells were lysed directly in the 12 well plates by adding 350 μl of Buffer RLT. Cell lysates were transferred to 2 ml tubes and were either frozen at -80°C or used immediately with the Qiacube for RNA extraction. Each sample was treated with RNase-Free DNase (Qiagen) to avoid DNA contamination. RNA was eluted in a volume of 30 μl. RNA was either frozen at -80°C or immediately taken to the next step of first strand cDNA synthesis. If RNA samples were frozen, they were thawed on ice to prevent degradation. Library preparations were made manually using the Illumina TruSeq™ RNA Sample Preparation Kit with no major deviations from this protocol.
Experimental Factor: cell line
fibroblast derived cell line
Experimental Factor: genotype
GATA6 R456C/+
Experimental Factor: cell type
induced pluripotent stem cell
Experimental Factor: phenotype
definitive endoderm cell
Experimental Factor: sampling time point
3