Illumina HiSeq 2000 sequencing; RNA-seq comparison: before/after MARIS protocol and library construction technique test
RNA-seq comparison: before/after MARIS protocol and library construction technique test
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. Processed sample was fixed with 4% paraformaldehyde, permabilized, antibody stained and mock sorted to collect all cells. After sorting, cells were pelleted by centrifugation at 3000 g for 5min at 4C. 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 50C and 15 minutes at 80C, we carried out the incubation for 3 hours at 50C. Cell lysates were frozen at -80C overnight before continuing the RNA isolation by the manufacturer's instructions. The aim of this experiment was to test how well 4 different RNA-seq library construction techniques perform on partially degraded RNA.
Experimental Factor: processing status
processed
Experimental Factor: library construction
DSN