Bulk RNA sequencing of pancreatic progenitor spheroids treated with CHIR99021 and control (DMSO) conditions
Bulk RNA sequencing of pancreatic progenitor spheroids treated with CHIR99021 and control (DMSO) conditions
hESC-derived PP-organoids (Gonçalves et al 2021) were dissociated into single cells with TrypLE and snap-frozen for RNA extraction. Total RNA was subjected to a modified version of SMARTseq-2 protocol published by Picelli et al. 2013 (doi: 10.1038/nmeth.2639). In short, total RNA was isolated with RNeasy Micro kit (Qiagen) and eluted in nuclease free water.  mRNA was isolated from on average 450 ng total RNA by poly-dT enrichment using the NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB) according to the manufacturer's instructions. Samples were then directly subjected to the workflow for strand-specific RNA-Seq library preparation (Ultra II Directional RNA Library Prep, NEB). For ligation NEB Next Adapter for Illumina of the NEB Next Multiplex Oligos for Illumina Kit were used. After ligation, adapters were depleted by an XP bead purification (Beckman Coulter) adding the beads solution in a ratio of 0.9:1 to the samples. Unique dual indexing was done during the following PCR enrichment (12 cycles) using amplification primers carrying the same sequence for i7 and i5 index (i5: AAT GAT ACG GCG ACC ACC GAG ATC TAC AC NNNNNNNN ACA TCT TTC CCT ACA CGA CGC TCT TCC GAT CT, i7: CAA GCA GAA GAC GGC ATA CGA GAT NNNNNNNN GTG ACT GGA GTT CAG ACG TGT GCT CTT CCG ATC T). After two more XP bead purifications (0.9:1), libraries were quantified using the Fragment Analyzer (Agilent).