GSM3938461: Day 7 shScramble spheroids; Mus musculus; RNA-Seq
Pancreas progenitor spheres were released from Matrigel on Day 7 using 100 uL per well of Dispase II (Sigma-Aldrich) dissolved at 1 mg/mL in PrEBM media (Lonza) for 2 hours at 37C. An additional 100 uL per well of RPMI with FBS was overlayed to stop the reaction by dilution, and spheres were subsequently mechanically released from Matrigel by pipetting and collected into a single Eppendorf tube. Spheres were pelleted at 750xg for 4 minutes, supernatant was removed, and 200 uL of TRIzol reagent (Thermo Fisher Scientific) was added for RNA extraction. Samples from Day 7 shScramble, shWdr5 and shDpy30 spheres were pooled from > 4 replicates into TRIzol to obtain > 500 ng RNA. cDNA libraries were prepared from Illumina TruSeq kits (RNA Sample Preparation Low Throughput (LT) and Stranded mRNA) using Ampure XP beads and validated on an Agilent Bioanalyzer High Sensitivity DNA chip before sequencing on Illumina sequencing platforms (HiSeq or NextSeq). Sequence reads were aligned to the mouse GRC38/mm10 genome using Salmon (Patro et al., 2017) and DESeq2 (Love et al., 2014) was used to determine differential gene expression.