Illumina HiSeq 2000 sequencing; RNA-seq of mouse embryonic pancreatic mesenchyme
RNA-seq of mouse embryonic pancreatic mesenchyme
Pancreatic tissues were dissected from Nkx3.2-Cre;YFP mouse embryos, at embryonic days 13.5, 15.5, and 17.5. To obtain single cells, dissected pancreatic tissues were digested with Collagenase P (0.4 mg/ml; Roche) and DNase (0.1 ng/ml; Sigma)(Epshtein et al., 2017b). For cell sorting, DAPI (Sigma-Aldrich) was added to label dead cells and live fluorescently-labeled (Nkx3.2/YFP-positive) cells were collected using FACS Aria (BD).  RNA was extracted from isolated tissue and cells using PureLink RNA micro kit (Invitrogen). Samples containing 200 ng of total RNA each that were used to generate libraries with the Illumina TruSeq RNA sample preparation kit. Each library was diluted to approximately 10 pM before loading and sequenced using a Hi-seq 2000 instrument generating 50 base pair reads. Library preparation and sequencing was performed by the Duke IGSP Genome Sequencing and Analysis Core. The 50 bp long reads were mapped to the mouse genome (NCBI37/mm9) and exon-exon junctions using TopHat (Trapnell et al., 2009) version 1.3.1 with default parameters. Genome information was downloaded from the UCSC Genome Browser (Dreszer et al., 2012). TopHat alignments were filtered to keep reads (and their locations) that could be mapped uniquely. Per gene, we extracted read counts by using SAMtools (Li et al., 2009) version 0.1.13. Read counts were normalized to the length of the gene and the total number of mapped reads to obtain a standardized count (RPKM, reads per kilobase of exon model per million mapped reads). P-values were calculated by comparing the normalized expression level of each gene to the normalized expression level of three housekeeping genes (Gusb, Rpl23, and Eif2a).
Experimental Factor: developmental stage
embryonic day 13.5