GSM8743153: Lep(ob/ob) pancreas, CCKR-I (lorglumide), 12-week age, male, 74; Mus musculus; RNA-Seq
Mouse pancreata were snap frozen in liquid nitrogen and manually pulverized using the BioPulverizer (BioSpec Products 59012MS). Total RNA was extracted from pulverized tissue (20-40 mg), homogenized via QIAshredder column, and purified using the RNeasy Mini Kit (Qiagen) with on column DNAse I digestion, per the manufacturer's protocol. RNA quality and quantity was assessed using an Agilent Bioanalyzer, and only high-quality RNA samples (RIN>7) were used in downstream analysis. RNA-seq libraries were prepared from mRNA purified from approximately 200ng total RNA with oligo-dT bead polyA selection (Illumina)  and sheared by incubation at 94C in the presence of Mg (Roche Kapa mRNA Hyper Prep Cat# KR1352). Following first-strand synthesis with random primers, second strand synthesis and A-tailing are performed with dUTP for generating strand-specific sequencing libraries. Adapter ligation with 3' dTMP overhangs are ligated to library insert fragments.  Library amplification amplifies fragments carrying the appropriate adapter sequences at both ends.  Strands marked with dUTP are not amplified. Indexed libraries are quantified by qRT-PCR using a commercially available kit (Roch KAPA Biosystems Cat# KK4854) and insert size distribution determined by either the Agilent Bioanalyzer. Samples with a yield of ≥ 0.5 ng/ul and a size distribution of 150-300bp are used for sequencing. Sequenced (>25M 100-bp paired-end (2x100) reads on NovaSeq 6000 (Illumina)) with the Yale Center for Genome Analysis (YCGA)