title: Illumina HiSeq 2500 sequencing; RNA-seq of wild-type and Utx- or Hnf1a-deficient mouse pancreas
design: RNA-seq of wild-type and Utx- or Hnf1a-deficient mouse pancreas
library_name: Hnf1aaKO3_s
library_construction_protocol: For RNA-seq, we implemented a method for purification of intact pancreatic RNA according to a modified Guanidinium salts method (MacDonald RJ et al. 1987). Briefly, a small piece of pancreatic tissue was dissected from P4 or adult pancreas and immediately homogenized with a Polytron (VWR) in pre-cooled 4M Guanidinium Thiocyanate with 112mM beta-mercaptoethanol, centrifuged at 5000g for 5 min at 4°C to remove insoluble debris. Conversion to FASTQ read format was done using Illumina's bcl2fastq algorithm.  RNA was precipitated from the supernatant with pre-cooled 75% Ethanol, 0.1M potassium acetate, pH 5.5, and 75mM acetic acid at -20°C for 2 hr. The precipitate was pelleted by centrifugation at 10,000g for 10 min at 4°C and resuspended at room temperature in 7.5 M Guanidinium HCl and 10.5 mM beta-mercaptoethanol. The RNA was re-precipitated twice with 0.1 M potassium acetate, pH 5.5, and 50% ethanol to remove residual RNAses, followed by purification with TRI Reagent RNA Isolation Reagent (Sigma-Aldrich). The concentration and quality of RNA was measured with a NanoDrop spectrophotometer (ND-1000, Thermo Scientific) and an Agilent 2100 Bioanalyzer. RNA integrity numbers ranged from 7.8 to 9.3.  One μg of total RNA was used to make RNA-seq libraries using the Truseq Stranded mRNA Sample Prep Kit (Illumina) following manufacturer's instructions.
attributes.tag: Experimental Factor: genotype
attributes.value: Ptf1aCre;Hnf1a LoxP/LoxP