Illumina HiSeq 2500 paired end sequencing; Strain-specific RNA-seq in Haster Stop/+ C57BL/6;PWK/PhJ F1 mice
Strain-specific RNA-seq in Haster Stop/+ C57BL/6;PWK/PhJ F1 mice
RNA was extracted from fresh whole liver samples. For pancreatic islets, the pancreas was inflated with an ice-cold collagenase P solution (1 mg/mL in Hanks' balanced salt solution (HBSS) buffer, Roche) injected through the main pancreatic duct. The pancreas was then dissected and incubated at 37ºC for 8 min with continuous agitation. The pancreatic tissue was disaggregated by gentle suction through a needle, and cell suspension was washed 4 times with cold HBSS-0.5% BSA. The islet pellet was resuspended in 7 mL Histopaque containing a 7:3 ratio of pre-cooled Histopaque 1077 (Merck) and Histopaque 1119 (Merck), and 7 mL of HBSS-0.5% BSA was layered on top of the Histopaque. The gradient was centrifuged at 950 g for 20 mins at RT. The interphase containing the islets was collected, washed 3 times with HBSS-0.5% BSA and islets were finally further enriched by gentle aspiration with a pipette under a stereomicroscope. Islets were allowed to recover for 2 days in culture in RPMI containing 11 mM glucose supplemented with 10% fetal calf serum and penicillin-streptomycin (1:100; Invitrogen) at 37°C and 5% CO2. RNA was prepared using RNeasy mini kit (Qiagen) including DNAse I (Qiagen) treatment. RNA-seq libraries were prepared using the Truseq Stranded mRNA Sample Prep Kit (Illumina) following manufacturer's instructions.