Illumina HiSeq 2000 sequencing; An RNASeq normal tissue atlas for mouse and rat
An RNASeq normal tissue atlas for mouse and rat
Male Wistar Han rats (Crl:WI(Han)) and male BL/6J mice (C57BL/6J) were obtained from Charles River Laboratories (Germany). Experimental protocols concerning the use of laboratory animals were reviewed by a German Federal Ethics Committee and approved by German governmental authorities. Animals were housed in groups of three on a 12-h light/dark cycle and fed ad libitum a standard pelleted rodent diet (Diet No. 3438, Provimi Kliba Switzerland) with free access to water. Rats with a body weight of 160-180 g and rats and mice at the age of 7-8 weeks were used for tissue sampling. Retrobulbar blood samples were collected in the morning at 9 a.m. under isofluran anesthesia immediately prior to dissection. Animals (n = 3 for each species) were sacrificed thereafter by intraperitoneal injection of pentobarbital (rats) or cervical dislocation (mice) and tissues (esophagus, stomach, duodenum, jejunum, ileum, colon, pancreas, liver, thymus, kidney, heart, brain, quadriceps muscle) were harvested and transferred immediately to RNA Later at 4 degrees celsius. Total RNAs were individually extracted using the Ambion Magmax™-96 total RNA isolation kit (Life Sciences) according to the manufacturer’s instructions. Briefly, 5 mg of tissue was placed in the lysis solution and homogenized in Qiagen Tissuelyzer™ for a period of 30 sec. Nucleic acids were captured onto magnetic beads, washed and treated with DNase. Total RNA was then eluted in 50 μl elution buffer. RNA quality and concentration was measured using an RNA Pico chip on an Agilent Bioanalyzer. The Sequencing library preparation has been done using 200 ng of total RNA input with the TrueSeq RNA Sample Prep Kit v2-Set B (RS-122-2002, Illumina Inc, San Diego, CA) producing a 275bp fragment including adapters in average size. In the final step before sequencing, eight individual libraries were normalized and pooled together using the adapter indices supplied by the manufacturer.