GSM1236408: Heat Stress Sample 3 (2162); Gallus gallus; RNA-Seq
Liver samples of  25 mg were homogenized in 0.6mL of TRI Reagent Solution (Ambion) and total mRNA was isolated using the Ambion MagMax-96 kit protocol. To ensure high-quality RNA for microarray hybridization,  samples were ethanol precipitated as follows. Samples were combined with 50 microliters of RNAse free water to 100  microliters. Ten microliters of 3M sodium acetate, 300 microliters of 100% ethanol, and 20 micrograms of glycogen were added to the water-RNA mixture. These samples were incubated at -80 degrees celsius overnight  and centrifuged at 48 celsius for 25 min at 14,000 rpm. The supernatant was aspirated and the pellets were washed with 500 microliters of 70% ethanol. This mixture was centrifuged again at 7,600g for 5 min at 48 celsius. The supernatant was removed and the pellet was redissolved in RNAse free water. The samples were stored at -80 degrees celsius until usage. Libraries were prepared according to Illumina's instructions accompanying the DNA Sample Kit (Part# 0801-0303). Briefly, DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (32 to 52 exo minus) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3’ end. After adapter ligation DNA was PCR amplified with Illumina primers for 15 cycles and library fragments of ~250 bp (insert plus adaptor and PCR primer sequences) were band isolated from an agarose gel. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Illumina Hi-Seq 2000 platform following the manufacturer's protocols.
GEO Accession
GSM1236408