GSM2977072: WT_4C-10; Mus musculus; RNA-Seq
Brown adipose tissues were removed, flash frozen on dry ice, and RNA was harvested using Trizol reagent and Rneasy mini kit. The SOLiD SAGE technology (Serial Analysis of Gene Expression) was utilized to determine mRNA expression levels by generating the SOLiD SAGE libraries of unique 27-bp sequence tags for all mRNAs and sequencing unique sequence tags isolated from the 3' ends of mRNAs using the Life Technologies 5500XL SOLiD system. Briefly, total RNA was bound to Dynabeads Oligo(dT) EcoP magnetic beads and transcribed into cDNA on the beads using SuperScript III Reverse Transcriptase and E.coli DNA polymerase. Double-stranded cDNA was digested with NlaIII restriction enzyme and ligated to Barcode adaptor A containing an EcoP15L restriction enzyme recognition site and a truncated internal adaptor sequence. An EcoP15I digestion was carried out to cleave the tag off from the magnetic beads. The tag consists of the adaptor sequence and 27 bp of unique sequence from the 3¢ end of a single mRNA. The 5¢ end of each tag was ligated to adaptor B that contains the P1 primer binding site for SOLiD emulsion PCR. Each library was amplified in a PCR reaction with a different barcoded SOLiD 3¢ Primer from the SOLiD RNA Barcoding kit and a SOLiD 5¢ Primer. SOLiD SAGE library sequencing was performed with next-generation sequencing using the Life Technologies 5500XL SOLiD system.