GSM2533335: A425WTStr; Mus musculus; OTHER
5-10μg total RNA was collected from homogenized tissue using Trizol (Ambion), and RNA quality was assessed by Bioanalyzer (Agilent). RNA was treated with TurboDNase (Ambion) and cleaned with Clean and Concentrator columns (Zymo). Cleaned RNA was fragmented in 1x SuperScript III first strand buffer (Invitrogen) at 94°C for 5 minutes 30 seconds and immediately placed on ice. SuperScript III (Invitrogen) reverse transcribed fragmented RNA from the custom Passeq oligo d(T) primer, and 20 minute incubation with RNAse I hydrolyzed remaining RNA (Life Technologies). Samples were run on a denaturing 8M urea, 10% acrylamide gel, and 160-200 nucleotide cDNA fragments were eluted overnight in elution buffer (300mM NaCl, 10mM EDTA). Spin-X columns (Corning) removed remaining gel pieces, and eluted cDNA was precipitated by incubation with 50% isopropanol at -20°C for 30 minutes followed by centrifugation at maximum speed for 45 minutes at 4°C. The pellet was washed with 70% ethanol and collected by spinning at maximum speed for 10 minutes 4°C. Dried pellets were resuspended in water. cDNA was circularized by incubation with CircLigase II (Epicentre) at 60°C for 4 hours. Phusion polymerase (NEB) amplified the insert and adaptors with PE 1.0 and PE 2.0 primers by incubation at 98°C for 30 seconds; 14 cycles of 98°C for 5 seconds, 62°C for 10 seconds, and 72°C for 10 seconds; and a final extension of 72°C for 2 minutes. The PCR products were run on a non-denaturing 10% polyacrylamide gel. The 215-254 nucleotide library was extracted and eluted and precipitated as above. Libraries were Bioanalyzed (Agilent) for quality control and sequenced with single-end 100 base-pair sequencing on the HiSeq 2000 machine (Illumina). Passeq