GSM5134365: MOplus_UPF2_KO2; Mus musculus; RNA-Seq
Total RNA extraction was performed using Trizol (Invitrogen). RNA quality (RNA integrity Number >7) was confirmed using RNA 6000 Nano Chips (Agilent Technologies). cDNA was generated from DNAseI-treated total RNA using SuperScript III First-Strand Synthesis System (Invitrogen)in accordance with the manufacturers' instructions. Reactions were in 20 ul volumes containing 1x IQ SyberGreen supermix and 0.3 mM of the respective forward and reverse primers using the CFX96 Real Time PCR Machine (Bio-Rad). Cycling conditions were: 95°C for 6min followed by 95°C for 30 s, primer-specific annealing temperature for 30 s, and extension at 72°C for 30 s. Primer sequences were: Upf2 ex2-3 Forward, 5′ GGCTGCTCAGCTGAAAGAGA-3′; Upf2 ex2-3 Reverse, 5′-TTTTCCTCTGGTCGGTTGTC-3′; Hprt Forward, 5′-AGTGTTGGATACAGGCCAGAC-3′; Hprt Reverse, 5′-CGTGATTCAAATCCCTGAAGT-3′ Poly-A enriched stranded RNA library preparation was performed using the TruSeq Stranded mRNA Library Prep kit (Illumina). Sequencing was performed on a Illumina Hi-Seq 2500 platform. Both library preparation and sequencing were performed at Novogene (China). For each sample, approximately 100M paired-end 100bp strand-specific reads were acquired.