GSM2224560: N1het_TercWT_2; Mus musculus; RNA-Seq
Hearts were washed with PBS, and three AV leaflets were harvested by micro-dissection from each of four N1WT/mTRWT, four N1+/-/mTRWT, three N1WT/mTRG2, and four N1+/-/mTRG2 mice. Leaflets were incubated in collagenase for 20 minutes at 37 °C to dissociate cells, and RNA was isolated using the Qiagen RNeasy Micro Kit. cDNA was prepared with the NuGen Ovation RNA-seq System V2 Kit. Total RNA was reverse transcribed to synthesize the first-strand cDNA using a combination of random hexamers and a poly-T chimeric primer. The RNA template was then partially degraded by heating, and the second-strand cDNA was synthesized using DNA polymerase. The double-stranded DNA was then amplified using single primer isothermal amplification (SPIA). In this linear cDNA amplification process, RNase H degraded RNA in DNA/RNA heteroduplex at the 5′-end of the double-stranded DNA, after which the SPIA primer bound to the cDNA and the polymerase started replication at the 3′-end of the primer by displacement of the existing forward strand. Random hexamers were then used to amplify the second-strand cDNA linearly. Finally, libraries from the SPIA-amplified cDNA were made using the NuGen Ovation Ultralow DR Kit. The mRNA-seq libraries were analyzed by Agilent Bioanalyzer and quantified using an Illumina Library Quantification Kit (KAPA Biosystems). Four mRNA-seq libraries were pooled per lane of paired-end 100 bp sequencing on an Illumina HiSeq 2500 instrument.
GEO Accession
GSM2224560