GSM5240520: Man1a2 homozygous dead NAC liver [NND_Liver_SIN238A13_S40]; Mus musculus; RNA-Seq
Total RNA in the lung and liver tissue was extracted using RNeasy Mini Kit (Qiagen, Valencia, CA) from 3 independent experiments per for alive Wild-type, alive Man1a2 heterozygous and dead Man1a2 null, and 2 independent experiments for dead Man1a2 heterozygous pups. Purified RNA was subjected to library synthesis using Illumina Truseq stranded mRNA kit. Purified RNA was fragmented using divalent cations under elevated temperature, and fragments copied into first stand cDNA using reverse transcriptase and random primers, followed by second strand cDNA synthesis using DNA polymerase I and RNase H. Aftering adding a single 'A' base, cDNA was ligated to the adapter, purified and enriched with PCR to create the final cDNA library. Libraries were validated using KAPA Biosystems primer premix kit with Illumina-compatible DNA primers and Qubit 2.0 fluorometer. Quality of libraries was examined using Agilent Bioanalyzer Tapestation 2200 and final libraries were pooled at a final concentration of 1.8 pM. Cluster generation and 150bp paired read dual-indexed sequencing was performed on Illumina Novaseq to obtain 20 million reads.