GSM2454141: RNA NMP T+; Mus musculus; RNA-Seq
Total RNA from approximately 2,500 cells was isolated using the RNeasy Micro kit (Qiagen) and genomic DNA was digested on column with the addition of an extra 1ul of RNase-free DNase I (Roche) to ensure complete digestion. RNA-Seq libraries with the RNA equivalent of approximately 1,000 cells were generated using the TotalScript RNA-Seq Kit (Epicentre) with random hexamers according to manufacturer’s instructions with slight modifications. Briefly, all reagents during the cDNA synthesis steps were scaled down in order to achieve a total volume of 39µl, allowing all of the material to be used in the subsequent tagmentation procedure. To further improve strand-specificity of the libraries, Actinomycin D was added to the first strand cDNA synthesis reaction at a 5ng/µl final concentration and the libraries were finally amplified using 14 cycles.