GSM5849657: Lung, tumor epithelial cells; Mus musculus; RNA-Seq
GSM5849657
Total RNA was isolated using RNeasy mini kit (Qiagen, Germany). Total RNA was quantified using a NanoDrop ND-2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) The total RNA samples are first treated with DNase I to degrade any possible DNA contamination. Then the mRNA is enriched by using the oligo(dT) magnetic beads (for_x000D_eukaryotes). Mixed with the fragmentation buffer, the mRNA is fragmented into short fragments (about 200 bp). Then the first strand of cDNA is synthesized by using random_x000D_hexamer-primer. Buffer, dNTPs, RNase H and DNA polymerase I are added to synthesize the second strand. The double strand cDNA is purified with magnetic beads. End_x000D_reparation and 3'-end single nucleotide A (adenine) addition is then performed. Finally, sequencing adaptors are ligated to the fragments. The fragments are enriched by PCR_x000D_amplification. During the QC step, Agilent 2100 Bioanaylzer and ABI StepOnePlus Real-Time PCR System are used to qualify and quantify of the sample library.