title: GSM8370965: SK-N-SH cells, DA+,rep3; Homo sapiens; RNA-Seq
library_construction_protocol: Lyse cells directly in a culture dish by adding 1 mL TRIzol reagent and mix thoroughly. add 200μL chloroform, vortex mix for 15 seconds. incubate the mix for 5 minutes at room temperature and two layer should form. centrifuge at 12000 g at 4 °C for 10 minutes. Carefully transfer the top layer into the new tubes.add 1:1V of isopropanol to each tube and mix by inversion. Centrifuge at 12000 g at 4 °C for 15 minutes. Discard the supernatant and wash the pellet with 1 mL 75%  ethanol. Centrifuge at 12000g at 4 °C for 10 minutes. Remove all of residential ethanol with a pipet and air dry for 5 min. redissolve RNA in 30-50 μL  DEPC treated water. The rRNA-depleted sequencing libraries from total RNA were prepared using Illumina TruSeq® Stranded Total RNA Gold preparation Kit. About 1 μg total RNA was used as input material, and Ribo-Zero Gold kit was used to remove both cytoplasmic and mitochondrial rRNA. Purified RNA was then fragmented into small pieces using divalent cations under elevated temperature. The cleaved RNA fragments were copied into first strand cDNA using reverse transcriptase and random primers, followed by second strand cDNA synthesis. These cDNA fragments were then subjected to end-repair, phosphorylation and 'A' base addition according to Illumina's library construction protocol.