GSM6122460: HS766T cells, DMSO treated, replicate 3; Homo sapiens; RNA-Seq
Approximately Two million cells in triplicate were harvested, rinsed with PBS and stored in RNAlater RNA stabilization solution RNA purification, reverse transcription, library construction and sequencing were performed at Mingma Technologies at Shanghai according to the manufacturer's instructions (Illumina). The mRNA-focused sequencing libraries from total RNA were prepared using Illumina TruSeq Stranded mRNA Library Prep kit. PolyA mRNA was purified from total RNA using oligo-dT-attached magnetic beads and then fragmented by fragmentation buffer. Taking these short fragments as templates, first strand cDNA was synthesized using reverse transcriptase and random primers. Then in the process of second strand cDNA synthesis, RNA template was removed and a replacement strand, incorporating dUTP in place of dTTP to generate ds cDNA, was synthesized. The incorporation of dUTP quenches the second strand during amplification, because the polymerase does not incorporate past this nucleotide. AMPure XP (Beckmen) beads were then used to purify the ds cDNA from the reaction mix. Next, the ds cDNA was subjected to end-repair, phosphorylation and 'A' base addition according to Illumina's library construction protocol. In the following process, Illumina sequencing adapters were added to both size of the dscDNA fragments. After PCR amplification for DNA enrichment, the AMPure XP Beads were used to clean up the target fragments of 200-300 bp. After library construction, Qubit 2.0 fluorometer dsDNA HS Assay (ThermoFisher Scientific) was used to quantify concentration of the resulting sequencing libraries, while the size distribution was analyzed using Agilent BioAnalyzer 2100 (Agilent).