GSM6069846: Ctrl, bio rep 3; Homo sapiens; RNA-Seq
RNA was extracted using a total RNA isolation kit (TR205-200, Tianmo). Agilent Bioanalyzer 2100 (Agilent Technologies, USA) was used for quality control and Qubit 3.0 Fluorometer (Life Technologies, USA) was used for quantification. RNA was fragmented into small pieces using divalent cations under 94 °C for 8 min. Cleaved RNA fragments were copied into first strand cDNA using reverse transcriptase and random primers, and followed by second strand cDNA synthesis using DNA Polymerase I and RNase H. These cDNA fragments then go through an end repair process, the addition of a single A' base, and then ligation of the adapters. The products were purified and enriched with PCR to create the final cDNA library. Purified libraries were quantified by Qubit 3.0 Fluorometer and validated by Agilent 2100 bioanalyzer to confirm the insert size and calculate the concentration.