GSM3613448: AI011_MCF10A_Control_rep1; Homo sapiens; RNA-Seq
RNA extracted from MCF-10A cell lines Total RNA was first evaluated for its quantity, and quality, using Agilent Bioanalyzer 2100. A RIN (RNA Integrity Number) of five or higher was required to pass the quality control. One to two nanograms of total RNA per sample were used for library preparation. cDNA was first synthesized using SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing (Takara Clontech Laboratories, Inc.). Dual indexed cDNA library was then prepared using Nextera XT DNA Library Prep Kit (Illumina, Inc.). Each library was quantified and its quality accessed by Qubit and Agilent Bioanalyzer, and multiple libraries were pooled in equal molarity. Average size of library insert was about 150b. Five microliters of 2 nM pooled libraries per lane were then denatured, neutralized and applied to the cBot for flow cell deposition and cluster amplification, before loading on to HiSeq 4000 for 75b paired-end sequencing (Illumina, Inc.). Each lane generated approximately 300-350 million reads. A Phred quality score (Q score) was used to measure the quality of sequencing. More than 90% of the sequencing reads reached Q30 (99.9% base call accuracy).