title: GSM5818957: MC3: Negative control_3; Homo sapiens; RNA-Seq
library_construction_protocol: Total RNA was extracted with Trizol (Invitrogen) and the concentration was calculated using Quant-IT RiboGreen (Invitrogen). Total RNA (0.5 µg) was used to construct a library using the Illumina TruSeq Stranded Total RNA Library Prep Gold Kit (Illumina). After removing the rRNA, fragmentation was performed with mRNA under elevated temperatures. First-strand cDNA was synthesized using SuperScript II reverse transcriptase (Invitrogen) and random primers. Second-strand cDNA synthesis was performed using DNA Polymerase I, Rnase H, and dUTP. These cDNA fragments go through an end repair process mediated by the addition of a single 'A' base and ligation of the adapters. The products were purified and enriched by PCR to create the final cDNA library. The libraries were quantified using KAPA Library Quantification Kits for Illumina Sequencing platforms according to the qPCR Quantification Protocol Guide (KAPA BIOSYSTEMS, #KK4854) and qualified using the TapeStation D1000 ScreenTape (Agilent Technologies, #5067-5582). Indexed libraries were then submitted to Illumina NovaSeq (Illumina), and the paired-end (2 × 100 bp) sequencing was performed by Macrogen Incorporated (Seoul, Republic of Korea).