GSM9010208: HCC1806_sgC1_rep2; Homo sapiens; RNA-Seq
Total RNA was purified using the Nucleospin RNA kit (Macherey-Nagel). RNA samples were quantified using Qubit 2.0 Fluorometer (ThermoFisher Scientific, Waltham, MA, USA) and RNA integrity was checked with 4200 TapeStation (Agilent Technologies, Palo Alto, CA, USA). Samples were initially treated with TURBO DNase (Thermo Fisher Scientific, Waltham, MA, USA) to remove DNA contaminants. The next steps included performing rRNA depletion using QIAseq® FastSelect™−rRNA HMR kit (Qiagen, Germantown, MD, USA ), which was conducted following the manufacturer's protocol. RNA sequencing libraries were constructed with the NEBNext Ultra II RNA Library Preparation Kit for Illumina by following the manufacturer's recommendations. Briefly, enriched RNAs are fragmented for 15 minutes at 94 °C. First strand and second strand cDNA are subsequently synthesized. cDNA fragments are end repaired and adenylated at 3'ends, and universal adapters are ligated to cDNA fragments, followed by index addition and library enrichment with limited cycle PCR. Sequencing libraries were validated using the Agilent Tapestation 4200 (Agilent Technologies, Palo Alto, CA, USA), and quantified using Qubit 2.0 Fluorometer (ThermoFisher Scientific, Waltham, MA, USA) as well as by quantitative PCR (KAPA Biosystems, Wilmington, MA, USA).