GSM8634967: ES-CM2; Homo sapiens; RNA-Seq
Total RNA was extracted using Qiagen Rneasy Plus Mini kit following manufacturer's instructions (Qiagen, Hilden, Germany) RNA samples were quantified using Qubit 4.0 Fluorometer (ThermoFisher Scientific, Waltham, MA, USA) and RNA integrity was checked with 4200 TapeStation (Agilent Technologies, Palo Alto, CA, USA). ERCC RNA Spike-In Mix kit (cat. 4456740) from ThermoFisher Scientific, was added to normalized total RNA prior to library preparation following manufacturer's protocol. 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. Strand specific RNA sequencing library was prepared by using NEBNext Ultra II Directional RNA Library Prep Kit for Illumina following manufacturer's instructions (NEB, Ipswich, MA, USA). Briefly, the enriched RNAs were fragmented for 8 minutes at 94 °C. First strand and second strand cDNA were subsequently synthesized. The second strand of cDNA was marked by incorporating dUTP during the synthesis. cDNA fragments were adenylated at 3'ends, and indexed adapter was ligated to cDNA fragments. Limited cycle PCR was used for library enrichment. The incorporated dUTP in second strand cDNA quenched the amplification of second strand, which helped to preserve the strand specificity. The sequencing library was validated on the Agilent TapeStation (Agilent Technologies, Palo Alto, CA, USA), and quantified by using Qubit 4.0 Fluorometer (ThermoFisher Scientific, Waltham, MA, USA) as well as by quantitative PCR (KAPA Biosystems, Wilmington, MA, USA). The sequencing libraries were multiplexed and clustered on a flowcell. After clustering, the flowcell was loaded onto the Illumina NovaSeq X instrument according to manufacturer's instructions. The samples were sequenced using a 2x150bp Paired-End (PE) configuration.