GSM9814505: sgMMADHC2, antimycin+oligomycin, rep2; Homo sapiens; RNA-Seq
Cells were harvested by pelleting at 600 x g for 2 mins and washed once with PBS. Total RNA was extracted usign the Qiagen Rneasy kit with on-column DNase (Qiagen ID. 79254) treatment as per manufacturer's instructions. RNA library preparation, sequencing and data analysis was conducted at Azenta Life Sciences (South Plainfield, NJ, USA) as follows: Library Preparation with PolyA selection and Illumina Sequencing: 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). 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 using random hexamer priming. 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 2.0 Fluorometer (ThermoFisher Scientific, Waltham, MA, USA) as well as by quantitative PCR (KAPA Biosystems, Wilmington, MA, USA). The sequencing libraries were multiplexed and clustered onto a flowcell on the Illumina NovaSeq instrument according to manufacturer's instructions. The samples were sequenced using a 2x150bp Paired End (PE) configuration.