GSM8436415: RNA-seq_CONTROL_PB1_KO; Homo sapiens; RNA-Seq
Illumina Compatible lowinputmRNA libraries were prepared using the Smart-Seq V4 Ultra LowInputRNA kit (Takara Bio, USA) and KAPA HyperPlus Library Preparation kit (Roche). Briefly, full length, double stranded cDNA was generated from 10ng of total RNA using Takara's SMART (SwitchingMechanismat 5' End ofRNATemplate) technology. The ds cDNA was amplified by 5 cycles of LD-PCR, then purified using Ampure Beads (Agencourt). Following bead elution, the cDNA was evaluatedforsize distribution and quantity using 4200 TapeStation High Sensitivity D1000 ScreenTape-Agilent Technologiesand the Qubit dsDNA HS Assay Kits(ThermoFisher) respectively. The cDNA was enzymatically fragmented, and 10ng of the fragmented cDNA was used to generate Illumina compatible libraries using the KAPA HyperPlus Library Preparation kit.  The KAPA libraries were purified and enriched with 8 cycles of PCR to create the final cDNA library. The libraries were assessed for size distribution using the Agilent Tapestation and quantified using the Qubit™ dsDNA HS Assay (ThermoFisher), then equimolar quantities of 33 libraries were combined to create the final library pool. The pooled library was quantified by qPCR using the KAPA Library Quantification Kit (KAPA Biosystems) and assessed for size distribution using the TapeStation 4200 (Agilent Technologies), then sequenced on the Illumina NovaSeq6000 SP flow cell using the 100nt paired end run format.