p20017-s162_522729_TotalRNA_S72_L002
The sequencing libraries were prepared with the KAPA RNA HyperPrep Kit with RiboErase (HMR) Globin kit (Roche Sequencing Solutions), from 500ng total RNA.  This kit is designed to deplete both ribosomal as well as globin RNA. Following manufacturers instructions, the first step depletes rRNA and globin RNA from total RNA.  After depletion, the remaining RNA is DNase digested to remove any gDNA contamination. Samples are then purified, fragmented and primed for cDNA synthesis.  Fragmented RNA is then reverse transcribed into first strand cDNA using random primers.  The next step removes the RNA template and synthesizes a replacement strand, incorporating dUTP in place of dTTP to generate ds cDNA.  Pure Beads (KAPA BIOSYSTEMS) are used to separate the ds cDNA from the second strand reaction mix resulting in blunt-ended cDNA.  A single A nucleotide is then added to the 3 ends of the blunt fragments.  Multiple indexing adapters, containing a single T nucleotide on the 3 end of the adapter, are ligated to the ends of the ds cDNA, preparing them for hybridization onto a flow cell.   Adapter ligated libraries are amplified by PCR, purified using Pure Beads, and validated for appropriate size on a 4200 TapeStation D1000 Screentape (Agilent Technologies, Inc.).   The DNA libraries are quantitated using KAPA Biosystems qPCR kit, and are pooled together in an equimolar fashion, following experimental design criteria.  Each pool is denatured and diluted to 350pM with 1% PhiX control library added.  The resulting pool is then loaded into the appropriate NovaSeq Reagent cartridge for 100 paired end sequencing, and sequenced on a NovaSeq6000 following the manufacturers recommended protocol (Illumina Inc.).