GSM5170815: CD28_KO_D3_2; Mus musculus; RNA-Seq
Total RNA was isolated from harvested cells using Trizol (Qiagen) using the miRNeasy Mini Kit (Qiagen) according to manufacturer's instructions. Quantity of RNA was evaluated using a Qubit 3.0 fluorometer and QC was performed using TapeStation Analysis Software A.02.02 The sequencing libraries are prepared with the RNA HyperPrep Kit with RiboErase (HMR)kit (Roche Sequencing Solutions), from 100ng total RNA.  Following manufacturer's instructions, the first step depletes rRNA from total RNA.  After ribosomal 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 200 cycle NovaSeq Reagent cartridge for 100 paired end sequencing, and sequenced on a NovaSeq6000 following the manufacturer's recommended protocol (Illumina Inc.).
GEO
GSM5170815