title: GSM9075116: C42_WT_R2; Homo sapiens; RNA-Seq
library_construction_protocol: Approximately, 1 X 106 cells were lysed with QIAzol lysis reagent, followed by the addition of chloroform. Aqueous and organic phases were later separated by centrifugation. Aqueous phases were transferred to a new tube, followed by the addition of 100% ethanol to it, and mixed by pipetting. The sample was later transferred into an RNeasy Mini spin column for collecting total RNA. On-column DNAse digestion was performed to remove residual genomic DNA. About 500ng of total RNA was used to make sequencing libraries with the mRNA HyperPrep kit (Roche) as per the manufacturer's instructions. First, Poly(A) RNA was captured using mRNA Capture Beads (KAPA BIOSYSTEMS). RNA was eluted, fragmented, and primed for cDNA synthesis. 1st strand of cDNA is later synthesized with random primers. Later, the RNA template gets removed and a replacement strand is synthesized by incorporating dUTP in place of dTTP to generate ds cDNA. Pure Beads (Roche) were used to separate the ds cDNA from the second strand reaction mix resulting in blunt-ended cDNA. A single 'A' nucleotide was 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, were ligated to the ends of the ds cDNA, preparing them for hybridization onto a flow cell. Adapter ligated libraries were amplified by PCR, purified using Pure Beads, and validated for appropriate size on a 4200 TapeStation D1000 Screentape (Agilent Technologies, Inc.). The DNA libraries were quantitated using KAPA Biosystems qPCR kit and were pooled together in an equimolar fashion, following experimental design criteria. Each pool was denatured and diluted to 350pM with 1% PhiX control library added. The resulting pool was then loaded into the appropriate NovaSeq Reagent cartridge, as determined by the number of sequencing cycles desired, and sequenced on a NovaSeq6000 following the manufacturer's recommended protocol (Illumina Inc.).