RNA-seq of homo sapiens, R1-DDR rep-2
Cells were lysed in QIAzol lysis reagent (Qiagen). Total RNA was extracted using a RNeasy mini kit (Qiagen) according to the manufacturers instructions. RNA-seq was performed by Arraystar, Inc. Briefly, total RNA from each sample was quantified using a NanoDrop ND-1000. Subsequently, 12 g of total RNA was used to prepare the sequencing library according to the following steps: 1. Total RNA was enriched by oligo (dT) magnetic beads (rRNA removed); and 2. RNA-seq library was prepared using the KAPA Stranded RNA-Seq Library Prep Kit (Illumina), which incorporates dUTP into the second cDNA strand and renders the RNA-seq library strand-specific. The completed libraries were qualified using an Agilent 2100 Bioanalyzer and quantified using the absolute quantification qPCR method. To sequence the libraries, the barcoded libraries were mixed, denatured to single stranded DNA in NaOH, captured on Illumina flow cell, amplified in situ, and subsequently sequenced for 150 cycles at both ends using the Illumina HiSeq 4000.