title: HT29 [wild type] - CRISPR/Cas9 gRNA: non-targeting [NT] - treatment: vehicle alone [DMSO] - [replicate #3]
design: Total RNA was purifed using Trizol (Ambion) and cleaned from impurities using a RNeasy MinElute Cleanup Kit (QIAGEN). The yield and integrity of the RNA preparation was evaluated using a BioAnalyzer (Agilent) and the RNA preparation was confirmed to have a high (>8.0) RNA integrity number (RIN) before being selected for RNA-sequencing (RNA-seq). The RNA-seq library was created starting from a total amount of 100 ng of total RNA, using a TrueSeq Stranded mRNA Library Prep Kit (Illumina), which uses poly-A to selectively pull-down messenger RNAs (mRNAs) before retro-transcribing them into complementary DNA (cDNA). The resulting cDNA library was sequenced using a NovaSeq 6000 (Illumina) sequencer (sequencing depth: 40 million DNA reads; read length: 100 bp). Sequencing reads were generated by paired-end (PE) sequencing, resulting into two independent FASTQ files for each sample (R1, R2) .