GSM5945989: HCT-116, NOMO KO-3; Homo sapiens; RNA-Seq
Total RNA was extracted from three NOMO-KO clones and three control clones for HCT-116 and HS-5 using RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. DNA was removed from the samples using RNase-free DNase Set (Qiagen, Hilden, Germany). RNA integrity was assessed using the Agilent 2100 Bioanalyzer (Agilent, Palo Alto, CA). 0.5 µg of total RNA was used to construct cDNA libraries with TruSeq stranded total RNA with Ribo-Zero kit (Illumina, San Diego, CA, USA). cDNA libraries were sequenced with NovaSeq600 (paired-end 150bp x 2) with a range of 61.8-78.7M reads/sample according to the manufacturer instructions. Library construction and sequencing were performed by Macrogen Inc. (Macrogen, Seoul, Korea). Adapter sequences and low-quality bases were trimmed using Trimmomatic (v.0.39). Raw and trimmed reads quality was assessed with the FASTQC tool (v.0.11.9). The surviving paired reads after trimming were mapped to hg19 human genome with the STAR aligner (v.2.7.9a). The reference genome sequence (hg19, Genome Reference Consortium GRCh37) and annotation data (v.87) were downloaded from the Ensembl website (https://www.ensembl.org/). Gene level counts were calculated using the union mode from the HTSeq package (v.0.12.4). Gene expression count normalization through the median of ratios method(26) and differential gene expression analyses were performed with the DESeq2 R package (v.1.30.1). Batch-effects were adjusted through the ComBat_seq function from the sva R package (v.3.38.0).