Escherichia coli rnase h

The RNA-Seq experiments were performed by Novogene (Beijing, China). The RNA-seq library was prepared for sequencing using standard Illumina protocols. Briefly, total RNAs from Caki-1 CTRL and CLDN7 cells were isolated using TRIzol reagent (Invitrogen) and treated with RNase-free DNase I (New England Biolabs, MA, USA), to remove any contaminating genomic DNA. RNA extraction was performed using Dynabeads oligo(dT) (Invitrogen Dynal). Double-stranded complementary DNAs were synthesized from 1 μg of total RNA using Superscript II reverse transcriptase (Invitrogen) and random hexamer primers. Escherichia coli RNase H (New England Biolabs) were added to remove RNA complementary to the cDNA. The cDNA was then fragmented by nebulization, and the standard Illumina protocol was followed thereafter to create the mRNA-seq library. The libraries were sequenced on an Illumina HiSeq 2000 platform. Sequencing reads were aligned to the human genome (hg19) using the TopHat program (v2.1.1) set to the default parameters. Total read counts for each protein-coding gene were extracted using HTSeq (version 0.6.0) and then loaded into R package DESeq2 to calculate the differentially expressed genes with a cut-off fold change of ≥1.5 and an FDR < 0.05. Gene expression was calculated using the RPKM (reads per kilobase transcriptome per million reads) method. Experiments were repeated three times.