Transcriptome dataset of HEK293T cells depleted of one of the subunits of the DNA-PK complex: Ku70, Ku80 or DNA-PKcs

DNA-PK is a heterotrimeric complex that consists of Ku70 (XRCC6), Ku80 (XRCC5) and DNA-PKcs (PRKDC) subunits. The complex is a major player in the repair of DNA double strand break (DSB) via the non-homologous end joining (NHEJ) pathway. This process requires all DNA-PK subunits, since Ku70/Ku80 heterodimer firstly binds to DNA ends at DSB and then recruits DNA-PKcs. Recruitment of the DNA-PKcs subunit to DSB leads to phosphorylation events near DSB and recruitment of other NHEJ-related proteins that restore DNA integrity. However, today a lot of evidence demonstrates participation of the DNA-PK components in other cellular processes, e.g. telomere length maintenance, transcription, metabolism regulation, cytosolic DNA sensing, apoptosis, cellular movement and adhesion. It is important to note that not all the subunits of the DNA-PK complex are necessary for these processes, and the largest number of independent functions has been shown for the Ku70/Ku80 heterodimer and especially the Ku70 subunit. To better understand the role of each DNA-PK subunit in the cell life, we have analyzed transcriptome changes in HEK293T cells depleted of Ku70, Ku80 or DNA-PKcs using NGS-sequencing. Here, for the first time, we present the data obtained from the transcriptome analysis.


a b s t r a c t
DNA-PK is a heterotrimeric complex that consists of Ku70 (XRCC6), Ku80 (XRCC5) and DNA-PKcs (PRKDC) subunits. The complex is a major player in the repair of DNA double strand break (DSB) via the non-homologous end joining (NHEJ) pathway. This process requires all DNA-PK subunits, since Ku70/Ku80 heterodimer firstly binds to DNA ends at DSB and then recruits DNA-PKcs. Recruitment of the DNA-PKcs subunit to DSB leads to phosphorylation events near DSB and recruitment of other NHEJ-related proteins that restore DNA integrity. However, today a lot of evidence demonstrates participation of the DNA-PK components in other cellular processes, e.g. telomere length maintenance, transcription, metabolism regulation, cytosolic DNA sensing, apoptosis, cellular movement and adhesion. It is important to note that not all the subunits of the DNA-PK complex are necessary for these processes, and the largest number of independent functions has been shown for the Ku70/Ku80 heterodimer and especially the Ku70 subunit. To better understand the role of each DNA-PK subunit in the cell life, we have analyzed transcriptome changes in HEK293T cells depleted of Ku70, Ku80 or DNA-PKcs using NGS-sequencing. Here, for the first time, we present the data obtained from the transcriptome analysis.
© 2021 The Author(s

Value of the Data
• These data are important for the investigation of transcriptional changes under Ku70, Ku80, or DNA-PKcs depletion in the cell. • These data can be used by investigators of cellular functions of the DNA-PK complex and its individual subunits. • These transcriptome data can be used to elucidate the independent cellular functions of Ku70, Ku80, and DNA-PKcs.

Transient knock-down
To transfect cells with siRNAs, 50 pmol of pre-annealed siRNA duplex (for siKu70 25 pmol of siKu70_352 duplex and 25 pmol of siKu70_1025 duplex, for siKu80 25 pmol of siKu80_179 duplex and 25 pmol of siKu80_423 duplex) in OptiMem transfection medium were mixed with Lipofectamine RNAiMAX reagent (Invitrogen) according to the manufacturer's protocol and then added to 293T cells seeded in a 12-well plate in 1 mL of growth medium for 72 h incubation. The used siRNAs are listed in Table 2 .

Total RNA Illumina sequencing
Total RNA fraction from all samples (3 repeats of HEK 293T wt independently transfected with siC, Ku70 with siKu70, Ku80 with siKu80 and DNA-PKcs transfected with siDNA-PKcs) was extracted using TRIzol reagent (Life Technologies) following the manufacturer's protocol 72h after siRNA transfection. 1 μg of total RNA was fragmented by incubation at 90 °C for 6 min in 40 μL of 100 mM Tris-HCl pH 8.0 and 8 mM MgCl 2 . After isopropanol precipitation, the RNA fraction was dissolved in 12 μL and subjected to ribosomal RNA depletion by NEBNext rRNA depletion kit (E6310L, New England Biolabs) according the manufacturer's protocol. mRNA sequencing libraries were prepared by NEBNext Ultra II Directional RNA library Prep kit for Illumina (E7760S, New England Biolabs) according to the manufacturer's protocol. NEBNext Multiplex Oligos for Illumina (Dual Index Primers Set1) (E7600S, New England Biolabs) were used for adaptor ligation. Quality control of the libraries was carried out on Agilent 2100 Bioanalyzer (Agilent Technologies). The libraries were sequenced on HiSeq 1500 platform (Illumina) at JSC Genoanalytica (Moscow, Russia).

Bioinformatic analysis
Quality of raw sequencing reads was checked by FastQC. Because of good quality of the reads they were mapped on genome without trimming. Raw sequencing data were mapped on hg38 human genome by STAR with default parameters. STAR was used for read counting for human genes obtained from the Ensemble database. Read coverage was normalized and compared among the samples using DESeq2 R library. Three independently transfected cell lines and the control cell line were compared. Negative binomial generalized linear models were used for differential expression analysis. We calculated the p-values adjusted on multiple testing (Benjamini and Hochberg method) and the logarithm of fold change of expression value (logFC) for all genes presented in the dataset (Supplementary file 1). The obtained data were visualized in R v. 3.6.0 as volcano-plots using ggplot2 library.

Ethics Statement
Human subjects research: Not applicable. Animal experiments: Not applicable. Social media platforms: Not applicable.

Funding
This work was supported by RSF grants 19-74-10021 (identification of differentially expressed genes in DNA-PKcs depleted cells) and 17-14-01107 (identification of differentially expressed genes in Ku70 and Ku80 depleted cells)

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships which have or could be perceived to have influenced the work reported in this article.