Superscript 2 first strand synthesis system

Total RNA was extracted using TRIZOL (Life Technologies) and reverse transcribed using the SuperScript II™ first-strand synthesis system (Life Technologies). Total RNA was quantified using a ND-1000 spectrophotometer (NanoDrop Technologies, Rockland, DE, USA) and contaminating genomic DNA removed with DNA-free™ reagent (Life Technologies) before reverse transcription.

Total RNA was extracted from terminally differentiated Atrx-flox and Atrx-ko cells by using the EuroGold TriFast (EuroClone, Pero, Milano, Italy) reagent, then the contaminating DNA was removed by DNase treatment (Turbo DNA-freeTM kit, Ambion, Austin, TX, USA) and cDNA was prepared by reverse transcription using the SuperScript II First-Strand Synthesis system (Life Technologies), following the manufacturer’s instructions. The obtained cDNA was, then, amplified by quantitative PCR (qPCR), using the primers for β-III tubulin (Beta-III tubulin Up and Beta-III tubulin Low, Table S1) and Gapdh (Gapdh F3 and Gapdh R3, Table S1) transcripts with SsoAdvance Universal SYBR Green supermix (Bio-Rad, Hercules, CA, USA) on CFX96 Real Time PCR system (Bio-Rad), according to the manufacturer’s protocols. The 2^−ΔΔCq method was applied to determine the relative quantitative levels, and the data were normalized with respect to Gapdh expression.

Total RNA was extracted from transfected cells using the Hybrid-RTM total RNA Kit (GeneAll Biotechnology, Seoul, Korea). Following cDNA synthesis using the Superscript II First-Strand Synthesis System (Life Technologies), qRT-PCR was executed with a dual system LightCycler (Roche Diagnostics). The SYBR Green-based comparative CT method was used to analyze the target gene expression in relation to HPRT expression (relative fold-change = 2^−ΔΔCT) [36 (link)]. Primers used are listed in Supplementary Table S1. All PCR primers were purchased from Cosmo Genetech (Seoul, Korea).

Total RNA was isolated using Trizol reagent (Life Technologies, Gaithersburg, MD, USA) in culture flasks and EP tubes. For reverse transcriptase (RT)-PCR, cDNA was synthesized by the Superscript II First Strand Synthesis System (Life Technologies, Gaithersburg, MD, USA). The cDNA was amplified by PCR using primers specific for COX-2 (sense: 5’-CAA AAGCTGGGAAGCCTTCT-3′: antisense: 5′- CCATCC TTGAAAAGGCGCAG-3′) and GADPH (as control) (sense: 5’-GGGTGTGAAC CATGAGAAGT-3′; antisense: 5′- GGCATGGACTGTGGTCATGA-3′). Prelimina- ry experiments were conducted to ensure that the PCR conditions were at the logarithmic phase of the reaction for each set of primers. PCR conditions were: 94 °C for 5 min followed by 94 °C for 30 s, 59 °C for 30 s, 72 °C for 60 s (forty cycles), and a final extension step at 72 °C for 5 min. PCR products were run on 2% agarose gels and analyzed using a gel documentation system (Ultra-Violet Product Limited, CA, USA). The relative expression levels were determined by comparing the density to the controls.

To validate RNA-seq results, DEGs were randomly selected and analyzed by reverse-transcription qPCR (rt-qPCR). cDNA was obtained by reverse transcription of 500 ng of total RNA using the SuperScript II First-Strand Synthesis system (Life Technologies, Carlsbad, CA, USA), following the manufacturer’s instructions. The obtained cDNA was, then, amplified by qPCR with SsoAdvance Universal SYBR Green supermix (Bio-Rad, Hercules, CA, USA) on a CFX Opus Real-Time PCR system (Bio-Rad), according to the manufacturer’s protocols. The 2^-ΔΔCq method was used to determine the relative quantitative levels, and the data were normalized with respect to the expression of β-actin, which showed a similar distribution of FPKM values with no statistically significant difference between the two experimental groups (Wilcoxon Test, p = 0.96; data not shown). List of primer sequences used in this study is reported in the Additional file 5: Table S5.