Illumina NovaSeq 6000 paired end sequencing; Utilization of redundant dBPs in cells with altered levels of MXE regulators
Utilization of redundant dBPs in cells with altered levels of MXE regulators
Human embryonic kidney 293 cells were cultured in RPMI containing 10% foetal bovine serum and penicillin/streptomycin at 37°C, 5% CO2. The cells were transfected with the indicated plasmids or siRNAs using JetPRIME (Polyplus) according to manufacturer's recommendations. Total RNA was prepared using TRIzol (Life Technologies) and a standard protocol. RNA was resuspended in RNase-free water and quantified with NanoDrop (ThermoScientific). RNA samples were treated with DNaseI (Promega) and reverse transcribed with a gene-specific primer using Maxima H Minus Reverse Transcriptase (Thermo Scientific). Complementary DNA was amplified in nested PCR reactions using taxon-specific PCR primers shown in Table S1 and Figure 3B. PCR products were separate on agarose gels to check their expected size. PCR products were extracted from agarose gel slices using GeneJET Gel Extraction Kit (ThermoScientific) and quantified by NanoDrop. cDNA samples were diluted and the integrity and concentration of the DNA was confirmed by Agilent 2100 Bioanalyzer (Agilent Technologies). Ampli-seq libraries were prepared using proprietary adapter ligation and amplification (Eurofins Genomics).