GSM4411948: HARPE on SCP1withTATA - replicate 2 in vitro; Homo sapiens; OTHER
For HARPE experiments transcribed in vitro, for each sample library, the products from twelve standard in vitro transcription reactions were combined. Standard reactions were performed as follows. DNA template (500 ng) was incubated with HeLa nuclear extract (prepared as described in Dignam et al. 1983) for preinitiation complex assembly at 30 °C for 1 hour in 46 μL of transcription buffer [20 mM HEPES-K+ (pH 7.6), 50 mM KCl, 6 mM MgCl2, 1.25% (w/v) polyvinyl alcohol, 1.25% (w/v) polyethylene glycol, 0.5 mM DTT, 3 mM ATP, 0.02 mM EDTA, and 2% (v/v) glycerol]. rNTPs (4 μL; 0.4 mM final concentration of each rNTP) were added to initiate transcription. [Where indicated, Sarkosyl was added to 0.2% (w/v) final concentration at 20 seconds after the addition of rNTPs.] The reaction was incubated at 30 °C for 20 min and terminated by the addition of 150 μL of Stop Mix [20 mM EDTA, 200 mM NaCl, 1% (w/v) SDS, 0.3 mg/mL glycogen]. Proteinase K (5 μL; 2.5 mg/mL) was added, and the mixture was incubated at 30 °C for 15 min. All in vitro transcription HARPE experiments were performed independently at least two times to ensure reproducibility of the data. RNA extraction and processing after transcription of HARPE libraries. RNA transcripts from cells or from in vitro transcription reactions were extracted with Trizol™ or Trizol™ LS (Thermo Fisher Scientific), respectively. Total RNA (40 µg for cell transfection experiments or the entire yield for in vitro experiments) was processed as follows. Contaminating plasmid DNA was removed with the TURBO DNA-free™ Kit - rigorous DNase treatment protocol (Thermo Fisher Scientific) as recommended by the manufacturer. The nucleic acids were precipitated with ethanol, and reverse transcription was performed with SuperScript™ III Reverse Transcriptase (Thermo Fisher Scientific) with the RT primer (5'- GTGACTGGAGTTCAGACGTGT) (Table 1) as recommended by the manufacturer. The reaction products were then treated with 30 U RNase H (New England Biolabs) for 20 min at 37°C. The nucleic acids were extracted with phenol-chloroform-isoamyl alcohol and precipitated with ethanol. The resulting cDNAs were then size-selected on a 6% polyacrylamide-8M urea gel by using radiolabeled size markers (Table 1) that enable the purification of cDNAs corresponding to transcription that initiates in the region from -5 to +6 relative to the A+1 in the Inr sequence. Size-selected cDNAs were used as templates to generate DNA amplicons for Illumina sequencing by using custom forward oligonucleotides containing the Illumina P5 and Read1-primer sequences preceding the sequence corresponding to nucleotides +1 to +16 of the promoter analyzed (Table 1). Reverse primers were selected from the NEBNext® Multiplex Oligos for Illumina® kits (NEB). NGS PCR amplicons were then size-selected on native 6% polyacrylamide gels prior to Illumina sequencing. Processing of plasmid DNA for Illumina sequencing. For in vitro experiments, the starting material used was the HARPE DNA libraries. For cell transfection experiments, post-transfection plasmid DNA extraction was performed as described in van Arensbergen et al. (2017). Briefly, cells were treated with trypsin, washed with PBS, and then incubated in 500 µL nuclear extraction buffer [10 mM NaCl, 2 mM MgCl2, 10 mM Tris-HCl (pH 7.8), 5 mM DTT, 0.5% NP40] on ice for 5 min. Nuclei were pelleted at 7,000 x g and washed twice with 1 mL nuclear extraction buffer. DNA was then extracted with ZymoPURE™ Plasmid Miniprep Kit (Zymo Research). Plasmid DNA samples were used as a template for the generation of DNA amplicons for Illumina sequencing. The forward oligonucleotides contain the Illumina P5 and Read1-primer sequences followed by a promoter-specific sequence (Table 1) that comprises nucleotides +1 through +16 (relative to the +1 TSS) for accurate DNA count assessment. Reverse primers were selected from the NEBNext® Multiplex Oligos for Illumina® kits (New England Biolabs), which match the Illumina Read2-primer sequence present on the HARPE plasmid. NGS PCR amplicons were then size-selected on native 6% polyacrylamide gels prior to Illumina sequencing. As described above, size-selected cDNAs or plasmid DNAs were used as templates to generate DNA amplicons for Illumina sequencing by using custom forward oligonucleotides containing the Illumina P5 and Read1-primer sequences preceding the sequence corresponding to nucleotides +1 to +16 of the promoter analyzed (Table 1). Reverse primers were selected from the NEBNext® Multiplex Oligos for Illumina® kits (NEB). NGS PCR amplicons were then size-selected on native 6% polyacrylamide gels prior to Illumina sequencing.
GEO Accession
GSM4411948