GSM9414924: Inr region HARPE (14nt, with DPR) - replicate 1 in vitro; Homo sapiens; OTHER
HARPE DNA/RNA extraction and processing: RNA transcripts from cells or from in vitro transcription reactions were extracted with Trizol or Trizol LS (Thermo Fisher Scientific), respectively. Total RNA (120 μg per condition for cell transfection experiments, or the entirety of each condition for in vitro transcription experiments) was processed as described in Vo ngoc et al. (2020, 2023). Briefly, the following steps were performed. First, contaminating plasmid DNA was removed with the TURBO DNA-free Kit (Thermo Fisher Scientific). Next, the RNA was subjected to reverse transcription with SuperScript III Reverse Transcriptase (Thermo Fisher Scientific) and treatment with RNase H (New England Biolabs). The resulting cDNA was size-selected on a 6% (w/v) polyacrylamide-8 M urea gel with fluorescently-labeled size markers. The size-selected cDNAs and HARPE plasmid DNAs were used as templates to generate DNA amplicons for Illumina sequencing (RNA and DNA datasets, respectively). In the cell-based HARPE experiments, the DNA datasets were obtained with plasmid DNA samples that were extracted post-transfection 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). HARPE library construction: Custom forward oligonucleotides containing the Illumina P5 and Read1-primer sequences preceding the sequences corresponding to nucleotides +19 to +34 (relative to the SCP1 +1 TSS; the SCP1 DPR and mutDPR promoter backbones required different primers) were used to amplify the randomized barcode regions. Custom forward oligonucleotides containing the Illumina P5 and Read1-primer sequences preceding the sequences corresponding to nucleotides −44 to −23 or −40 to −23 (relative to the SCP1 +1 TSS; the SCP1 TATA and mutTATA promoter backbones required different primers) were used to pair each barcode with its corresponding Inr variant. 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 and corresponding cDNA. NGS PCR amplicons were size-selected on native 6% (w/v) polyacrylamide gels prior to Illumina sequencing.