Journal: STAR Protocols

Article Title: CapZyme-Seq: A 5′-RNA-Seq Method for Differential Detection and Quantitation of NAD-Capped and Uncapped 5′-Triphosphate RNA

doi: 10.1016/j.xpro.2019.100002

Figure Lengend Snippet:

Article Snippet: Micellula DNA Emulsion PCR Kit , Chimerx , Cat#3600-02.

Techniques: Recombinant, Staining, Reverse Transcription, Sequencing, Illumina Sequencing, Emulsion

Journal: Nucleic Acids Research

Article Title: Identifying high-affinity aptamer ligands with defined cross-reactivity using high-throughput guided systematic evolution of ligands by exponential enrichment

doi: 10.1093/nar/gkv534

Figure Lengend Snippet: Convergence of the in vitro selection begins at round 5. ( A ) Schematic representation of in vitro selection process and its key improvements: at amplification step we employed emulsion PCR to preserve variability of the selected library by physical separation of amplification of individual DNA molecules; at fractionation step we introduced parallel binding there identical aliquotes of the same library had been reacted with different targets (depicted in black), for example (H)uman protein, (M)urine ortholog of it and (C)ontrol, to deduce aptamers’ binding patterns in highly parallel format. Bound RNA portions from hIL-10 SELEX rounds 2–5 were isolated, mass sequenced and analyzed for cluster formation. ( B ) Fractions of single copy aptamers (unenriched, open bars), aptamers with multiple copies but did not belonging to any cluster (unclustered, gray bars) and aptamers belonging to a cluster plotted (clustered, black bars). ( C ) Total number of individual clusters (blue bars, left Y axis) and average number of members per cluster (red bars, right Y axis) in each round.

Article Snippet: After preparing the master mix PCR reaction solution, it was separated into 100-μl aliquots, and each aliquot was mixed with 600 μL of ice-cold oil fraction assembled from components supplied in the emulsion PCR kit (EuRx Ltd, Gdansk, Poland), according to the manufacturer's instructions.

Techniques: In Vitro, Selection, Amplification, Emulsion, Fractionation, Binding Assay, Isolation

Journal: bioRxiv

Article Title: Functional regulatory variants implicate distinct transcriptional networks in dementia

doi: 10.1101/2021.06.14.448395

Figure Lengend Snippet: Characterization of technical features impacting MPRA performance (Supplemental Text). ( A ) Assessment of sequence-level features of the 366 variants (“Dropout”) that failed to pass quality control thresholds through both MPRA experimental stages (Methods). Violin plots display GC-content, CpG content, sequence complexity measures (-log 2 compressibility and log 2 Shannon’s entropy), predicted secondary structure (calculated using RNAfold), and longest mono- or di-nucleotide repeat per tested oligo for the 366 dropout variants vs. a random sample of 366 variants that passed QC (“Tested”). Dropout variants were enriched for GC content extremes (>75% or <25%), increased CpG skew, had decreased mean sequence complexity, and on average contained longer runs of nucleotide repeats (mean 13.2 vs 7.2 nucleotides) (2-tailed Mann-Whitney-U test). Predicted secondary structure did not differ for dropout variants (p > 0.05). ( B ) Simulated power analysis, using MPRA-determined empirical variance and different quantiles of empirical effect sizes, revealed that adequate power (0.8) to detect significant allelic skew at a broad range of effect-sizes is achieved at a barcode complexity of ∼40 barcodes/allele. ( C ) Plots mean correlations (Spearman’s rho) of inter-replicate allele expression for four separate MPRA experiments. Inter-replicate reproducibility is inversely proportional to the number of PCR cycles needed to amplify library mRNA before sequencing.

Article Snippet: Step 1: We amplified and attached 20 bp degenerate barcodes to the oligo library by emulsion PCR (Chimerx, 3600-01).

Techniques: Sequencing, MANN-WHITNEY, Expressing

Journal: bioRxiv

Article Title: Functional regulatory variants implicate distinct transcriptional networks in dementia

doi: 10.1101/2021.06.14.448395

Figure Lengend Snippet: Technical schematic for massively parallel reporter assay (MPRA). ( A ) We selected all bi-allelic noncoding genome-wide significant (p > 5 × 10 -8 ) or locus lead SNPs and LD partners (r 2 > 0.8) from three GWAS (Methods). ( B ) The reference and alternate alleles of each variant were centered within 162 bp of genomic context (hg19) and synthesized on an Agilent array. ( C ) Oligos were amplified by PCR using primers complementary to flanking shared adaptor sequences. This step also attached 20 nucleotide random barcodes and restriction enzyme sites. ( D ) Oligos were digested and cloned into an expression library, followed by paired-end 2x150 bp sequencing to map barcodes to associated oligos. ( E ) A minimal promoter and eGFP gene was inserted between the oligo and barcode, with the oligo upstream of the minimal promoter and the barcode in the 3’UTR of eGFP. ( F ) The expression library was transfected into HEK293T cells for 24 hours. ( G ) following mRNA extraction, mRNA and plasmid barcodes were amplified, pooled, and sequenced using indexed single-end sequencing. ( H ) mRNA barcode counts were normalized to DNA counts, and the median normalized barcode count was taken as an activity summary score for each allele.

Article Snippet: Step 1: We amplified and attached 20 bp degenerate barcodes to the oligo library by emulsion PCR (Chimerx, 3600-01).

Techniques: Reporter Assay, Genome Wide, Variant Assay, Synthesized, Amplification, Clone Assay, Expressing, Sequencing, Transfection, Plasmid Preparation, Activity Assay