Journal: Cell Reports

Article Title: B cell receptor repertoire kinetics after SARS-CoV-2 infection and vaccination

doi: 10.1016/j.celrep.2022.110393

Figure Lengend Snippet:

Article Snippet: KAPA Hyper Prep Kit , KAPA Biosystems , Cat# KK8502, Cat# KK8503.

Techniques: Recombinant, Library Amplification, Flow Cytometry, Cell Counting, Software

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: HyPR-seq: Single-cell quantification of chosen RNAs via hybridization and sequencing of DNA probes

doi: 10.1073/pnas.2010738117

Figure Lengend Snippet: HyPR-seq enables single-cell quantification of selected RNAs. (A) At a probe binding site, initiator probes bind adjacent to one another on an RNA of interest, creating a binding site for a hairpin oligo. A final readout oligo is annealed and ligated, forming a template for PCR amplification containing transcript information, UMI, and handles for PCR and sequencing. (B) Experimental workflow for HyPR-seq. (C) Comparison of three single-cell methods in K562 cells. (Top) HCR measurements of GAPDH and GATA1 using probes targeting the same binding sites used for HyPR-seq. (Middle) Histogram of UMIs per cell for three probes targeting GAPDH and GATA1 from HyPR-seq. (2,148 cells) and (Bottom) 10X Genomics Chromium 3′ scRNA-seq (207,324 cells). (Scale bar, 10 μm.) All experiments performed in K562 cells. (D) HyPR-seq counts per cell for each of the three probes targeting GAPDH (left columns), GATA1 (center columns), or GFP (not expressed, right side). Error bars represent 95% confidence interval (CI) of the mean from two replicates (from 4,605 cells). For more analysis of probe variability, See SI Appendix, Fig. S4D. (E) Counts per cell at a constant depth of 1,000 total UMIs per cell for three genes (GATA1, PGK1, and TUBB) as assayed by 10X Genomics Chromium 3′ scRNA-seq (26) and HyPR-seq. Counts for HyPR-seq are shown for the 22-gene experiment in K562 cells and 2 simulated experiments (based on these counts) measuring different numbers of total genes. Horizontal bars show the caps of the (vertical) error bars, which represent 95% CI of the mean from two replicates. (F) Counts per cell for 19 genes in K562 cells as measured by 10X Genomics Chromium 3′ scRNA-seq (x axis) and HyPR-seq (y axis). Error bars for HyPR-seq measurements show 95% CI of the mean from two replicates (4,605 total cells). (G) In THP1 cells ± LPS, fold changes of 18 genes assayed by bulk RNA-seq (x axis) and HyPR-seq (y axis). Error bars represent 95% CI of the mean from three (RNA-seq) or four (HyPR-seq) replicates (13,957 total cells). (H) To assess mixing, K562 cells were transduced to express two different barcode RNAs, each detectable by three HyPR probes. Scatterplot shows UMI counts for each barcode per droplet (2,727 total droplets). Unassigned cells are in gray.

Article Snippet: Once the droplets were formed, the cartridge containing the emulsions was placed under the UV lamp (6.5 J/cm2 at 365 nm) about 3 to 8 cm away from the bulb for 5 min. After UV exposure, ∼50 µL of droplets per well was transferred to 96-well plates (Eppendorf #951020362) and sealed with foil (Bio-Rad #181-4040) using a plate sealer (PX1 Plate Sealer #181-4000) for droplet PCR amplification (Eppendorf Mastercycler Pro #E90030010).

Techniques: Binding Assay, Amplification, Sequencing, RNA Sequencing Assay

Journal: International Journal of Molecular Sciences

Article Title: Impact of DNA Extraction and 16S rRNA Gene Amplification Strategy on Microbiota Profiling of Faecal Samples

doi: 10.3390/ijms26115226

Figure Lengend Snippet: DNA concentration of amplicons obtained during bacterial library preparations with two protocols, home brew and VeriFi. Amplicons were quantified by Nano Drop ® ND-1000. Median values are indicated by the line in the box plot. The box extends from the 25th to the 75th percentile and whiskers indicate the minimum and maximum values. t -test was used to calculate p -values ( p -value *); ( A ) box plot showing DNA concentrations obtained during the amplification of the 16S rRNA V3–V4 regions, p -value = 0.026; ( B ) box plot showing DNA concentrations obtained during the barcoding step using Nextera TM primers, p -value = 1.6 × 10 −9 .

Article Snippet: VeriFi Library Amplification Mix for NGS Kit (PCR Biosystems simplifying research, London, UK, named VeriFi Kit) : The first PCR reaction was performed using the following reaction mixture: 1X VeriFiTM Hot Start Polymerase, 3 mM MgCl 2 , 1 mM dNTPs (PCR Biosystems simplifying research, London, UK), and 10 μM of each primer mix.

Techniques: Concentration Assay, Amplification

Journal: International Journal of Molecular Sciences

Article Title: Impact of DNA Extraction and 16S rRNA Gene Amplification Strategy on Microbiota Profiling of Faecal Samples

doi: 10.3390/ijms26115226

Figure Lengend Snippet: Sequencing parameters of amplicons obtained using the two protocols of amplification, home brew and VeriFi. The box plots report the median value and the 25th and the 75th percentiles. Wilcoxon rank test was used to calculate p -values. ( A ) Box plot showing the number of reads per sample obtained for the three kits evaluated. ( B ) Figure showing the ASV frequencies for the three kits evaluated. ( C ) Box plot showing the number of chimeras obtained for the three kits evaluated.

Article Snippet: VeriFi Library Amplification Mix for NGS Kit (PCR Biosystems simplifying research, London, UK, named VeriFi Kit) : The first PCR reaction was performed using the following reaction mixture: 1X VeriFiTM Hot Start Polymerase, 3 mM MgCl 2 , 1 mM dNTPs (PCR Biosystems simplifying research, London, UK), and 10 μM of each primer mix.

Techniques: Sequencing, Amplification

Journal: International Journal of Molecular Sciences

Article Title: Impact of DNA Extraction and 16S rRNA Gene Amplification Strategy on Microbiota Profiling of Faecal Samples

doi: 10.3390/ijms26115226

Figure Lengend Snippet: Analysis of alpha and beta diversity. Alpha diversity was based on the Shannon ( A ) and Simpson ( B ) indices. The pairwise Wilcoxon rank test, used to compare the two protocols of amplification, home brew and VeriFi, indicated no statistically significant differences. Beta diversity was calculated using the Bray–Curtis distance and represented by PCoA plot ( C ). PERMANOVA test is statistically significant, p -value = 0.035, R 2 = 0.082. The Bray–Curtis distance was represented by box plot ( D ). **, p value < 0.001.

Article Snippet: VeriFi Library Amplification Mix for NGS Kit (PCR Biosystems simplifying research, London, UK, named VeriFi Kit) : The first PCR reaction was performed using the following reaction mixture: 1X VeriFiTM Hot Start Polymerase, 3 mM MgCl 2 , 1 mM dNTPs (PCR Biosystems simplifying research, London, UK), and 10 μM of each primer mix.

Techniques: Amplification

Journal: International Journal of Molecular Sciences

Article Title: Impact of DNA Extraction and 16S rRNA Gene Amplification Strategy on Microbiota Profiling of Faecal Samples

doi: 10.3390/ijms26115226

Figure Lengend Snippet: Protocol-dependent variations in the relative abundance of microbiota profiles at the phylum ( A , B ) and genus ( C , D ) levels. ( A , C ) Histograms of the relative abundances of the 7 most abundant phyla and 20 most abundant genera obtained by the sequencing of amplicons obtained during bacterial library preparations with two protocols, home brew and VeriFi. ( B , D ) Heatmap showing, for each sample, the relative abundance of the 7 most abundant phyla and 20 most abundant genera for each sample.

Article Snippet: VeriFi Library Amplification Mix for NGS Kit (PCR Biosystems simplifying research, London, UK, named VeriFi Kit) : The first PCR reaction was performed using the following reaction mixture: 1X VeriFiTM Hot Start Polymerase, 3 mM MgCl 2 , 1 mM dNTPs (PCR Biosystems simplifying research, London, UK), and 10 μM of each primer mix.

Techniques: Sequencing

Journal: Journal of Comparative Physiology. B, Biochemical, Systemic, and Environmental Physiology

Article Title: Noradrenergic tone is not required for neuronal activity-induced rebound sleep in zebrafish

doi: 10.1007/s00360-023-01504-6

Figure Lengend Snippet: Key resources

Article Snippet: Nuclease-free water for RNA isolation and for PCR amplification for MiSeq , Omega Bio-tek , S1392200.

Techniques: Concentration Assay, Nucleic Acid Electrophoresis, Isolation, Amplification, Reverse Transcription, Blocking Assay, Activity Assay, SYBR Green Assay, Fluorescence, Suspension, CRISPR, Recombinant, Injection, Sequencing, Negative Control, Software, Spectrophotometry