Energy spread of ultracold electron bunches extracted from a laser cooled gas

Ultrashort and ultracold electron bunches created by near-threshold femtosecond photoionization of a laser-cooled gas hold great promise for single-shot ultrafast diffraction experiments. In previous publications the transverse beam quality and the bunch length have been determined. Here the longitudinal energy spread of the generated bunches is measured for the first time, using a specially developed Wien filter. The Wien filter has been calibrated by determining the average deflection of the electron bunch as a function of magnetic field. The measured relative energy spread $\frac{\sigma_{U}}{U} = 0.64 \pm 0.09\%$ agrees well with the theoretical model which states that it is governed by the width of the ionization laser and the acceleration length

Automated high throughput nucleic acid purification from formalin-fixed paraffin-embedded tissue samples for next generation sequence analysis

<div><p>Curation and storage of formalin-fixed, paraffin-embedded (FFPE) samples are standard procedures in hospital pathology laboratories around the world. Many thousands of such samples exist and could be used for next generation sequencing analysis. Retrospective analyses of such samples are important for identifying molecular correlates of carcinogenesis, treatment history and disease outcomes. Two major hurdles in using FFPE material for sequencing are the damaged nature of the nucleic acids and the labor-intensive nature of nucleic acid purification. These limitations and a number of other issues that span multiple steps from nucleic acid purification to library construction are addressed here. We optimized and automated a 96-well magnetic bead-based extraction protocol that can be scaled to large cohorts and is compatible with automation. Using sets of 32 and 91 individual FFPE samples respectively, we generated libraries from 100 ng of total RNA and DNA starting amounts with 95–100% success rate. The use of the resulting RNA in micro-RNA sequencing was also demonstrated. In addition to offering the potential of scalability and rapid throughput, the yield obtained with lower input requirements makes these methods applicable to clinical samples where tissue abundance is limiting.</p></div

Automated high throughput FormaPure-based extraction protocol.

<p>(<b>A</b>) Work flow illustration of sample acquisition, upstream sample processing and extraction. Note that a separate high temperature incubation step is added to facilitate the reversal of remaining crosslinks. The upstream processes are manual in the original protocol whereas those steps are modified to be suitable for automation in the modified protocol. The in-house on-deck heating blocks were instrumental in rendering the lysis/deparaffinization steps automatable. Acquisition of samples in SBS format matrix tubes with their automated capping and decapping were also further measures that allowed the entire process to be amenable for automated liquid handling. (<b>B</b>) gDNA yield. Historical gDNA yield data from the Qiagen/High Pure protocol (Q; n = 142) using equivalent sizes of numerous FFPE samples of lymphoma origin was compared with that of the FormaPure protocol (F; n-91). (C) RNA yield. Comparison of the Qiagen-High Pure (Q-H), and FormaPure (F) protocols are shown. N = 142 for Q-H and N = 44 for F.</p

Suitability of the FormaPure extracted RNA for FFPE strand-specific RNA-seq.

<p>(<b>A)</b> Strand-specific libraries were generated from four different FormaPure extracted human FFPE samples (FFPE A-D) and UHR fresh RNA. Two different total (DNase-treated) RNA input amounts were used (100 and 200 ng, respectively). Final library yield (nM) (left panel) and % duplicates (middle panel) as well as the distribution of aligned reads to various regions of the transcriptome (right panel) are shown graphically. These libraries were sequenced as a pool at PE75 bp. (<b>B</b>) Comparison of Qiagen and FormaPure extraction protocols using mouse FFPE scrolls. Final library yield (nM) (Left panel) and % duplicates, % aligned, and the distribution of aligned reads to various regions of the transcriptome (middle panel) as well as number of genes with 1x coverage (right panel) are shown graphically.</p