An article published in GenomeBiology presents FIPRESCI: a droplet microfluidics based indexing for multi-sample and large-scale single-cell transcriptomics profiling.
Nuclei isolationfor 5 min at 4 °C. The cell pellets were resuspended in 100–1000 µL chilled lysis buffer , 10 mM NaCl , 3 mM MgCl, 0.1% NP40 , 0.1% Tween-20 , 0.01% digitonin , 1 U/µl Sigma Protector RNase inhibitor with 1% BSA ), and mixed 10 times with a pipette. After incubation for 3–5 min on ice, 1–10 ml chilled Wash Buffer was added to the lysed cells.
for 5 min at 4 °C. The cell pellets were re-suspended in 100 µl 1 × PBS and fixed in 900 µl of ice-cold methanol at − 20 °C for 10 min. After two additional washes with 1 ml of ice-cold PBS-BSA- RNase inhibitor, the permeabilized cells were filtered through a cell strainer then re-suspended in 5–500 µl of ice-cold PBS-BSA-SUPERase and counted by using a LUNA Automated Cell Counter .
described. For 10X Genomics Chromium processing, 100,000 cells or nuclei were loaded per microfluidic channel to generate single-cell Gel Bead-In-Emulsions using Chromium Next GEM Single Cell VJ Reagent Kits v1.1 according to User Guide: CG000207 Rev E.For testing the tagmentation activity of Tn5 on RNA/DNA hybrids within permeabilized cells or nuclei, we performed a set of experiments with different 16 reaction buffers and variations according to the previously published studies .
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ACT-Discover: identifying karyotype heterogeneity in pancreatic cancer evolution using ctDNA - Genome MedicineBackground Liquid biopsies and the dynamic tracking of somatic mutations within circulating tumour DNA (ctDNA) can provide insight into the dynamics of cancer evolution and the intra-tumour heterogeneity that fuels treatment resistance. However, identifying and tracking dynamic changes in somatic copy number alterations (SCNAs), which have been associated with poor outcome and metastasis, using ctDNA is challenging. Pancreatic adenocarcinoma is a disease which has been considered to harbour early punctuated events in its evolution, leading to an early fitness peak, with minimal further subclonal evolution. Methods To interrogate the role of SCNAs in pancreatic adenocarcinoma cancer evolution, we applied whole-exome sequencing of 55 longitudinal cell-free DNA (cfDNA) samples taken from 24 patients (including 8 from whom a patient-derived xenograft (PDX) was derived) with metastatic disease prospectively recruited into a clinical trial. We developed a method, Aneuploidy in Circulating Tumour DNA (ACT-Discover), that leverages haplotype phasing of paired tumour biopsies or PDXs to identify SCNAs in cfDNA with greater sensitivity. Results SCNAs were observed within 28 of 47 evaluable cfDNA samples. Of these events, 30% could only be identified by harnessing the haplotype-aware approach leveraged in ACT-Discover. The exceptional purity of PDX tumours enabled near-complete phasing of genomic regions in allelic imbalance, highlighting an important auxiliary function of PDXs. Finally, although the classical model of pancreatic cancer evolution emphasises the importance of early, homogenous somatic events as a key requirement for cancer development, ACT-Discover identified substantial heterogeneity of SCNAs, including parallel focal and arm-level events, affecting different parental alleles within individual tumours. Indeed, ongoing acquisition of SCNAs was identified within tumours throughout the disease course, including within an untreated metastatic tumour. Conclusions T
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Six Yorkshire firms gain place in top 100 fastest-growing companies in the UKSix companies from Yorkshire and the Humber have taken a place in the Growth Index 2023, the annual ranking of the 100 fastest-growing companies in the UK.
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Estimating the risk of irreversible post-SSRI sexual dysfunction (PSSD) due to serotonergic antidepressants - Annals of General PsychiatryBackground Sexual dysfunction is a common side effect of Serotonergic antidepressants (SA) treatment, and persists in some patients despite drug discontinuation, a condition termed post-SSRI sexual dysfunction (PSSD). The risk for PSSD is unknown but is thought to be rare and difficult to assess. This study aims to estimate the risk of erectile dysfunction (ED) and PSSD in males treated with SAs. Methods A 19-year retrospective cohort analysis was conducted using a computerized database of the largest HMO in Israel. ED was defined by phosphodiesterase-5 inhibitors prescriptions. 12,302 males aged 21–49 met the following criteria: non-smokers, no medical or psychiatric comorbidities or medications associated with ED, no alcohol or substance use. Logistic regression was used for estimation of ED risk in SA-treated subjects compared to non-SA-treated controls, assessed with and without the effects of age, body mass index (BMI), socioeconomic status (SES), depression and anxiety, yielding crude and adjusted odds ratios (cOR and aOR, respectively). Results SAs were associated with an increased risk for ED (cOR = 3.6, p | 0.000001, 95% CI 2.8–4.8), which remained significant after adjusting for age, SES, BMI, depression and anxiety (aOR = 3.2, p | 0.000001, 95% CI 2.3–4.4). The risk for PSSD was 1 in 216 patients (0.46%) treated with SAs. The prevalence of PSSD was 4.3 per 100,000. Conclusions This work offers a first assessment of the small but significant risk of irreversible ED associated with the most commonly prescribed class of antidepressants which should enhance the process of receiving adequate informed consent for therapy.
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The transcriptional and regulatory identity of erythropoietin producing cells - Nature MedicineSingle-cell analyses in a reporter mouse model and human tissues identify a rare cell subset that produces erythropoietin in vivo, opening potential new avenues for research in erythropoiesis and oxygen homeostasis.
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Single duplex DNA sequencing with CODEC detects mutations with high sensitivity - Nature GeneticsConcatenating Original Duplex for Error Correction (CODEC) is a method that concatenates both strands of each DNA duplex to enable highly sensitive mutation detection in a range of analytes with fewer reads and lower error rates than current methods.
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Screening hydrogels for antifibrotic properties by implanting cellularly barcoded alginates in mice and a non-human primate - Nature Biomedical EngineeringThe throughput of the in vivo screening of hydrogels for antifibrotic properties can be increased by tagging the biomaterials with cells and reading their genotype via next-generation sequencing.
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