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SRX24605986: GSM8279794: BT474, Illumina, sRNA-seq, rep2; Homo sapiens; ncRNA-Seq
1 ILLUMINA (Illumina NovaSeq 6000) run: 20M spots, 2G bases, 690Mb downloads

External Id: GSM8279794_r1
Submitted by: Dr. Duttke Lab, School of Molecular Biosciences, Washington State University
Study: Efficient small DNA fragment sequencing and miRNA,?small RNA or csRNA-seq libraries using AVITI
show Abstracthide Abstract
Next-Generation Sequencing (NGS) catalyzed breakthroughs across various scientific domains. Illumina's sequencing by synthesis method has long been essential for NGS but emerging technologies like Element Biosciences' sequencing by avidity (AVITI) represent a novel approach. It has been reported that AVITI offers improved signal-to-noise ratios and cost reductions. However, the method relies on rolling circle amplification which can be impacted by polymer size, raising questions about its efficacy sequencing small RNAs (sRNAs) libraries including microRNAs (miRNAs), piwi-interacting RNAs (piRNAs), and others that are crucial regulators of gene expression and involved in various biological processes. In addition, capturing capped small RNAs (csRNA-seq) has emerged as a powerful method to map active or “nascent” RNA polymerase II transcription initiation in tissues and clinical samples. Finally, we present an accelerated and optimized protocol for small fragment sequencing with AVITI that aligns seamlessly with available sRNA library kits to meet this need. Overall design: Capped small (cs)RNA-sequencing for human A375 and BT474 cells, as well as blood samples from cattle and bison using Illumina and AVITI sequencing.
Sample: BT474, Illumina, sRNA-seq, rep2
SAMN41455494 • SRS21343556 • All experiments • All runs
Organism: Homo sapiens
Library:
Name: GSM8279794
Instrument: Illumina NovaSeq 6000
Strategy: ncRNA-Seq
Source: TRANSCRIPTOMIC
Selection: size fractionation
Layout: SINGLE
Construction protocol: Cells were washed once with ice cold DPBS (Gibco), rested on ice for 5 minutes, washed one more time with ice cold DPBS and then lysed in 1 ml TROZOL. RNA isolated as described by the manufacturer. csRNA-seq was performed as described in (S. H. Duttke et al. 2019). Small RNAs of ~20-60 nt were size selected from 0.4-3 µg of total RNA by denaturing gel electrophoresis. A 10% input sample was taken aside and the remainder enriched for 5'-capped RNAs. Monophosphorylated RNAs were selectively degraded by 1 hour incubation with Terminator 5'-Phosphate-Dependent Exonuclease (Lucigen). Subsequently, RNAs were 5'dephosphorylated through 90 minutes incubation in total with thermostable QuickCIP (NEB) in which the samples were briefly heated to 75°C and quickly chilled on ice at the 60 minutes mark. Input (sRNA) and csRNA-seq libraries were prepared as described in (Hetzel et al. 2016) using RppH (NEB) and the NEBNext Small RNA Library Prep kit, amplified for 11-14 cycles.
Runs: 1 run, 20M spots, 2G bases, 690Mb
Run# of Spots# of BasesSizePublished
SRR2908177920,042,8702G690Mb2024-06-26

ID:
32924748

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