Simultaneous mapping of RNA ends (SMORE-seq) provides a method for determining the 5′ ends of transcripts (38 (link)). This technique allows for precise mapping of transcription start sites, and, can also allow the detection of short antisense RNAs that are difficult to detect by standard RNA-seq methods. In particular, this technique can be used to identify antisense transcripts arising from between tandemly arranged genes, which we term UAN-RNAs (for sites of (promoter) u pstream a ntisense n on-coding RNA transcription). SMORE-seq was performed as previously described (38 (link)). Briefly, RNA was incubated with tobacco acid pyrophosphatase (TAP, Epicentre) to remove 5′ caps. TAP was inactivated by heating at 65°C and RNA was purified. A control reaction omitting TAP was also carried out. Purified RNAs were then used for construction of Illumina RNA-seq libraries using the NEBNext Small RNA kit following manufacturer's instructions and as previously described (38 (link)). For our analysis, we combined data from our previously published study (GSE49026) with newly generated datasets.
Nebnext small rna kit
Manufactured by Illumina
The NEBNext Small RNA kit is a laboratory equipment product designed for the preparation and analysis of small RNA samples. It provides a streamlined workflow for the isolation, library preparation, and sequencing of small RNA molecules such as microRNAs, siRNAs, and other non-coding RNAs. The kit includes the necessary reagents and protocols to efficiently process small RNA samples for downstream applications.
Automatically generated - may contain errors
4 protocols using nebnext small rna kit
1
Simultaneous Mapping of RNA Ends (SMORE-seq)
2
Ribosome Methylation Sequencing Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For RiboMethSeq, 150 ng polysomal RNA was fragmented under denaturing conditions using an alkaline buffer (pH 9.4) to obtain an average size of 20 to 40 nt. Fragments were end-repaired and ligated to adapters using NEBNext Small RNA kit for Illumina. Sequencing was performed on Illumina HiSeq1000. Reads were mapped to the yeast rDNA sequences, and the RMS score (fraction methylated) was calculated as MethScore (for ±2 nt) (88), equivalent to “ScoreC” (30 ).
3
Sequencing of Circulating miRNAs from Serum
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA, including miRNAs, was extracted from 600 µL serum using the MirVana Paris Kit (Ambion). From this, the miRNA libraries were prepared using the NEBNext smallRNA kit for Illumina (E7330L) following the manufacturer’s instructions. Size selection was carried out using Blue Pippin. Individual libraries were QC’d using Tapestation (Agilent) before being pooled and sequenced on Hiseq2500 (Illumina) at the Oxford Genomics Centre (Wellcome Centre for Human Genetics, University of Oxford). For analysis, read1 of the FASTQ was trimmed using fastx_clipper (https://github.com/agordon/fastx_toolkit ) and aligned using Bowtie2 (Langmead & Salzberg 2012 (link)) to GRCh37, and miRNA counts were obtained using htseq-count (Anders et al. 2015 (link)) against the annotation from miRBase v20. The raw gene count matrix was imported into the R/BioConductor environment (https://www.r-project.org/ ; Huber et al. 2015 (link)) for further processing and analysis with the edgeR package (Robinson et al. 2010 (link), McCarthy et al. 2012 (link)). Genes with very low expression (i.e. those with ≤10 reads, after normalising for library size, in the four-paired samples of the test set) were excluded. Multiple testing correction was performed by using edgeR’s default Benjamini–Hochburg method for controlling the false discovery rate.
4
Quantitative RNA Methylation Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RiboMethSeq was essentially performed as previously reported (32 (link)). Briefly, 150 ng of total RNA was fragmented under denaturing conditions using an alkaline buffer (pH 9.4) to obtain an average size of 20 to 40 nucleotides. Fragments were end repaired and ligated to adapters using a NEBNext Small RNA kit for Illumina. Sequencing was performed on an Illumina HiSeq1000. Reads were mapped to the yeast rDNA and snoRNA sequences, and the MethScore (fraction methylated) was calculated as MethScore (for ±2 nucleotides) (93 (link)), equivalent to “ScoreC” in Birkedal et al. (31 (link)). Statistical significance was determined by Student’s t test (P < 0.05).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!