Questions tagged [rna-sequencing]

Determining the sequence of RNA. This technique is used for discovery and quantification of mRNAs and ncRNAs.

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Good poly-A filtering rules or tools

I am aligning a large number of ESTs. It seems poly-A tails show in many different ways. In addition to occurring at the very end, they can be flanked by the cloning sequence one one end, or have ...
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What does the final (Illumina) RNA-seq library contain? ss cdNA or ds cDNA?

I guess the oligos on Illumina's flow cell only hybridize to to single-stranded cDNAs (sscDNA). However, when I read the TruSeq protocol (googled: TruSeq® RNASample Preparation v2 Guide) at the PCR ...
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When to decide that two similar sequences are different genes or not?

Working on RNA-Seq data directed me to ask this question. In RNA-Seq jobs, after de novo assembly we have lots of transcripts with different rates of similarity (0 to almost 100 percent) that we ...
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783 views

Understanding Illumina Adapters

I am currently working on a project where I need to trim the adapters off of some single end read RNA-Seq data, and I want to know which sequences to cleave. Illumina TruSeq adapters were used. I ...
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33 views

Gene expression for mouse feeder cells (inactivated MEFs)

I'm looking for a paper with gene expression data for mouse feeder cells, inactivated by gamma radiation or mitomycin C. Ideally I'd like RNA-seq data but I'll use microarray data if that's all there ...
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Too few transcripts from transcriptome assembler Oases

I am trying to run Oases for transcriptome assembly. The result is far from expected, so I would like to ask whether I am running it in a right way? Thanks. Here is my running command: ...
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35 views

How to map the N6-dimethyladenosine by primer extension?

I am trying to map N6-dimethyladenosine on rRNA using primer extension (low dNTPs assay) method. But i am not able to detect map my position. I went through some articles they mentioned like they ...
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How many samples to run an association study on B-Cell receptors

We are trying to find biomarkers on the B-Cell receptor VDJ regions. To do this we plan to run Single cell RNA-Seq (using the solution from 10x genomics https://www.10xgenomics.com/solutions/vdj/) to ...
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Should I use glycogen in isopropanol to precipitate RNA or does glycogen have harmful consequences on downstream protocols such as RNA seq?

I have heard that one can use glycogen in isopropanol to better visualise the RNA pellet after centrifugation. What are the advantages and disadvantages of adding glycogen in isopropanol for RNA ...
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227 views

Is DNase treatment necessary before RNA sequencing?

Is it necessary to treat total RNA with DNase before sequencing it? In particular, if the library prep relies on a poly-A enrichment, is it necessary to remove DNA, knowing that genomic DNA does not ...
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22 views

RNA extraction from soil

I have been trying to extract RNA from soil without success. I have used commercial kits (The Plant DNeasy kit) because I did not want to buy a complete new kit (Soil) to do 5 extractions. Is there ...
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75 views

The advantages of using a strand specific cDNA in RNA-Seq?

May I ask what are the advantages of using a strand specific cDNA library in an RNA-seq experiment? as far as I know it can make us able to read the code region but what about other advantages?
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Blastx: from gi to description

I have done a blastx and use the outfmt 6 option, because I want to work in the tables format. When I do this I get subject ids of the type sp|Q7XJS0|ASHR1_ARATH, sp|Q39547|CUCM1_CUCME, et cetera....
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RNA-Seq library normalization and experimental setup

I'm working on an RNA-Seq project and I am trying to figure out library normalization. I'm aware of using the geometric means (e.g. cuffdiff) of the fpkm for the normalization. However, I was ...
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88 views

Can we take advantage of nanopore sequencing systematic errors to predict secondary structure motifs?

One of the methods of single-molecule sequencing, Nanopore sequencing, is based on traversal of DNA strand through a nanopore. Nucleotide is determined by measurement of ion current (when nucleotide ...
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Getting read size less then specified in parameter file Flux Simulation

I have tried to use flux simulation tool to generate simulated RNA-seq data. I gave the following parameter file to flux-simulation shell script ...
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3 end RNA seq library construction

I have tried to make cDNA libraries from FFPE tumor blocks (highly degraded RNA) using the 3' end RNA seq protocol provided by West lab. Here is the link for the same. http://med.stanford.edu/labs/...
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558 views

Is the default kmer 25 value in Trinity still ok for assembly of 150bp reads from Illumina HiSeq 2500?

I will soon be receiving Illumina HiSeq 2500 data (150bp PE stranded reads). In the past we have been using Trinity as our assembler of choice, but it uses a default kmer value of 25. I believe this ...
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59 views

RT-qPCR of selected differential expressed genes: which dilutions in calibration curve?

I've seen in MIQE guidelines that calibration curves a needed when performing RT-qPCR. I'll use genomic DNA to perform these calibration curves. My question is that I don't know the range of ...
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How to map A single N1-methyladenosine (m1A) by primer extension?

I want to map a single N1-methyladenosine (m1A) modification by primer extension. I have silenced a gene which is responsible for guiding modification of m1A then I confirmed the silencing. My next ...
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26 views

How to reduce the number of sequences in a Multiple Sequence Alignment?

I have a Multiple Sequence Alignment, where there are around 5000 sequences. There also exists many sequences where, there are so much of non-sequenced regions (for instance, AU----CGGGCA--NNNNNNNNNN)....
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1answer
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Can Cas13 be used with multiple crRNAs in the same reaction?

CRISPR-Cas13 equipped with crRNA (complementary to transcripts of interest) can be designed to target ssRNA transcripts in cells. Upon successful crRNA and ssRNA binding, a fluorescent domain on ...
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What is the advantages of using single primer in PCR ( Being found in library preparation step of Smart-seq2)

I am curious about the advantages of using single primer in PCR. I found this strategy in single cell sequencing method Smart-seq 2. Will it raise the yield of PCR? If so, Why? Does anybody have ...
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How to differentiate transportable element and non-coding RNA in genome?

I am searching for unannotated non-coding RNA in my genome at the same time i want to omit the transposable element from my genome. My sequencing libraries are paired-end and i am searching in the IGV ...
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35 views

Why do the RNAseq reads map to only a certain region of a viral genome?

I am looking at RNAseq data and mapping them to 3 RNA segments from Cucumber Mosaic Virus. I trimmed the adapters from the fastq files and converted them to fasta, which I searched against a virus ...
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Is it okay to scale the RNA-Seq counts after log-transformation?

On an RNA-Seq dataset, I want to apply a clustering algorithm which requires zero-mean uni-variance gene expression levels. I am wondering whether it makes sense to use "scale" function in R after ...