RNA Sequencing - Revision history

RTEvans at 16:48, 1 November 2023

2023-11-01T16:48:35Z

← Older revision		Revision as of 16:48, 1 November 2023
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	#Extract and purify RNA samples according to [[RNA extraction from Actinobacteria]]. Add [https://www.thermofisher.com/order/catalog/product/4456740#/4456740 ERCC RNA Spike-In Mix] if necessary.		#Extract and purify RNA samples according to [[RNA extraction from Actinobacteria]]. Add [https://www.thermofisher.com/order/catalog/product/4456740#/4456740 ERCC RNA Spike-In Mix] if necessary.
	#Check the quality of RNA samples using [https://www.chem.agilent.com/store/productDetail.jsp?catalogId=5067-1511&catId=SubCat2ECS_227716 Agilent RNA 6000 Nano Kit] or by running them on an agarose gel.		#Check the quality of RNA samples using [https://www.chem.agilent.com/store/productDetail.jsp?catalogId=5067-1511&catId=SubCat2ECS_227716 Agilent RNA 6000 Nano Kit] or by running them on an agarose gel.
	#Deplete rRNA using [https://www.illumina.com/products/by-type/accessory-products/ribo-zero-plus-rrna-depletion.html Illumina Ribo-Zero Plus rRNA Depletion Kit] or a similar product		#Deplete rRNA using [https://www.illumina.com/products/by-type/accessory-products/ribo-zero-plus-rrna-depletion.html Illumina Ribo-Zero Plus rRNA Depletion Kit] or a similar product, or using the [[rRNA depletion with Streptomyces coelicolor A3(2) specific probes]] protocol.
	#Create Illumina RNA-seq libraries using [https://www.illumina.com/products/by-type/sequencing-kits/library-prep-kits/truseq-rna-v2.html TruSeq RNA Library Prep Kit].		#Create Illumina RNA-seq libraries using [https://www.illumina.com/products/by-type/sequencing-kits/library-prep-kits/truseq-rna-v2.html TruSeq RNA Library Prep Kit].
	#Sequence the libraries using an Illumina system such as [https://www.illumina.com/systems/sequencing-platforms/novaseq.html NovaSeq 6000 Sequencing System].		#Sequence the libraries using an Illumina system such as [https://www.illumina.com/systems/sequencing-platforms/novaseq.html NovaSeq 6000 Sequencing System].

H Otani at 00:18, 25 March 2020

2020-03-25T00:18:08Z

← Older revision		Revision as of 00:18, 25 March 2020
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			==Instructions==

			This procedure is an example and the optimal library preparation and data analysis procedures can be different depending on the type of the DNA sequencer used and purpose of the experiment.

	#Extract and purify RNA samples according to [[RNA extraction from Actinobacteria]]. Add [https://www.thermofisher.com/order/catalog/product/4456740#/4456740 ERCC RNA Spike-In Mix] if necessary.		#Extract and purify RNA samples according to [[RNA extraction from Actinobacteria]]. Add [https://www.thermofisher.com/order/catalog/product/4456740#/4456740 ERCC RNA Spike-In Mix] if necessary.
	#Check the quality of RNA samples using [https://www.chem.agilent.com/store/productDetail.jsp?catalogId=5067-1511&catId=SubCat2ECS_227716 Agilent RNA 6000 Nano Kit] or by running them on an agarose gel.		#Check the quality of RNA samples using [https://www.chem.agilent.com/store/productDetail.jsp?catalogId=5067-1511&catId=SubCat2ECS_227716 Agilent RNA 6000 Nano Kit] or by running them on an agarose gel.

H Otani: Created page with "#Extract and purify RNA samples according to RNA extraction from Actinobacteria. Add [https://www.thermofisher.com/order/catalog/product/4456740#/4456740 ERCC RNA Spike-In..."

2020-03-25T00:07:56Z

Created page with "#Extract and purify RNA samples according to RNA extraction from Actinobacteria. Add [https://www.thermofisher.com/order/catalog/product/4456740#/4456740 ERCC RNA Spike-In..."

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#Extract and purify RNA samples according to [[RNA extraction from Actinobacteria]]. Add [https://www.thermofisher.com/order/catalog/product/4456740#/4456740 ERCC RNA Spike-In Mix] if necessary.
#Check the quality of RNA samples using [https://www.chem.agilent.com/store/productDetail.jsp?catalogId=5067-1511&catId=SubCat2ECS_227716 Agilent RNA 6000 Nano Kit] or by running them on an agarose gel.
#Deplete rRNA using [https://www.illumina.com/products/by-type/accessory-products/ribo-zero-plus-rrna-depletion.html Illumina Ribo-Zero Plus rRNA Depletion Kit] or a similar product
#Create Illumina RNA-seq libraries using [https://www.illumina.com/products/by-type/sequencing-kits/library-prep-kits/truseq-rna-v2.html TruSeq RNA Library Prep Kit].
#Sequence the libraries using an Illumina system such as [https://www.illumina.com/systems/sequencing-platforms/novaseq.html NovaSeq 6000 Sequencing System].
#Trim low quality subsequences from reads using [http://hannonlab.cshl.edu/fastx_toolkit/ FASTX-Toolkit] (single-end) or [https://research.bioinformatics.udel.edu/genomics/ngsShoRT/ ngsShoRT] (single- or paired-end).
#Align reads with the reference genome using [http://daehwankimlab.github.io/hisat2/ HISAT2] or [http://bowtie-bio.sourceforge.net/bowtie2/index.shtml Bowtie2].
#Convert the SAM files into BAM files and sort and index the BAM files using [http://www.htslib.org/ Samtools].
#Count the number of reads overlapping with each feature such as CDS or tRNA gene using [http://subread.sourceforge.net/ featureCounts] or [https://htseq.readthedocs.io/en/master/ HTSeq].
#Perform a differential gene expression analysis using a Bioconductor package such as [https://bioconductor.org/packages/release/bioc/html/DESeq2.html DESeq2].