Cronn Lab:Protocols

http://openwetware.org/wiki/Random_Lab_Methods

Illumina GA Data Management

Short read toolbox. Many of our projects use short-read data from Illumina Genome Analyzer and HiSeq. Brian Knaus from our lab developed a number of scripts for managing and analyzing short-read files and data for the GA1 and GA2 platforms.

Illumina GA DNA-Seq

DNA_Seq Prep. Our research group has developed several methods for sequencing small genomes (mitochondria, chloroplasts, BACS) in multiplex using Illumina GA2. This page provides details on DNA-Seq library construction.

Illumina GA RNA-Seq

RNA_Seq Prep. We do mRNA-sequencing using methods developed by Todd Mockler's group at Oregon State University. This page provides details on RNA-Seq library construction.

Illumina GA Hyb-Seq

Hyb_Seq Prep. Like many groups, we've developed customized approaches to enrich rare genomic targets for high-throughput sequencing. Our method for isolating chloroplast genomes by Hyb-Seq is detailed here.

Whole Genome Amplification

WGA Prep. We use phi29-based whole-genome amplification in a variety of different applications. Our standard phi29 WGA method is detailed here.

Purifying DNA with Agilent AMPure Beads

AMPure_Mods. By altering the ratio of DNA:AMPure beads, it's possible to alter the size of the retained bands. We use AMPure beads to clean DNA bands, as well as reduce or eliminate the abundance of small DNAs (oligos, double-stranded adapters, primer dimers).

Random Lab Methods

Random Lab Methods. RNA extraction, DNA extraction, gels, short cuts... find it here

Rapid Isolation of RNA from Conifer Needles

Conifer_RNA_prep. Conifers join a long list of 'recalcitrant' plants that are difficult for RNA extraction, and fail using "traditional" RNA extraction kits. We use a modification of the method by Tai et al, 2004.

Isolation of poly(A) mRNA with Sera-Mag Oligo(dT) beads

mRNA_Prep. There are many methods for isolating mRNA from total RNA. We have had excellent results with Sera-Mag Oligo(dT) beads. We use this approach for constructing Illumina mRNA-Seq libraries, but it should be useful for any application that demands rRNA-depleted mRNA.

Preparation of strand-specific mRNA-Seq libraries with the Illumina TruSeq RNA Sample kit

directional-RNAseq_Prep. There are many methods available for making strand-specific mRNA libraries. We chose to adapt one of the most reliable methods identified by Levin et al. (2010) - dUTP labelling followed by dUTP degredation - for use in the Illumina TruSeq mRNA kit. This method produces mRNA-Seq libraries that are highly enriched for the complementary sequence of the native mRNA.

Rapid Isolation of DNA from Conifer Needles

DNA_Seq Prep. Conifers join a long list of 'recalcitrant' plants that are difficult for DNA extraction. We use a modification of the Fast-Prep method.