Journal: Cold Spring Harbor Perspectives in Biology

Article Title: Group II Intron RNPs and Reverse Transcriptases: From Retroelements to Research Tools

doi: 10.1101/cshperspect.a032375

Figure Lengend Snippet: Three methods of using thermostable group II intron reverse transcriptase (TGIRT) enzymes for RNA-seq library construction (TGIRT-seq). (A) TGIRT-seq of eukaryotic mRNAs using an anchored oligo(dT)42 primer for initiation of cDNA synthesis. The primer of sufficient length to stably anneal to mRNA poly(A) tails at high temperature is extended by TGIRTs at 60°C to synthesize cDNA copies of the mRNAs. After second-strand synthesis, dsDNAs are fragmented and RNA-seq adapters are added by conventional methods, such as the transposon-based Illumina Nextera method. (B) TGIRT-seq Small RNA/CircLigase method. The TGIRT initiates by template switching from an initial template-primer substrate comprised of an RNA oligonucleotide containing Illumina R1 and R2 adapter sequences annealed to a 5′-labeled DNA primer that leaves a single-nucleotide 3′ overhang (N, an equimolar mixture of A, C, G, and T) that can base-pair to the 3′ nucleotide of target RNAs. The single base-pair is sufficient to direct TGIRT template switching, even at 60°C (Mohr et al. 2013b; Qin et al. 2016). After reverse transcription, cDNAs are purified on a denaturing polyacrylamide gel to select specific size classes, circularized with CircLigase (Epicentre), and minimally polymerase chain reaction (PCR) amplified with primers that add capture sites and barcodes for Illumina sequencing. (C) TGIRT Total RNA-seq method for construction of comprehensive RNA-seq libraries without size selection. The TGIRT initiates from an initial template-primer substrate similar to that in panel B but containing only an Illumina R2 adapter sequence. After reverse transcription, cDNAs are cleaned up by using a MinElute column to remove unincorporated adapters and a second oligonucleotide containing the reverse complement of an Illumina R1 adapter is ligated to the 3′ end of the cDNA using thermostable 5′ AppDNA/RNA ligase (New England Biolabs). After an additional MinElute cleanup, the cDNAs with adapters on both ends are amplified by PCR with primers that add Illumina capture sites and barcodes. Before sequencing, the libraries are cleaned up using Ampure beads to remove adapter dimers (not shown).

Article Snippet: In this variation, the initial template/primer substrate used for template switching contains only an Illumina R2 sequence, and after cDNA synthesis a second adapter containing the reverse complement of an Illumina R1 sequence is ligated to the 3′ end of the cDNA by an efficient single-stranded DNA ligation ( Nottingham et al. 2016 ; Qin et al. 2016 ).

Techniques: RNA Sequencing Assay, Stable Transfection, Labeling, Purification, Polymerase Chain Reaction, Amplification, Sequencing, Selection

Journal: Sports

Article Title: CrossFit ® : ‘Unknowable’ or Predictable?—A Systematic Review on Predictors of CrossFit ® Performance

doi: 10.3390/sports11060112

Figure Lengend Snippet: Overview in predictors of performance outcomes for CrossFit ® athletes.

Article Snippet: , , , , 2016 CrossFit ® Open 16.5 , R 2 = 0.94.

Techniques: Modification