Journal: RNA

Article Title: High-throughput sequencing of human plasma RNA by using thermostable group II intron reverse transcriptases

doi: 10.1261/rna.054809.115

Figure Lengend Snippet: TGIRT-seq overview. ( A ) RNA-seq library construction via TGIRT template-switching. TGIRT template-switching reverse transcription reactions use an initial template–primer substrate comprised of an RNA oligonucleotide, which contains an Illumina Read 2 primer-binding site (R2 RNA) and has a 3′-blocking group, annealed to a complementary DNA primer (R2R DNA), which leaves an equimolar mixture of A, C, G, and T (denoted N) single-nucleotide 3′ overhangs. In the protocol used in the present work, the initial R2 RNA-R2R DNA substrate was mixed with target RNA and TGIRT enzyme in the reaction medium, with the enzyme added last, and then pre-incubated for 30 min at room temperature prior to initiating reverse transcription reactions by adding dNTPs. The reactions were incubated for 15 min at 60°C and terminated by alkaline treatment, as described in Materials and Methods. The cDNA products were then purified with a MinElute Reaction Cleanup Kit (QIAGEN) and ligated at their 3′ ends to a 5′-adenylated/3′-blocked DNA oligonucleotide complementary to an Illumina Read 1 primer (R1R) by using a Thermostable 5′ AppDNA/RNA Ligase (New England Biolabs). The ligated cDNAs were repurified and amplified by PCR for 12 cycles to add Illumina flow cell capture sites (P5 and P7) and barcode sequences for sequencing. ( B ) Mapping pipeline for RNA-seq data sets constructed with TGIRT enzymes. After trimming adapter sequences and reads with low quality base calls by using cutadapt, reads of ≥18 nt were mapped by TopHat and Bowtie2 (default settings) to a human genome reference sequence (Ensembl GRCh38 release 76) supplemented with additional rRNA gene contigs and other sequences (Pass 1) (see Materials and Methods). Unmapped reads from Pass 1 were then remapped to the same human genome reference sequence using Bowtie2 local alignment (default settings) to recover reads from RNAs with post-transcriptionally added nucleotides [e.g., 3′ CCA, poly(U)] or short introns (e.g., tRNA introns; Pass 2). Concordant read pairs that mapped uniquely with MAPQ ≥15 from Passes 1 and 2 were combined and mapped to genomic features. Reads that mapped to tRNA genes were filtered and combined with the reads that remained unmapped after the Bowtie2 local alignment, and remapped to human tRNA reference sequences (UCSC genome browser website) to achieve optimal recovery and mapping of tRNA reads. tRNA reads with MAPQ ≥1 were combined with mapped genome reads from the prior steps for downstream analysis.

Article Snippet: In contrast, in the new TGIRT-seq method developed here, the cDNAs with an attached R2R adapter sequence are processed into RNA-seq libraries without size selection by ligating a 5′-adenylated (5′ App) DNA oligonucleotide containing the R1R adapter to the cDNA 3′ end with Thermostable 5′ AppDNA/RNA Ligase (New England Biolabs).

Techniques: RNA Sequencing, Reverse Transcription, Binding Assay, Blocking Assay, Incubation, Purification, Amplification, Sequencing, Construct

Journal: Scientific Reports

Article Title: A small molecule ApoE4-targeted therapeutic candidate that normalizes sirtuin 1 levels and improves cognition in an Alzheimer’s disease mouse model

doi: 10.1038/s41598-018-35687-8

Figure Lengend Snippet: APP cleavage products after 56-day treatment of E4FAD mice. ( a ) The mean for sAPPα in the Hip of A03-treated E4FAD mice was only slightly higher than that for vehicle-treated mice (AU = Arbitrary Units). ( b ) sAPPβ was not changed in the Hip by A03 treatment. ( c ) The mean for Aβ1-42 in the Hip of A03-treated mice was lower than E4FAD Veh, but not significantly so. ( d ) There was a very modest difference in the means for the sAPPα/sAPPβ ratio in the Hip. ( e ) There was a trend ( p = 0.0895) for an increase in the sAPPα/Aβ1-42 ratio in A03-treated mice. All statistical analysis was performed using a two-tailed, unpaired t-test to compare E4FAD groups only. All data graphed as the mean ± SEM.

Article Snippet: The sAPPα and sAPPβ AlphaLISAs were similar to the SirT1 AlphaLISA (see Supplementary Fig. ) but the former uses anti-sAPPα antibody AF1168 and 2B3-biotinylated antibodies and the latter an anti-sAPPβ antibody (IBL Cat. #18957) and an anti-N-terminal APP antibody (R&D Systems AF1168) instead of anti-SirT1 antibodies.

Techniques: Two Tailed Test