Journal: Molecular microbiology

Article Title: Mycobacteriophage Fruitloop gp52 inactivates Wag31 (DivIVA) to prevent heterotypic superinfection

doi: 10.1111/mmi.13946

Figure Lengend Snippet: RNA was isolated from three samples, a Fruitloop lysogen of M. smegmatis, and a culture of M. smegmatis infected with Fruitloop at a multiplicity of infection of three, either 30 mins or 150 mins are infection (as indicated on the left). Following strand-specific RNAseq analysis, sequence reads were mapped to the Fruitloop genome (shown below); lysogen reads are shown in green, 30 mins after infection in blue, and 150 mins after infection are shown in red. Reads mapping to the forward and reverse strands are indicated on the right. Note that different scales are used to display the different samples, 0–500 reads for the lysogen, 0–2000 reads for the 30 mins infection sample, and 0–5000 reads for the 150 mins infection sample, as indicated. The positions of several genes are indicated below the genome map.

Article Snippet: Following the removal of DNA with a TURBO DNA-free kit (Ambion) and removal of rRNA using a RiboZero rRNA removal kit (Gram-positive bacteria, Illumina), RNA was library prepped using the strand specific TruSeq RNAseq kit (Illumina) according to the manufacturer’s instructions, as described previously ( Dedrick et al. , 2017 ).

Techniques: Isolation, Infection, Sequencing

Journal: Molecular microbiology

Article Title: Mycobacteriophage Fruitloop gp52 inactivates Wag31 (DivIVA) to prevent heterotypic superinfection

doi: 10.1111/mmi.13946

Figure Lengend Snippet: A. Ratio of RNAseq reads in the absence and presence of Fruitloop gp52 mapping to each of 48 individual phages used for pooled infections. The ratios were normalized for the reads mapping to the host genome for the strain expressing Fruitloop gp52 relative to the strain that does not. For some phages, the numbers of reads/genome were low (<50) and are shown in yellow. B. M. smegmatis cells expressing Fruitloop gp52 (as indicated) were infected with phage Hedgerow and RNA was isolated 150 mins after infection and used as input for semi-quantitative RT-PCR, in the absence or presence of reverse transcriptase (RT), as shown. Following reverse transcription, three different amounts of cDNA (0.5 μl, 1.58 μl, and 5.0 μl) were used as input for the PCR. Upper panel shows amplification of Hedgerow gene 15, and lower panel shows amplification of M. smegmatis gene Msmeg_6947. 100bp DNA markers are shown, with the 300 bp fragment indicated. C–E. Similar reactions as in panel B, except for infection by phage Fruitloop (panel C), Wildcat (panel D), and Rosebush (panel E); upper panels show amplification of Fruitloop gene 101 (panel C), Wildcat gene 101 (panel D), and Rosebush gene 15 (panel E). F. Similar reactions to those in panel A but using M. smegmatis cells expressing the Fruitloop gp52 I70S mutant. Amplicon sizes are 335 bp for Hedgerow and Rosebush 15, 303 bp for Fruitloop 101, 282 bp for Wildcat 101, and 301 bp for Msmeg_6947.

Article Snippet: Following the removal of DNA with a TURBO DNA-free kit (Ambion) and removal of rRNA using a RiboZero rRNA removal kit (Gram-positive bacteria, Illumina), RNA was library prepped using the strand specific TruSeq RNAseq kit (Illumina) according to the manufacturer’s instructions, as described previously ( Dedrick et al. , 2017 ).

Techniques: Expressing, Infection, Isolation, Quantitative RT-PCR, Reverse Transcription, Amplification, Mutagenesis

Journal: Cell reports

Article Title: m 6 A mRNA methylation-directed myeloid cell activation controls progression of NAFLD and obesity

doi: 10.1016/j.celrep.2021.109968

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: The fragmented mRNA was purified by RNase MiniElute Cleanup kit (Cat#:74204, QIAGEN), and the fragment sizes were centered on ~100nt by agarose get validation.

Techniques: Recombinant, SYBR Green Assay, Reporter Assay, Isolation, Gene Expression, Software