Rna nano kit

Transcriptional profiles of transposon mutants grown under planktonic conditions were recorded as previously described [26 ]. Briefly, planktonic bacteria were cultivated in 10 ​ml LB to early stationary phase (OD₆₀₀ of 2) under shaking conditions (37 ​°C, 180 ​rpm). Three independent cultures were pooled and an equal volume of RNAprotect (Qiagen) was added prior to cell harvest. RNA was extracted using the RNeasy Mini Kit (Qiagen) and Qiashredder columns (Qiagen) according to the manufacturer’s instruction. RNA quality was checked using the RNA Nano Kit on an Agilent Bioanalyzer 2100 (Agilent Technologies). Ribosomal RNA was removed using the Ribo-Zero Bacteria Kit (Illumina) and cDNA libraries were generated with the ScriptSeq v2Kit (Illumina). The samples were sequenced in single-end mode on an Illumina HiSeq 2500 device (1 ​× ​50 bp reads). Mapping was performed using a stampy pipeline [38 (link)] with the PA14 genome as a reference. Differential gene expression analysis was performed as described in the previous section.

Total RNA of entire newt knee joints was isolated of arbitrarily grouped pools of 2 or 3 operated or injected animals with both low and high clinical score to facilitate detection of genes expressed at early and later time points. Their respective contralateral controls were also processed. RNA extraction was performed by tissue homogenization in ice-cold Trizol reagent using a swing mill (Retsch MM200, Haan, Germany), following the manufacturer's instructions (Invitrogen, Karlsruhe, Germany) as described previously. Afterwards, RNA quality was checked on an Agilent 2100 Bioanalyzer with the RNA Nanokit (Agilent Technologies, Santa Clara, CA, USA).

RNA concentration and integrity were determined by Bioanalyzer using the RNA Nano Kit (Agilent Technologies, Santa Clara, CA). RNA-seq libraries were prepared using the TruSeq RNA Sample Prep Kit (Illumina, San Diego, CA) per the manufacturer's protocol. Briefly, mRNA was purified from total RNA using oligo dT magnetic beads, fragmented, and then reverse transcribed via random hexamers and SuperScript II Reverse Transcriptase (Invitrogen, Carlsbad, CA). Second strand cDNA was synthesized using a proprietary master mix. The double stranded cDNA was then blunt ended, the 3′ ends were adenylated, and indexed Illumina adaptors were ligated. DNA fragments were enriched by PCR using the following conditions: initial denaturation of 98 °C for 30 s, 15 cycles of 98 °C for 10 s, 60 °C for 30 s, and 72 °C for 30 s, followed by a final extension of 72 °C for 5 min. RNA-seq libraries were visualized by Bioanalyzer (Agilent) and quantified by qPCR. Libraries were bar-coded and pooled by quantity and sequenced via 50 bp single reads on a HiSeq2000 (Illumina). Reads were trimmed to include only bases 9–57 for quality purposes. Alignment was done to mm9 using Tophat with the Bowtie 1 option [22] (link), [23] (link). The number of reads intersecting with each gene was calculated, one was added, and counts were log2 transformed. DESeq was used for differential expression testing [24] (link).