Bioanalyzer high sensitivity kit

We performed whole-genome sequencing to identify the insertion-sites of transgene in the mouse genome. Paired-end 150 bp-long sequencing reads were aligned to a modified version of the reference genome (GRCm38/mm10) using the bowtie aligner (bowtie-bio.sourceforge.net). The reference was modified to include a copy of the original cloning vector. Sequencing reads that uniquely mapped both to the mouse genome and to the cloning vector sequence were used to localize the insertion sites. Raw-sequencing reads in the form of.fastq files were deposited in the European Nucleotide Archive with the accession (E-MTAB-5502). To generate the library, DNA was extracted from liver using the DNA Mini kit (Qiagen) and fragmented using Bioruptor (Diagenode). Briefly, 380 ng DNA in 100 microL in 500 microL tubes were subjected to sonication with the following parameters: power setting low, cycle 30 sec on/90 sec off, time 8 minutes in ice-cold, degassed water, resulting in average 550 bp size fragments. The library was prepared using the TruSeq Nano DNA kit (Illumina). DNA and library quantifications were performed with the Quant-iT PicoGreen dsDNA Kit (Invitrogen). Library quality was assessed by using the High sensitivity Bioanalyzer kit (Agilent).

5.7 μl of retro‐transcription mix (see reference for details) was added to each sample. Retro‐transcription was carried out according to the original Smart‐seq2 protocol and cDNA was then pre‐amplified for 15 cycles (Picelli et al, 2014 (link)). After PCR pre‐amplification, cDNA was purified using Ampure XP magnetic beads according to the manufacturer's instructions, in a ratio of 0.8 to 1 with cDNA, resuspended in 17.5 μl of buffer EB (Qiagen) and stored at −20°C. Quality and concentration of the cDNA generated were assessed using High‐Sensitivity Bioanalyzer kit (Agilent).

A 5-mL sample of the culture was taken from at least two chemostat experiments with 4 replicates in total and mixed with RNAlater, which was incubated overnight at 4°C and then stored at −80°C. RNA-seq libraries were prepared as previously described for in vitro samples (5 (link)). rRNA was depleted using the MICROBExpress bacterial mRNA enrichment kit (Sigma). The depleted RNA was fragmented for 2 min with the NEBNext magnesium RNA fragmentation module kit, and cDNA libraries were prepared using the NEBNext multiplex small RNA library prep kit (New England BioLabs). The final RNA concentration for each sample was assessed with a Qubit dsDNA HS (double-stranded DNA high-sensitivity) assay kit (Thermo) and the high-sensitivity Bioanalyzer kit (Agilent). Libraries were sequenced at the Molecular Evolution Core at the Georgia Institute of Technology on an Illumina NextSeq500 platform using 75-bp single-end runs.