NextSeq 500 sequencing; Cappable-seq of Sinorhizobium meliloti ECF/anti-σ-free free strains overexpressing heterologous ecf genes compared to an empty vector control strain
Cappable-seq of Sinorhizobium meliloti ECF/anti-σ-free free strains overexpressing heterologous ecf genes compared to an empty vector control strain
Rm1021 ECF/anti-σ-free strain derivatives were grown to an OD600 of 0.1-0.3 in MOPS buffered minimal medium with gentamicin (6 µg/mL). The OD600 was adjusted to 0.1 and IPTG was added at a final concentration of 500 µM. Strains were grown for ~ 8-9 hours until cell density reached an OD600 of 0.8 and collected by centrifugation at 10,000 rcf for 5 min. Cell pellets were frozen in liquid nitrogen and kept at -80 °C. Assays were performed in triplicates for each strain. For total RNA isolation, the Qiagen miRNeasy Kit was used starting from a pellet of 8 mL cultured cells. The cell pellet was resuspended in 1 mL QIAzol lysis reagent and homogenized in a FastPrep® sample preparation system using Lysing Matrix B containing 0.1 mm silica beads (MP biomedicals) and the following settings: 3x 6500 rpm for 20 sec, 15 sec break. The supernatant was transferred into a new tube and RNA isolation was performed according to the manufacturer's instruction including the optional on column DNaseI digestion. RNAs from biological triplicates were pooled in equal amounts before library preparation. 5'-triphosphorylated RNAs were capped with a 3'-desthiobiotin-TEG-guanosine 5'-triphosphate (DTBGTP) using the vaccinia capping enzyme (VCE) (NEB). Biotinylated RNAs were captured on streptavidin beads and eluted to obtain the 5'-fragments of primary transcripts. The eluted RNAs were poly(A)-tailed using poly(A) polymerase. To distinguish primary transcripts from contaminating processed or degraded RNA molecules with a 5'-monophosphate, they were differentially labelled with identifying sequence tags (TSS-tag and PSS-tag, respectively): the 5' Illumina TruSeq sequencing adapters, which carry sequence tags ATTACTCG and TCCGGAGA, were ligated to the 5'-monophosphate groups of processed transcripts. The samples were then treated with CapClip Acid Pyrophosphatase (Cellscript) in order to convert 5' triphosphate structures into 5'-monophosphate ends. To the newly formed 5'-monophosphate groups 5' Illumina TruSeq sequencing adapters, carrying sequence tag CGCTCATT and GAGATTCC were ligated. The resulting cDNAs were PCR-amplified to yield 10-20 ng/µL (16 cycles) using a high fidelity polymerase. The full length cDNAs were degraded using dsDNA fragmentase (NEB), 100-300 bp long fragments were isolated and then bound to streptavidin magnetic beads. The bound cDNAs were blunted and 3' Illumina sequencing adapter were ligated to the 3' ends of the cDNA fragments. The bead bound cDNAs were PCR-amplified and barcoded. Barcoded cDNA libraries were pooled in equimolar amounts. The cDNA pool was size fractionated in the size range of 200-500 bp with the Agencourt AMPure kit.