Illumina MiSeq paired end sequencing; Identification of RNA-RNA interactions in Bacillus Subtilis by in vivo UV crosslinking
Identification of RNA-RNA interactions in Bacillus Subtilis by in vivo UV crosslinking
Bacteria were grown to the required O.D before 10 O.D 600 nm units were harvested by centrifugation (4000 g, 5 minutes, 4C). Bacteria were resuspended in 2 ml PBS either containing no AMT (to identify background and levels of spurious interactions) or 0.7mM AMT. Bacteria were incubated for 10 minutes at 37C for 10 minutes before being transferred to a 6 well plate. The bacteria were exposed to UV 365 nm at 0.120 Jcm-2in a for 10 minutes before being added to 1 ml of ice cold killing buffer (20 mM Tris-HCl [pH 7.5], 5 mM MgCl2, 20 mM Na-azide). The bacteria were harvested by centrifugation at X g, the supernatant was discarded and the pellet flash frozen in liquid nitrogen. We determined the in vivo RNA interactome of B. subtilis grown in M9 minimal media supplemented with 0.3% glucose at three points in the growth curve (exponential phase O.D.600nm 0.5, stationary phase O.D.600nm 1.4 and just after lysis had started to occur, and in LB at mid-exponential phase (O.D.600nm of 1.0). Samples were prepared in duplicate. ) Growth experiments were performed in LB, M9 medium supplemented with glucose at a final concentration of 0.3%. The RNA was extracted by resuspending the cell pellet in 800 µl LETS buffer (10 mM Tris-HCl [pH 8.0], 50 mM LiCl, 10 mM EDTA, 1% sodium dodecyl sulfate [SDS]) and bead beating in a FastPrep using 0.1 µm glass beads for three rounds of 40 seconds. The tubes were transferred to ice in between cycles. The tubes were briefly spun to remove the bubbles created during bead beating. Two rounds of phenol chloroform isoamyl alcohol extraction and one round of choloroform isoamyl alcohol extraction were carried out. Before the addition of 10 % v/v NaAcetate and 1 ml Isopropanyl and precipitation of RNA overnight at -20C. The RNA was pelleted by centrifugation at maximum speed at 4C and the pellet was washed with 70% Ethanol before being air dried and resuspended in water. The RNA was quantified using the Qubit kit (Fisher Life Science). 10 µg of RNA was treated with Turbo DNase (Fisher Scientific) to remove contaminating DNA. Ribosomal RNA was removed using Ribozero (Illumina) according to the manufacturer's instructions. To form the chimeric RNAs between RNAs crosslinked with AMT the protocol described by Sharma et al was followed. The only modification was the use of CircDNAligase (Epicentre) instead of CircRNAligase as this has been discontinued. Following uncrosslinking at UV 254 nm, RNA was purified and resuspended in 10 µl H2O and processed through the TruSeq RNAseq kit (Illumina) according to the manufacturers instructions.