NextSeq 500 sequencing; Genome-wide transcription start sites mapping in Methylorubrum grown using dichloromethane and methanol
Genome-wide transcription start sites mapping in Methylorubrum grown using dichloromethane and methanol
Methylorubrum extorquens wild-type strain DM4 were harvested at mid-phase (OD at 600nm of 0.15). Methylorubrum extorquens wild-type strain DM4 was grown aerobically at 30°C in in 1.2L Erlenmeyer  flasks  closed  with  gas-tight  screw  caps  with Mininert R valves (Supelco) in M3 media (Roselli et al , 2013: https://doi.org/10.1371/journal.pone.0056598) supplemented with methanol or DCM provided at 10mM as the sole source of carbon and energy in independent biological  duplicates. For 220-mL cultures, one-carbon growth substrates were supplied at 10 mM final concentration, by adding either aqueous solutions of 2.75 mL of filter-sterilized aqueous stock solution of methanol (800 mM), or 141μL of neat dichloromethane. Cells were harvested upon reaching mid-exponential phase. Growth was stopped by addition of 27.5 mL stabilization buffer. This buffer was prepared by mixing 5 mL of phenol and 5mL of 1 M sodium acetate pH 5.5, then after centrifugation at 1,800 g for 3 min, 5 mL of the lower phase was mixed with 95 mL of absolute ethanol. Resulting cell suspensions were centrifuged at 5,000 rpm for 5 min at 4◦C, and suspended in 10 mL TE containing 2 mg.mL−1lysozyme (Euromedex). After 15 min incubation at 37◦C, each cell suspension was centrifuged 10 min at 4◦C, the obtained pellet resuspended in 10 mL of Trizol (Invitrogen), and 2.5 mL of chloroform was added. RNA was precipitated with isopropanol and washed with ethanol (70%), then resuspended in DEPC-water. RNA were then treated with a TURBO DNA-free kit (Ambion) to remove residual genomic DNA. Differential (dRNA-seq) library construction. Ribosomal RNAs-depleted RNA samples were poly(A)-tailed using poly(A) polymerase for library construction in a strand specific manner. Briefly, after a treatment with 5' monophosphate-dependent RNA exonuclease (TEX), the four samples were split in two: one part was treated with RNA 5' polyphosphatase (5'PP) to remove the 5'PPP structures while the other part was left untreated, and used for “+” and “-“ library construction, respectively. RNA adapter was ligated to RNA with a 5' monophosphate end. Oligo(dT)-adapter primer was used to perform synthesis of first strand cDNA with M-MLV reverse transcriptase. Resulting cDNAs were PCR-amplified with high fidelity DNA polymerase and barcoded using 3' sequencing adaptors. After cDNAs purification using the Agencourt AMPure XP kit (Beckman Coulter Genomics) and quality-checked using capillary electrophoresis (Shimadzu MultiNA microchip electrophoresis system), the cDNA were pooled and ready to be sequenced.