Computational protocol: Allopatric integrations selectively change host transcriptomes, leading to varied expression efficiencies of exotic genes in Myxococcus xanthus

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Protocol publication

[…] In this study, we exploit the ssRNA-seq method to identify the transcriptional template strands of M. xanthus DZ2 and three transformants ZE-5, ZE-9 and ZE-14 at a whole genome level using the Illumina-platform high throughput sequencing. First of all, the total RNA of the M. xanthus strains were extracted according to the protocol provided by the SV Total RNA isolation system kit (Promega, USA). Residual genomic DNA was removed by treatment with recombinant DNase I (RNase-free; Ambion, USA) according to the manufacturer’s instructions. The quality of the total RNA was verified by agarose gel electrophoresis, and the concentration was determined using a NanoDrop ND-1000 spectrophotometer (NanoDrop technologies, USA). Then, libraries were created by modifying the previously described dUTP second strand method []. We fragmented 200 ng of M. xanthus polyA+ RNA by heating at 98°C for 40 min in 0.2 mM sodium citrate, pH 6.4 (Ambion, USA). The fragmented RNA was concentrated to 5 μL, mixed with 3 μg random hexamers, incubated at 70°C for 10 min, and then cooled on ice. The RNA mixtures were further added with 4 μL of 5× first-strand buffer, 2 μL of 100 mM DTT, 1 μL of 10 mM dNTPs, 4 μg of actinomycin D (USB), 200 U SuperScript III, and 20 U SUPERase-In (Ambion, USA), incubated at room temperature for 10 min followed by 1 h at 55°C to synthesize the first-strand cDNA. First-strand cDNA was cleaned up by extraction twice with phenol: chloroform: isoamyl alcohol (25:24:1), followed by ethanol precipitation with 0.1 volumes 5 M ammonia acetate to remove dNTPs and re-suspension in 104 μL ddH2O. Second-strand cDNA was synthesized by adding 4 μL 5× first-strand buffer, 2 μL 100 mM DTT, 4 μL 10 mM dNTPs with dTTP replaced by dUTP (Sigma-Aldrich, USA), 30 μL 5× second strand buffer, 40 U Escherichia coli DNA polymerase, 10 U E. coli DNA ligase, 2 U E. coli RNase H and incubating at 16°C for 2 h. A paired-end library for Illumina sequencing was prepared according to the instructions provided with the following modifications. First, five times less adapter mixture was ligated to the cDNAs. Second, 1 U USER (New England Biolabs, USA) was incubated with 180- to 480-bp size-selected, adapter-ligated cDNA at 37°C for 15 min followed by 5 min at 95°C before PCR. Third, PCR was performed with Phusion High-Fidelity DNA Polymerase with GC buffer (New England Biolabs, USA) and 2 M betaine (Sigma, USA). Fourth, PCR primers were removed using 1.8× volume of AMPure PCR Purification kit (BeckmanCoulter Genomics, USA). Transcriptome sequencing was performed at the BGI Corporation. Reagents were all from Invitrogen (Carlsbad, USA) except as noted.To assay the growth curves, M. xanthus colonies were first cultured overnight in CYE medium. Then, the cultures were transferred at a ratio of about 2:100 into 5 mL fresh CYE medium with a start OD600 of 0.15, and assayed of the OD600 values periodically.The reference genome (M. xanthus DK1622) and the gene annotation was retrieved from GenBank (accession no. NC_008095.1). After removing the reads containing sequencing adapters and low-quality reads (reads containing Ns >10%), the remaining 90 bp clean reads with high quality were aligned with the reference genome using Bowtie software []. Then the RPKM method was used to normalize the transcript level, which was expressed as the number of reads per kilobase of exon region per million mapped reads (RPKM). Go annotation of the genes was performed using Blast2GO software and visualized by WEGO software. […]

Pipeline specifications

Software tools Bowtie, Blast2GO, WEGO
Application RNA-seq analysis
Organisms Myxococcus xanthus
Chemicals Epothilones