GSM4454324: PSPPH_0805-KB-1; Pseudomonas savastanoi pv. phaseolicola 1448A; RNA-Seq
TruSeq RNA sample prep kit protocol (Illumina) The ChIP assay was performed as previously described.For the FLAG-tagged plasmids, the open reading frame of each regulator coding gene was amplified by PCR from Psph1448A genome and cloned into pHM1 plasmid. Wild-type Psph1448A containing empty pHM1 or pHM1-RR-FLAG was cultured in 20 ml KB medium until mid-log phase (OD600 = 0.6). Cultured bacteria were then reated with 1% formaldehyde for 10 min at 28 ℃. Cross-linking was stopped by the addition of 125 mM glycine. Bacterial pellets were washed twice with a Tris buffer (20 mM Tris-HCl [pH 7.5] and 150 mM NaCl), re-suspended in 500 μl IP buffer (50 mM HEPES–KOH [pH 7.5], 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, and mini-protease inhibitor cocktail (Roche), and then subjected to sonication to produce 100-300 bp DNA fragments. Insoluble cellular debris was removed by centrifugation at 4 ℃ and the supernatant was used as the input sample in IP experiments. Both control and IP samples were washed with protein A beads (General Electric) and then incubated with 50 μl agarose- conjugated anti-FLAG antibodies (Sigma) in IP buffer. Washing, crosslink reversal, and purification of the ChIP DNA were conducted. DNA fragments (150-250 bp) were selected for library construction and sequencing libraries were prepared using the NEXTflex™ ChIP-Seq Kit (Bioo Scientific). The libraries were sequenced using the HiSeq 2000 system (Illumina). Two biological replications have been performed for all ChIP-seq experiments. ChIP-seq reads were mapped to the Pseudomonas syringae pv. phaseolicola 1448A genome (NC_005773.3) using Bowtie2 (version 2.3.4.3). Uniquely mapped reads were retained for the subsequent analyses. Binding peaks (q < 0.01) were identified using MACS2 software (version 2.1.0). Reproducible binding sites in both replicates were identified by BEDtools software (version 2.27.1)and used for the following analysis. Subsequently, the enriched loci for each regulator were annotated using the R package ChIPpeakAnno (version 3.18.2) . Target genes were annotated by peaks located in gene promoters defined as upstream of transcription start site (TSS), overlapping with TSS or encompassing the whole gene. To examine the effect of histidine kinases on the transcriptome, 2 ml of mid-log-phase (OD600 = 0.6) bacterial cultures or 2 ml cultures of mid-log-phase induced in MM with 6 hours (Psph1448A and transcriptional regulator mutant strains) were collected by centrifugation (12,000 rpm, 4 ℃). A RNeasy mini kit (Qiagen) was used for subsequent RNA purification with DNaseI (NEB) treatment. After removing rRNA by using the MICROBExpress kit (Ambion), mRNA was used to generate the cDNA library according to the NEBNext® UltraTM II RNA Library Prep Kit protocol (Illumina), which was then sequenced using the HiSeq 2000 system (Illumina). Two biological replications have been performed for all RNA-seq experiments. For each RNA-seq sample, raw reads were mapped to the Pseudomonas sringae pv. phaseolicola 1448A genome (NC_005773.3) using STAR (version 2.6.1), and uniquely mapped reads were kept for further analyses. Differentially expressed genes were identified using DESeq2 (BH-adjusted P < 0.05 and log2 Fold Change| > 0.5).
GEO Accession
GSM4454324