title: GSM3476660: RhpRS-MM-1; Pseudomonas savastanoi pv. phaseolicola 1448A; RNA-Seq
library_construction_protocol: The wild-type, ΔrhpS and ΔrhpRS strains were cultured in KB medium until the OD600 = 0.6 before transferring to MM liquid medium for 6 hours. Then, 2 ml of bacterial cultures were collected by centrifugation (12,000 rpm, 4℃). RNA purification was conducted by an RNeasy mini kit (Qiagen). 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 (NEB), which was then sequenced using the HiSeq 2000 system (Illumina). Bacterial RNA-seq reads were mapped to the Pseudomonas savastanoi pv. phaseolicola 1448A genome (NC_005773.3) by using STAR, Only the uniquely mapped reads were kept for the subsequent analyses. The gene differential expression analysis was performed using Cuffdiff software (version 2.0.0) . GO enrichment analyses were conducted on all differentially transcribed genes using DAVID . TruSeq RNA sample prep kit protocol (Illumina) ChIP-seq:  An empty pHM2, pHM2-rhpRpsph-HA or pHM2-rhpRpsph(D70A)-HA plasmid was transformed into the wild-type or ΔrhpS strain, then cultured in KB medium until the OD600 = 0.6 before transferring to MM liquid medium for 6 hours. The strains were treated with 1% formaldehyde for 10 min at 37℃. Cross-linking was stopped by the addition of 125 mM glycine. The bacteria were centrifugated and the pellets were washed twice with Tris-buffer (20 mM Tris-HCl [pH 7.5], 150 mM NaCl), and then resuspended in 500 μl IP buffer (50 mM HEPES-KOH [pH 7.5], 1 mM EDTA, 150 mM NaCl, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, mini-protease inhibitor cocktail). Next, the DNA was subjected to sonication to produce 100 to 300 bp DNA fragments. The insoluble cellular debris was removed by centrifugation at 4℃, and the supernatant was saved as the input sample in the IP experiments. Both the control and IP samples were washed by protein A beads (General Electric) and mixed with 50 μl agarose conjugated anti-HA antibodies (Sigma) in IP buffer. The following washing, crosslink reversal and purification of ChIP-DNA were conducted as previously described. DNA fragments (150 to 250 bp) were selected for library construction, and libraries were constructed by using the NEXTflexTM ChIP-Seq Kit (Bioo Scientific). The libraries were sequenced using the HiSeq 2000 system (Illumina). ChIP-seq results were mapped to the Pseudomonas savastanoi pv. phaseolicola 1448A genome (NC_005773.3) by using Bowild-typeie (Version 1.2.1.1). Only the uniquely mapped reads were kept for the subsequent analyses. Binding peaks (P < 1e-5) were identified using MACS software (version 2.1.0). All the experiments have two repeats and the reported peaks are found in both experiments RNA-seq:The wild-type, ΔrhpS and ΔrhpRS strains were cultured in KB medium until the OD600 = 0.6 before transferring to MM liquid medium for 6 hours. Then, 2 ml of bacterial cultures were collected by centrifugation (12,000 rpm, 4℃). RNA purification was conducted by an RNeasy mini kit (Qiagen). 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 (NEB), which was then sequenced using the HiSeq 2000 system (Illumina). Bacterial RNA-seq reads were mapped to the Pseudomonas savastanoi pv. phaseolicola 1448A genome (NC_005773.3) by using STAR, Only the uniquely mapped reads were kept for the subsequent analyses. The gene differential expression analysis was performed using Cuffdiff software (version 2.0.0) . GO enrichment analyses were conducted on all differentially transcribed genes using DAVID . NEXTflex™ ChIP-Seq Kit (Bioo Scientific)  and TruSeq RNA sample prep kit protocol (Illumina)
attributes.tag: GEO Accession
attributes.value: GSM3476660