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Sample GSM3109111

Query DataSets for GSM3109111

Status

Public on Sep 14, 2018

Title

StdF

Sample type

SRA

Source name

Bacterial cells constitutively expressing stdEF::3xFLAG

Organism

Salmonella enterica subsp. enterica serovar Typhimurium str. SL1344

Characteristics

genotype/variation: PLtetO-stdEF::3xFLAG
chip antibody: Monoclonal anti-FLAG M2 (Sigma F1804)

Treatment protocol

Bacterial cells were collected and cross-linked with 1% formaldehyde at 37ºC for 25 min, followed by quenching of the unused formaldehyde with 450 mM glycine for an additional 5 min of incubation. Cross-linked cells were harvested and washed with 10 mL of TBS pH7.6 (2.42 g/l Trizma base, 8 g/l NaCl). The washed cells were resuspended in 1 mL of lysis buffer (10 mM Tris-HCl -pH 8, 20% sucrose, 50 mM NaCl, 10 mM EDTA) and after an additional centrifugation step, the cells were resuspended in 0.5mL of lysis buffer with lysozyme (20mg/ml; Sigma Chemical Co.). The cells were incubated for 30 min at 37°C and then treated with 4 mL of IP buffer (50mM HEPES-KOH-pH7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Na deoxycholate, 0.1% SDS and 1mg/mL PMSF). The lysate was then sonicated using a Bioruptor (Diagenode) with 5 cycles of 7 minutes (30″ON/30″OFF) at high setting. Cell debris was removed by centrifugation for 20 min at 4°C and the resulting supernatant was used as cell extract for the immunoprecipitation. The range of the DNA size resulting from the sonication procedure was 100-500 bp, and the average DNA size was 300 bp.

Growth protocol

20ml of the bacterial strain PLtetO-stdEF::3xFLAG, incubated with shaking (200rpm) at 37ºC until OD600∼2.

Extracted molecule

genomic DNA

Extraction protocol

To immunoprecipitate StdE-DNA and StdF-DNA complexes, 800 μL of chromatin, 20 μL of Ultralink Immobilized protein A/G beads (Pierce) and 2 μL of the corresponding antibody were used (anti-StdE and anti-FLAG). Control sample (mock-IP) was performed with no antibody added. They were then incubated for 90 minutes at room temperature on a rotating wheel. Beads were transferred to a Spin-X column tube (Costar) and centrifuged at 3000rpm for 1min. Beads were gently re-suspended in 500 μL of IP buffer and incubated on wheel for additional 3min. Beads were washed with 500 μL of IP salt buffer, IP wash buffer and TE pH8.0 by resuspending and centrifuging the sample. The column was transferred to a fresh tube and the beads were resuspended in 100 μL of elution buffer (50 mM Tris-HCl at pH 7.5, 10 mM EDTA, and 1% SDS) and incubated at 65ºC for 20min. After centrifuging at 3000rpm for 1min, the flow-through was treated with 10μL of 40mg/mL Pronase (Roche) made up in TBS. The samples were heated at 42ºC for 2h and 65ºC for 6 hours. The reactions were then kept at 4ºC overnight. The samples were cleaned up by using a PCR clean up Kit (Promega) and resuspended in 50 μL of H2O.
NEBNext® Ultra DNA Library Prep Kit for Illumina; 50 nt SE sequencing.

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina HiSeq 2500

Description

Sample 2
The bacteria constitutively express both stdE and stdF. StdF has the 3xFLAG tag.
processed data file: F_CEF_anotacion_picos.bed
processed data file: F_EFT_anotacion_picos.bed
processed data file: ChIP-seq_results.xlsx

Data processing

BAM files reported by the sequencing facility of the Functional Genomics Core Facility were converted to FASTQ format with the BAM2FASTQ tool (http://www.hudsonalpha.org/gsl/information/software/bam2fastq).
The quality of the sequence reads was examined using FASTQC (Andrews, 2010) that reported the presence of Illumina adapters. The adapters were trimmed with the FASTX_CLIPPER tool of the FASTX-Toolkit suite (http://hannonlab.cshl.edu/fastx_toolkit/). Reads shorter than 40 nt were discarded.
Peaks were called using CisGenome version 2.0 (Ji et al., 2008) using default parameters.
The IGV browser (Thorvaldsdóttir et al., 2013) was used for data visualization.
Genes closest to a ChIP peak were identified using the bedtools suite (Quinlan & Hall, 2010). Peak boundaries sequences were extracted from the reference genome using the fastaFromBed utility from the BEDTools suite (Quinlan & Hall, 2010).
NCBI GCA_000210855.2 genome assembly of S. enterica SL1344 was used as reference genome. Mapping was performed with Bowtie (Langmead et al., 2009) allowing only two mismatches for unique alignment.
Genome_build: GCA_000210855.2 (ASM21085v2)
Supplementary_files_format_and_content: E_CEF_anotacion_picos.bed: Peaks in BED format. The obtained peaks for the IP sample (Sample 1) is compared with the peaks for the control sample (mock IP: Sample 3).
Supplementary_files_format_and_content: E_EFT_anotacion_picos.bed: Peaks in BED format. The obtained peaks for the IP sample (Sample 1) is compared with the peaks for the control sample (mock IP: Sample 4).
Supplementary_files_format_and_content: F_CEF_anotacion_picos.bed: Peaks in BED format. The obtained peaks for the IP sample (Sample 2) is compared with the peaks for the control sample (mock IP: Sample 3).
Supplementary_files_format_and_content: F_EFT_anotacion_picos.bed: Peaks in BED format. The obtained peaks for the IP sample (Sample 2) is compared with the peaks for the control sample (mock IP: Sample 4).
Supplementary_files_format_and_content: ChIP-seq results.xlsx: Peaks in Excel format. The obtained peaks for the IP samples (Sample 1 and 2) are compared with the peaks for the control samples (mock IP: Sample 3 and 4).

Submission date

Apr 23, 2018

Last update date

Sep 14, 2018

Contact name

Lucía García

E-mail(s)

lgarcia9@us.es

Organization name

Universidad de Sevilla

Street address

Avda. Reina Mercedes