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Sample GSM5767769

Query DataSets for GSM5767769

Status

Public on Apr 01, 2022

Title

DMR314_t90_uninduced

Sample type

SRA

Source name

anti-ScpB ChIP-seq of Bs cells carrying an inducible version of Apo-Soj conducted 90 min after re-suspension in sporulation salts w/o xylose

Organism

Bacillus subtilis

Characteristics

chip antibody: anti-ScpB

Treatment protocol

At respective time points the samples were crosslinked with formaldehyde (1 % final) for 30 min at room temperature with shaking every 10 mins. Each sample pellet was washed twice in PBS, prior to rapid freezing and storage at −80°C until further processing. Each sample pellet was resuspended in 2 ml cold PBS and adjusted to 4 OD600 units (4 ml at OD600=1). Adjusted sample pellets were resuspended in TSEMS buffer (50 mM Tris pH 7.4, 50 mM NaCl, 10 mM EDTA pH 8.0, 0.5 M sucrose and PIC (Sigma)) supplemented with 10 mg/ml lysozyme from chicken egg white (Sigma) and incubated for 30 min at 37°C with vigorous shaking. Resulting protoplasts were collected by centrifugation, washed twice with TSEMS, resuspended in 1 ml TSEMS and split into 3 aliquots. Pelleted samples were rapidly frozen in liquid nitrogen for storage at −80°C until further processing. For immunoprecipitation (IP), the sample pellets were resuspended in 2 ml buffer L (50 mM HEPES-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA pH 8.0, 1 % (v/v) Triton X-100, 0.1 % (w/v) Na-deoxycholate, 0.1 mg/ml RNaseA and PIC (Sigma)) and transferred to 50 ml round bottom tubes. Sonication was performed using a Bandelin Sonoplus with an MS72 tip (90 % pulse and 35 % power output) for 3 rounds of 20 sec. Lysates were transferred to 2 ml tubes and, after centrifugation for 10 min at 21,000 g at 4°C, 800 μl of the supernatant was collected for IP and 200 μl was kept as whole-cell extract (WCE) at −20°C. Prior to IP, the anti-scpB antibody serum (Gruber Lab) was incubated with Protein G coupled Dynabeads (Invitrogen) in a 1:1 ratio for 2.5-3.5 hrs at 4°C with rotation. The antibody-bead mixture was then washed and resuspended in buffer L, prior to adding 50 μl to each IP sample, which were incubated for 2.5-3 hrs at 4°C with rotation. Next, the samples were washed with the following buffers: buffer L, buffer L5 (buffer L containing 500 mM NaCl), buffer W (10 mM Tris-HCl pH 8.0, 250 mM LiCl, 0.5 % (v/v) NP-40, 0.5 % (w/v) Na-deoxycholate, 1 mM EDTA pH 8.0), and buffer TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0). Finally, for crosslink reversal, the IP samples were resuspended in 520 μl buffer TES (50 mM Tris-HCl pH 8.0, 10 mM EDTA pH 8.0, 1 % (w/v) SDS) and transferred to 1.5 ml screw-cap tubes. WCE samples were thawed and 300 μl of TES and 20 μl of 10 % SDS were added. Both tubes were incubated overnight at 65°C with vigorous shaking.

Growth protocol

For the single time point ChIP-seq, 10 ml overnight cultures of strains DMR178, DMR179 and DMR181 grown in CH media were diluted to OD600 = 0.1 in 50 ml fresh CH. For the time-course ChIP-seq, 10 ml overnight cultures of strains DMR312, DMR314 and DMR363 grown in CH media were diluted to OD600 = 0.1 in 100 ml fresh CH. Each strain was then grown at 30°C or 37°C until the OD600 reached 0.8. Cultures were then pelleted and resuspended into an equal volume of A+B sporulation salts, which marked t0 of sporulation. All subsequent time points for sporulation (in min or h) are measured similarly (e.g. t90 min marks 90 minutes after re-suspension in sporulation salts). For the time-course ChIP-seq,upon re-suspension in A+B sporulation salts, each sample was split into 2x 50 ml samples, with expression of the ectopic copy of soj being induced in one of each pair by the addition of 0.5 % (final) xylose.

Extracted molecule

genomic DNA

Extraction protocol

To purify the DNA, 500 μl phenol:chloroform:isoamyl alcohol mix (Sigma) was added to the IP and WCE tubes, mixed vigorously and centrifuged for 10 min at 13,000 rpm (at room temperature). 450 μl of aqueous phase was collected and precipitated with 1 ml 100 % ethanol in the presence of 45 μl NaOAc (Sigma) and 1.2 μl of Glycoblue (Invitrogen) for 20 min at −20°C. DNA pellets were collected by centrifugation and resuspended in 100 μl EB (Qiagen) by vigorous shaking for 10 min at 55°C. Final purification was performed using a PCR purification kit (Qiagen), eluting the DNA in 50 μl EB. Prior to deep sequencing, the success of the IP was verified with qPCR.
For deep sequencing, the DNA libraries were prepared by the Genomic Facility at CIG, UNIL, Lausanne. Briefly, the DNA was fragmented by sonication (Covaris S2) until the DNA was sheared to 220-250 bp. The Ovation Ultralow Library Systems V2 kit (NuGEN) including 15 cycles of PCR amplification was used to prepare the DNA libraries. 12-15 million sequence reads per sample were obtained on a HiSeq4000 (Illumina) with 150 bp read length.

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina HiSeq 4000

Description

sporulation_1kb_ChIPseq_DMR312_314_363.txt

Data processing

End trimming using trimmomatic Version 0.39
Reads were mapped using bowtie2 (--very sensitive-local mode)
Downstream data analysis was conducted using SeqMonk (Babraham Institute), with a bin size of 1 kb. Data was visualized using R.
Genome_build: NC_000964.3
Supplementary_files_format_and_content: txt format for read count in 1 kb bins

Submission date

Jan 01, 2022

Last update date

Apr 01, 2022

Contact name

Anna Anchimiuk

E-mail(s)

stephan.gruber@unil.ch

Organization name

University of Lausanne

Department

Department of Fundamental Microbiology