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| Status |
Public on Feb 09, 2024 |
| Title |
Δhns KDS1 rep1 - ChIP |
| Sample type |
SRA |
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| Source name |
DD427
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| Organism |
Vibrio cholerae |
| Characteristics |
strain: DD427
genotype: {delta}hns::frt-spec-frt
media: LB
chip antibody: anti-RNA polymerase antibody (NeoClone WP023)
time of_harvest: logarithmic phase
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| Growth protocol |
All standard overnight bacterial cultures were grown from a single colony in 2 mL volume with aeration at 37C in standard LB Miller liquid growth medium, supplemented with streptomycin (100 µg/mL). For cultures requiring mitomycin C treatment, an overnight culture grown in standard conditions was diluted to an OD600 of 0.003 into 140 mL of pre-warmed LB Miller liquid medium supplemented with mitomycin C to a final concentration of 20 ng/mL. Cultures were grown to a final OD600 of 0.3.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
IPOD sample preparation. IPOD samples were collected as previously described (Freddolino PL et al, PLoS Biology, 2022) with minor modifications. Overnight cultures were diluted to a starting OD600 of approximately 0.003 in a final volume of 140 mL of LB medium in a 1 L flask. The diluted cultures were grown at 37C with shaking at 250 RPM until a final OD600 of 0.3. 28.9 mL of culture was collected at each time point and mixed with 300 μL 1 M sodium phosphate buffer (pH 7.4) and 810 μL formaldehyde (37%, fresh) in a 50 mL conical tube, then allowed to crosslink for 5 minutes shaking at room temperature (notably, unlike in Freddolino PL et al, no rifampicin was added prior to crosslinking). After 5 minutes, the crosslinking reactions were quenched with 6 mL of 2M glycine and returned to the room temperature shaker. After 10 minutes of shaking, tubes were placed in ice for 10 minutes and then pelleted at 7,000x g at 4C for 4 minutes. Pellets were washed twice with 10 mL of ice-cold phosphate-buffered saline and the final cell pellets were snap-frozen in a dry ice-ethanol slurry and stored at -80C. Cell lysis, digestion, and lysate clarification. The original IPOD-HR protocol is from ref. (Freddolino PL et al, PLoS Biology, 2022); below we summarize the procedures applied here. Frozen cell pellets were resuspended in 600 μL of 1X IPOD lysis buffer (10 mM Tris HCl, pH 8.0; 50 mM NaCl) with 1x protease inhibitors tablet (Roche Complete Mini, EDTA free, Roche Diagnostics GmbH, Mannheim, Germany) and 1.5 μL lysozyme (ThermoScientific, REF90082, 50 mg/mL) incubated for 15 minutes at 30C with gentle shaking and then placed on ice. Resuspended cells were sonicated with a Branson sonicator at 25% power with three bursts of 10s ON and 10s OFF while in an ice water bath. Sonicated cells were then digested with 6μL RNaseA (SIG 10109169001, Sigma-Aldrich/Roche, 10mg/mL), 5.4 μL of 100mM MnCl2, 4.5 μL of 100mM CaCl2, 9 μL of DNase I, RNAse-free (ThermoScientific, #89836, 1U/μL) and incubated on ice for 30 minutes, after which reactions were quenched with 50 μL 500mM EDTA (pH 8.0); we aimed to obtain fragment sizes of about 150 bp. 400 μL of 1x IPOD lysis buffer without protease inhibitors and lysozyme was added to the digested mixture and vortexed. The mixture was clarified by centrifugation for 10 minutes at 15,700 x g at 4C and transferred to new tubes. The clarified lysate was partitioned into three different tubes: 50 μL for INPUT (for baseline reference), 400 μL for IPOD (for total protein occupancy), and the rest for CHIP (for RNA polymerase occupancy). 450 μL of ChIP elution buffer (50mM Tris (pH 8.0), 10mM EDTA, 1% SDS) was added to the INPUT sample and kept on ice until reverse cross-linking step (mentioned in the next step). Cell lysis, digestion, and lysate clarification. The original IPOD-HR protocol is from ref. (Freddolino PL et al, PLoS Biology, 2022); below we summarize the procedures applied here. Frozen cell pellets were resuspended in 600 μL of 1X IPOD lysis buffer (10 mM Tris HCl, pH 8.0; 50 mM NaCl) with 1x protease inhibitors tablet (Roche Complete Mini, EDTA free, Roche Diagnostics GmbH, Mannheim, Germany) and 1.5 μL lysozyme (ThermoScientific, REF90082, 50 mg/mL) incubated for 15 minutes at 30C with gentle shaking and then placed on ice. Resuspended cells were sonicated with a Branson sonicator at 25% power with three bursts of 10s ON and 10s OFF while in an ice water bath. Sonicated cells were then digested with 6μL RNaseA (SIG 10109169001, Sigma-Aldrich/Roche, 10mg/mL), 5.4 μL of 100mM MnCl2, 4.5 μL of 100mM CaCl2, 9 μL of DNase I, RNAse-free (ThermoScientific, #89836, 1U/μL) and incubated on ice for 30 minutes, after which reactions were quenched with 50 μL 500mM EDTA (pH 8.0); we aimed to obtain fragment sizes of about 150 bp. 400 μL of 1x IPOD lysis buffer without protease inhibitors and lysozyme was added to the digested mixture and vortexed. The mixture was clarified by centrifugation for 10 minutes at 15,700 x g at 4C and transferred to new tubes. The clarified lysate was partitioned into three different tubes: 50 μL for INPUT (for baseline reference), 400 μL for IPOD (for total protein occupancy), and the rest for CHIP (for RNA polymerase occupancy). 450 μL of ChIP elution buffer (50mM Tris (pH 8.0), 10mM EDTA, 1% SDS) was added to the INPUT sample and kept on ice until reverse cross-linking step (mentioned in the next step). Interphase extraction and nucleic acid purification. 400 μL of 100 mM Tris Base and 800 μL of 25:24:1 phenol:chloroform:isoamyl alcohol (Sigma Aldrich, #77617) was added to the 400 μL of clarified lysate kept for the IPOD-HR sample, vortexed for 10s, and then incubated for 10 minutes at room temperature. To obtain a separation of the organic and aqueous layers and formation of the white interphase disc, which is enriched in protein-DNA complexes, the mixture was spun for 2 minutes at 21,130 x g at room temperature. The aqueous and organic layers around the interphase were removed while minimizing disturbance of the interphase disc. The extracted disc was washed once in 350 μL TE (10 mM Tris, pH 8.0; 1 mM EDTA), 350 μL 100 mM Tris base, and 700 μL 24:1 chloroform:isoamyl alcohol. The layers were again separated by spinning for 2 minutes at 21,130 x g at room temperature. After removal of liquid around the interphase disc, a final wash of 700 μL TE and 700 μL 24:1 chloroform:isoamyl alcohol was applied. After vortexing and spinning the final wash as before, as much of the liquid around the white disc as possible was removed and discarded. The interphase disc was resuspended in 500 μL of ChIP elution buffer and vortexed. The resuspended interphase of IPOD sample and INPUT sample (kept on ice from above step) were incubated at 65C overnight to reverse-crosslink (6-16 hours). RNA Polymerase Chromatin Immunoprecipitation. The rest of the clarified lysate (about 500 μL) was mixed with 1 volume of 2x immunoprecipitation (IP) buffer (200mM Tris (pH 8.0), 600mM NaCl, 4% Triton X-100) and incubated with 10μg of anti-E. coli RNA polymerase antibody (NeoClone WP023, NeoClone, Madison, WI, Lot: 2019G15-002) overnight with rocking at 4C. After overnight incubation, 50 μL of protein G beads (New England Biolabs (NEB), S1430S) per sample was equilibrated in 1x IP buffer. 50 μL of equilibrated protein G beads was added to each antibody-lysate mixture and then incubated with rocking 2 hours at 4C. After incubation, the beads were washed once in 1mL of the following buffers: IP wash buffer A, IP wash buffer B, IP wash buffer C, 1x IP buffer, and 1x TE. 1x wash buffer A (100mM Tris, pH 8.0; 250mM LiCl; 2% Triton X-100; 1mM EDTA) 1x wash buffer B (10mM Tris, pH 8.0; 500mM NaCl; 1% Triton X-100; 0.1% sodium deoxycholate; 1mM EDTA) 1x wash buffer C (10mM Tris, pH 8.0; 500mM NaCl; 1% Triton X-100; 1mM EDTA) After the wash steps, beads with the immunoprecipitated material were resuspended in 500 μL of ChIP elution buffer by rocking for 5 minutes; the samples were then incubated for 30 minutes at 65C with vortexing every 5 minutes. The beads were then separated and discarded to retain only the eluted immunoprecipitated sample. The samples were then left to reverse-crosslink overnight (6-16 hours) at 65C. DNA extraction post reverse cross-linking. The DNA from INPUT, IPOD, and ChIP samples were isolated and purified following identical steps. After overnight incubation to reverse the formaldehyde crosslinks, each sample was incubated with 10 μL of RNAse A (Roche Diagnostics GmbH, #SIG10109169001, 10mg/mL) for 2 hours at 37C. Then we followed by adding 10 μL Proteinase K (ThermoScientific, #EO0491, 20mg/mL) and incubating for 2 hours at 50 oC. The DNA of each sample was isolated by phenol-chloroform extraction using one volume of 25:24:1 phenol:chloroform:isoamyl alcohol, then re-extracted with one volume of 24:1 chloroform:isoamyl alcohol. At the last stage the samples went into DNA LoBind tubes. The isolated DNA was then precipitated by adding 1/25th volume of 5M NaCl as a precipitating salt, 1/300th volume of GlycoBlue (Invitrogen, #AM9515, 15mg/mL) as a co-precipitant, and two volumes of ice cold 1:1 isopropanol:ethanol. The DNA was precipitated at 4C for one hour and at -20C for more than 1 hour or overnight. Chilled samples were then centrifuged for 15 minutes at 16,100 x g at 4C to pellet the DNA. All the liquid was then removed without disturbing the DNA pellet. Pellets were then washed in freshly diluted 95% ethanol, vortexed and then centrifuged for 5 minutes at 16,100 x g at 4C. Finally, the liquid was removed, and the remaining ethanol was left to evaporate for 30 minutes or less. The following volumes of TEe (10 mM Tris, pH 8.0; 0.1 mM EDTA) were added to resuspend the DNA from the three sample types: 200 μL for INPUT pellets, 50 μL for IPOD, and 30 μL for ChIP. All samples were quantified with QuantiFluor dsDNA System (Promega, #2670) using a BioTek Synergy plate reader. Input samples were assessed for fragment sizes on 2% agarose gel electrophoresis.
Illumina library preparation for IPOD-HR. The purified and quantified DNA from INPUT, IPOD, and RNA Polymerase ChIP samples were then prepared for Illumina sequencing using NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® (#7645S/L) with NEBNext® Multiplex Oligos for Illumina® Unique Dual Index UMI Adaptors DNA Set 1 (#E7395S) or NEBNext® Multiplex Oligos for Illumina® Dual Index Primers Set 1 (#E7600S) and Dual Index Primers Set 2 (#E7780S). Omega Biotek Mag-Bind DNA purification or Axygen purification beads were used for all SPRI bead cleanup steps. The bead purification at the post-adaptor ligation stage was modified as follows: 1.8x volume of DNA purification beads and 0.7x isopropanol were added instead of the normal 0.9x volume of beads. Each sample prepared for sequencing was quantified and assessed for fragment size and for the presence of any adaptor present on 2% agarose gel electrophoresis. The samples were pooled into libraries, which were sequenced on a NextSeq 550 instrument at the Michigan Advanced Genomics Core.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
NextSeq 550 |
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| Data processing |
For IPOD, INPUT and ChIP-seq: ipod singularity container version 2.7.2 was used (available at https://github.com/freddolino-lab/ipod). Peak calling was done with version 2.8.1
Assembly: We obtained an initial genome reference sequence for KDS1 by sequencing high molecular weight genomic DNA purified from the strain. Raw reads are available at SRA########. Initial assembly was performed on the Nanopore reads using NECAT (version 0.0.1 20200803), resulting in two contigs which clearly correspond to the two V. cholerae chromosomes. The assembly was then polished using pilon (version 1.24) using default arguments, with all available reads from INPUT samples (aligned using bowtie2) as the short read inputs. Manual finishing was performed using a set of iterative rounds of breseq (version 0.37.0) runs to identify remaining discrepancies between the Illumina short read data and the in-progress assembly and resolving them by applying differences with gdtools. We then manually assigned the starting position of each chromosome to match those of commonly used El Tor reference genomes. Annotation of the newly obtained assembly was performed using prokka (version 1.14.5) with arguments --genus Vibrio --species cholerae --accver 2, using a draft genome obtained from the ***** as a reference for potential proteins. We then assigned VC numbers to all identifiable genes matching the reference El Tor strain (EMBL reference sequences AE003852.1 and AE003853.1 for chromosomes 1 and 2, respectively). Annotated genes from the El Tor reference were aligned to those of our prokka-annotated KDS1 assembly, using nucmer (v. 3.1) with default settings. We further required that for each potential match, the starting and ending positions of the reference El Tor version and the KDS1 version in the identified features differed by no more than 20 nucleotides.
Supplementary files format and content: Files with suffix _IPOD_rzchipsub_rep*.bedgraph are four-column bedgraph files with the first column representing the reference genome, second – start coordinate in 5nt increments and third – end coordinate in 5nt increments, and the fourth column represents the occupancy robust z-score of quantified total protein occupancy with RNA ChIP-seq subtracted. The robust z-scores are given for each 5 nucleotides on the genome. The rep* represents the replicate number for the sample
Supplementary files format and content: Files with suffix _IPOD_vs_inp_rzlograt_rep*.bedgraph are four column bedgraph files with the first column representing the reference genome, second -- start coordinate in 5nt increments and third – end coordinate in 5nt increments, and the fourth column represents the robust z-score of quantified total protein occupancy. The robust z-scores are given for each 5 nucleotides on the genome. The rep* represents the replicate number for the sample
Supplementary files format and content: Files with suffix _CHIP_vs_inp_rzlograt_rep*.bedgraph are four column bedgraph files with the first column representing the reference genome, second – start coordinate in 5nt increments and third – end coordinate in 5nt increments, and the fourth column represents the robust z-score of quantified RNA polymerase ChIP-seq. The robust z-scores are given for each 5 nucleotides on the genome. The rep* represents the replicate number for the sample
Supplementary files format and content: EPODs: file KDS1_IPOD_rzchipsub_mean_epods_strict.bedgraph represents called strict EPODs by the IPOD pipeline and represents the extended protein occupancy regions based on protein occupancy with RNA polymerase ChIP-seq subtracted in each of the conditions (IPOD-HR). The file is a bedgraph file where the first column is the reference genome chromosome, second – start and third – end coordinates on the genome in 5 nucleotide intervals, and fourth – 1 or 0 for whether the position is an EPOD (1) or not (0)
Supplementary files format and content: inverted EPODs (nEPODs): file KDS1_IPOD_vs_inp_rzlograt_mean_inverted_epods_strict.bedgraph represents called strict inverted EPODs by the IPOD pipeline and represents the extended negative protein occupancy regions based on total protein occupancy negative scores in each of the conditions (IPOD). The file is a bedgraph files where the first column is the reference genome chromosome, second – start and third -- end coordinates on the genome in 5 nucleotide intervals, and fourth – 1 or 0 for whether the position is an inverted EPOD (i.e. nEPOD; 1) or not (0)
Supplementary files format and content: KDS1_IPOD_vs_inp_rzlograt_inverted_strict_merged_epods.narrowpeak and KDS1_IPOD_rzchipsub_strict_merged_epods.narrowpeak are narrowpeak files where the columns represent: col 1 – reference genome chromosome, col 2 – start and col 3 – end coordinates of merged EPODs from each replicate of IPOD-HR samples. The files are generated from IPOD pipeline, see https://github.com/freddolino-lab/ipod for details.
Supplementary files format and content: Peak calls: files with suffix _idr_passed.narrowpeak are called peak files generated from our IPOD pipeline, see https://github.com/freddolino-lab/ipod for details.
Library strategy: IPOD-HR which include ChIP-seq and IPOD
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| Submission date |
Dec 17, 2023 |
| Last update date |
Feb 09, 2024 |
| Contact name |
Yulduz Rakibova |
| E-mail(s) |
yulduzr@umich.edu
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| Organization name |
University of Michigan
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| Department |
Biological Chemistry
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| Street address |
1150 W Medical center drive
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| City |
Ann Arbor |
| State/province |
MI |
| ZIP/Postal code |
48109 |
| Country |
USA |
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| Platform ID |
GPL31913 |
| Series (2) |
| GSE250405 |
TsrA regulates gene expression of horizontally acquired elements in Vibrio cholerae via both H-NS dependent and independent mechanisms [IPOD-HR] |
| GSE250408 |
TsrA regulates gene expression of horizontally acquired elements in Vibrio cholerae via both H-NS dependent and independent mechanisms |
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| Relations |
| BioSample |
SAMN38873749 |
| SRA |
SRX22917039 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7978697_delHNS_CHIP_vs_inp_rzlograt_rep2.bedgraph.gz |
11.6 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
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