GSM7978723: ΔtsrAΔhns KDS1 rep1 - INPUT; Vibrio cholerae; OTHER
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.