title: GSM8792904: RpoC ChIP of  S. Typhimurium SL1344 rpoC-3xFLAG cells growing in LB at 37°C OD=0,1 (EEP condition) Replicate 3; Salmonella enterica subsp. enterica serovar Typhimurium str. SL1344; ChIP-Seq
library_name: GSM8792904
library_construction_protocol: Hi-C: Frozen pellets were thawed on ice, resuspended in 200 μL of 1× Tris-EDTA (TE) buffer (Sigma-Aldrich, Cat# T928) containing a complete protease inhibitor cocktail (Roche, Cat# 11873580001). Then, 1 µl of ready-lyse lysozyme (LGC Biosearch, Cat# LU-R1804M) was added and the mixture was incubated at RT for 20 minutes.  Next, 10 µl of 10% SDS (Sigma-Aldrich, Cat#  05030) was added to the cells and incubated for 10 minutes at RT. Subsequently, the lysed cells were transferred into a 2 mL Eppendorf tube containing 800 μL of digestion mix: 100 μL of 10× NEB1 buffer (New England Biolabs, Cat# B7001S), 100 μL of 10% Triton (Sigma-Aldrich, Cat# T8787), and 600 μL of sterile water. After removing 80 μL of non-digested control, 20 μL of HpaII enzyme (New England Biolabs, Cat# R0171L) were added to the digestion mix. Both tubes were then incubated for 3 hours at 37°C with agitation. Following DNA digestion, 80 μL of the digested control were removed and kept on ice with the non-digested control. The remaining digestion mix was centrifuged for 20 minutes at 16,000 × g, 4°C, and the pellet was re-suspended in 80 µL of sterile water. The 80 µL of digested DNA was then added to the biotinylation mix containing 10 µL of 10× ligation buffer, 1 µL of 10 mM dAGTP mix, 3 µL of 1 mM biotin-14-dCTP (Jena Biosciences, Cat# E-NU-956-BIO14-S), and 2 µL of DNA polymerase I, large (Klenow) fragment (New England Biolabs, Cat# M0210L), and incubated for 1 hour at 37°C with agitation. Following biotinylation, proximity ligation of DNA fragments was performed by adding the ligation mix: 24 µL of 10× ligation buffer (without ATP), 2.4 µL of 100 mM ATP, 2.4 µL of 10 mg/mL BSA (New England Biolabs, Cat# B9000S), and 3.2 µL of of T4 DNA ligase (Thermo Fisher Scientific, Cat# EL0013). Ligation was performed at 25°C for 3 hours with gentle agitation. The crosslink was then reversed by incubating the samples (including the non-digested and digested controls) overnight at 65°C in the presence of proteinase K (20 mg/mL; Eurobio Scientific, Cat# GEXPROK01-B5), EDTA (500 mM; Sigma-Aldrich, Cat# 03690), and 10% SDS. The next day, DNA was purified by phenol extraction and ethanol precipitation. Briefly, one volume of phenol:chloroform:isoamyl alcohol (25:24:1) (Thermo Fisher Scientific, Cat# 10308293) was added to the samples. Following centrifugation, the upper aqueous phase was transferred to a new 1.5 mL tube, and DNA was precipitated with 1/10 volume of 3 M Na-acetate (pH 5.2) and two and a half volumes of cold ethanol. After incubation at −80°C for 30 minutes, the DNA was pelleted, washed once with 70% ethanol, and dried using an Eppendorf concentrator plus speed vac. The DNA was resuspended in 130 µL of 1× TE buffer containing 10 μg/mL RNase A (Thermo Fisher Scientific, Cat# EN0531) ChIP-seq: frozen cell pellets, fixed with 1% formaldehyde, were thawed on ice and re-suspended in 500 μl of lysis buffer I: 20% sucrose (Sigma-Aldrich, Cat# S7903), 10 mM Tris-HCl (pH 8), 50 mM NaCl, and 10 mM EDTA, supplemented with 4 μl of ready-lyse lysozyme. The mixture was incubated at 37°C for 30 minutes. Next, 500 μl of lysis buffer II (50 mM Tris-HCl (pH8), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100 and complete protease inhibitor cocktail) was added. The cells were transferred to a 1 mL Covaris tube and sonicated using a Covaris S220 focused Ultrasonicator. Sonication parameters were set to 210 seconds for EEP samples and 270 seconds for ESP samples (peak power = 140 W; duty factor = 5%; cycles per burst = 200). Following sonication, the samples were centrifuged at 18,000 × g for 30 minutes at 4°C. The supernatant was transferred into a new 1.5 mL tube, with 50 μL set aside as input and stored at −20°C until further use. Immunoprecipitation of FLAG-tagged proteins was performed by incubating the cell extracts with 100 μL of ANTI-FLAG M2 affinity gel (Sigma-Aldrich, Cat# A2220) overnight at 4°C on a rotator. The beads were washed twice with 400 μL of TBS (Tris-HCl 50mM pH 7.4, 150mM NaCl) containing 0.05% Tween 20, followed by three washes with 400 μL of cold TBS. Each wash step was performed under rotation for 10 minutes at 4°C. The 3xFLAG-protein-DNA complexes were eluted by adding 2.5 volumes of 3xFLAG peptide solution (Sigma-Aldrich, Cat# F4799) relative to the total packed gel volume. The samples were incubated for 90 minutes on a rotator at 4°C, centrifuged for 30 seconds at 5000 × g at 4°C, and the supernatant was transferred to 1.5 mL LoBind DNA tubes. A second elution step was performed similarly. The IP and input samples were treated with RNase A (10 μg/mL) for 1 hour at 37°C and then incubated overnight at 65°C with proteinase K (final concentration: 50 μg/mL) to reverse the formaldehyde crosslinks. DNA was then purified using a Qiagen MinElute PCR Purification Kit (Qiagen, Cat# 28004) and stored at -20°C until further processing. RNA-seq:Frozen pellets of sorted cells were thawed on ice and resuspended in 1 mL of TRIzol (Qiagen, Cat# 79306), followed by the addition of 400 µL of chloroform. After a 3-minute incubation, the mixture was centrifuged at 20,000 × g for 15 minutes at 4°C. The upper aqueous phase containing RNA was carefully transferred to a new tube, and 600 µL of isopropanol was added to precipitate the RNA at room temperature. Following a 30-minute centrifugation at 20,000 × g at 4°C, the supernatant was discarded. The RNA pellet was washed with 500 µL of 70% ethanol, air-dried, and resuspended in RNase-free water by shaking at 900 rpm for 5 minutes. RNA was treated with DNase according to the manufacturer's instructions (Invitrogen™ TURBO DNA-free™ Kit, Cat# AM1907). RNA concentrations were determined using either a Nanodrop spectrophotometer or a Qubit fluorometer (Invitrogen). RNA quality was assessed with an Agilent 2100 Bioanalyzer, and samples were stored at −20°C. For Hi-C and ChIP-seq libraries preparation, samples were first sheared to a target fragment size of 300 bp using a Covaris S220 instrument. Then, Hi-C libraries were prepared as described in (Ckrockram et al, STAR protocol, 2021). For ChIP-seq libraries, after sonication, IP and input samples were purified using 1.6x volume of AMPure XP beads according to manufacturer instructions (Beckman Coulter, Cat# A63881). DNA was eluted into 40 μL of elution buffer (EB, 10 mM Tris-HCl pH 7.5). After adjusting the volume of the samples to 80 μL with sterile water, DNA fragments were end-repaired by adding 40 μL of the following mix: 12 μL 10× T4 DNA ligase buffer (New England Biolabs, Cat# B0202S), 4 μL 10 mM dNTP mix, 5 μL T4 DNA polymerase (3 U/μL, New England Biolabs, Cat# M0203L), 5 μL T4 Polynucleotide Kinase (10 U/μL, Thermo Fisher Scientific, Cat# EK0032), 1 μL DNA polymerase I, large (Klenow) fragment (5 U/μL, New England Biolabs, Cat# M0210L), and 13 μL H2O . The reaction was incubated at room temperature (RT) for 30 minutes, purified using 1.6× AMPure XP beads, and DNA was eluted in 30 μL of EB. Purified DNA was then dA-tailed by adding 5 μL of 10× NEBuffer 2 (New England Biolabs, Cat# B7002S), 10 μL of 1 mM dATP (Sigma-Aldrich, Cat# D4788), 3 μL of Klenow Fragment (3'–5' exo-), (5 U/μL, New England Biolabs, Cat# M0212L), and 2 μL of H2O. The reaction was incubated at 37 °C for 30 minutes, followed by heat inactivation at 65 °C for 20 minutes to deactivate the Klenow Fragment (3'–5' exo-). After purification with 1.6× volume of AMPure XP beads, DNA was eluted in 12 μL of EB. Illumina adapters were ligated using the NEBNext Quick Ligation Module (New England Biolabs, Cat# E6056S) for 15 minutes at RT. The ligation reaction was then purified using AMPure XP beads. First, the sample volume was adjusted to 50 μL with water, and a double size selection step was performed with the beads at a ratio of 0.7×-1.1×. For 3xFLAG-H-NS and 3xFLAG-RpoC libraries, PCR amplification was performed in a single 15-cycle reaction using  5 µl of library, 10 µl of 2 µM Illumina primers P1 and P2 mix, 10 µl Phusion 2× High Fidelity Master Mix (New England Biolabs, Cat#  M0531S) and 10 µl of H2O (for both IP and input). For the 3xFLAG-HilD and 3xFLAG-H-NS on sorted populations, three PCR reactions were performed for IP samples and one for the input sample. PCR products were purified using Qiagen MinElute PCR purification columns, and primer dimers were removed using AMPure XP beads. The concentration of Hi-C and ChIP-seq libraries was quantified using a Qubit fluorometer (Invitrogen) with the Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific, Cat# 10606433). Libraries were diluted to 0.5 ng/μL, and their quality was assessed on an Agilent 4150 TapeStation system using Agilent High Sensitivity D5000 ScreenTape (Agilent, Cat# 5067-5592). RNA-seq libraries.....