title: GSM4120364: IP/WT1; Brevundimonas subvibrioides; ChIP-Seq
library_construction_protocol: Wild-type cells were grown to mid-log stage and molecular crosslinking was performed by adding 10 mM sodium phosphate (pH 7.6) and 1% formaldehyde at room temperature for 10 min, followed by incubation on ice for 30 min. Crosslinking was stopped by adding glycine to final concentration of 100 mM and incubated for 5 mins at room temperature followed by 15 min on ice. Cells were centrifuged at 5000 x g at 4oC for 5 mins. The supernatant was removed, and cells were resuspended in 1 ml of 1X phosphate buffered saline (PBS, pH 7.4). This step was repeated 2 more time and cells were finally resuspended in 500 µL of TES buffer (10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 100 mM NaCl) to which 2 µL of 20,000 U/µL lysozyme was added and the solution was then incubated for 15 min at room temperature. ChIP buffer (16.7 mM Tris-HCl (pH 8.1), 167 mM NaCl, 1.1% Triton X-100, 1.2 mM EDTA) containing Protease inhibitors (Roche cOmplete EDTA-free tablets) solution (prepared as per manufacture's instruction) was prepared and 500 µL was added. After incubating for 10 mins at 37°C, the lysates were sonicated on ice to generate DNA fragments of 0.3–0.5 kbp (assessed by agarose gel electrophoresis) followed by centrifugation at 14000 x g for 5 mins at 4oC. Supernatant was collected and the protein concentration in the supernatant was measured by Pierce BCA protein assay kit (Thermo Scientific). A protein solution containing 500 µg was diluted to a final volume of 1 mL using ChIP buffer (containing protease inhibitor) with 0.01% SDS, and pre-cleared with 80 µL of Protein-A agarose (Invitrogen)(pre-blocked with 100 µg bovine serum albumin (BSA) overnight) for 1 hr at 4oC in a shaking platform. After centrifugation (3000 x g, 1 min), supernatant was collected and 10% of the supernatant was stored at -80 °C and used as total chromatin input DNA. Anti-GcrA sera (1:500 dilution) was added to the remaining supernatant with 80 µl of protein-A agarose (Invitrogen) (pre-blocked with 100 µg BSA overnight) and incubated at 4°C overnight. The pellet was washed with low salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl (pH 8.1), 150 mM NaCl) followed by centrifugation (5000 x g, 2 mins) at 4oC and the supernatant was discarded. This washing step was repeated with high salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl (pH 8.1), 500 mM NaCl), LiCl wash buffer (0.25 M LiCl, 1% NP-40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris-HCl (pH 8.1)) and finally twice with TE buffer (10 mM Tris-HCl (pH 8.1),1 mM EDTA). Elution was performed twice from the beads with 250 µL of freshly prepared elution buffer (1% SDS, 0.1 M NaHCO3) followed by addition NaCl to a final concentration of 300 mM as well as 2 µl of RNase A (10mg/ml) (Thermo scientific). Reverse crosslinking was done overnight by incubating at 65 °C. Samples were then incubated at 45 °C for 2 hr with 5 µL of Proteinase-K (20 mg/ml) in the presence of 40 mM EDTA (pH 8.0) and 40 mM Tris-HCl (pH 6.8). Phenol:chloroform:isoamyl alcohol (25:24:1) was used for DNA extraction which was followed by addition of 1/10 volume of 3M sodium acetate (pH 5.2), 100 µg glycogen and 1 volume of cold isopropanol. The solution was stored -20°C overnight. Next day, centrifugation (16000 x g, 2 min) was done to pellet glycogen containing DNA and washed with 75% ethanol followed by centrifugation (16000 x g, 2 min) twice and finally resuspended in 100 µl of TE buffer (pH 8.0). The raw Illumina 2x75bp pair-end reads were quality checked using FastQC v0.10.1, followed by adapter trimming and quality clipping by Trimmomatic 0.35. Any reads with start, end or the average quality within 4 bp windows falling below quality scores 18 were trimmed. The clean reads were aligned to the reference genome Brevundimonas subvibrioides ATCC 15264  by Bowtie 2 version 2.2.9.  Library insert size was checked by Picard Tool (https://broadinstitute.github.io/picard/). Library complexity was checked by NRF (nonredundancy fraction), defined as the number of unique start positions of uniquely mappable reads divided by number of uniquely mappable reads. IGVtools and bamCompare from deepTools were employed for comparing two BAM files based on the number of mapped reads. First the genome is partitioned into bins of equal size and then the number of reads in each bin is counted. The log2 value for the ratio of number of reads per bin of each sample was reported for IGV visualization and compared between each pair. With 95% correlation, three biological replicates were combined for peak identification. MACS2 was used for peaks calling with 0.05 FDR cutoff.
attributes.tag: GEO Accession
attributes.value: GSM4120364