GSM8257092: liver, WT, ZT6, BRG1, ChIP, rep2; Mus musculus; ChIP-Seq
For all ChIPs 2-3 biological replicates for each condition were pooled by weighing a total of ~220-250mg of snap-frozen liver tissue. Which was homogenized in 10mL PBS containing 2mM DSG (ProteoChem, 79642-50-5) using IKA T10 standard ULTRA TURRAX on level 4 for 15 sec followed by dounce homogenization in a 15mL douncer for 20 strokes with a tight pestle. The samples were incubated for 40 min at RT under rotation and centrifuged for 5 min at 900g. Supernatant was discarded, and the pellet was resuspended in 10mL PBS, centrifuged for 5 min at 900g, and supernatant was discarded. The pellet was resuspended in 10mL PBS containing 1% formaldehyde (Thermo Scientific, 28908) and incubated at RT for 5 min under rotation. 1mL 1.25M glycine was added, and samples were incubated for 5 min at RT under rotation to stop the crosslinking. The samples were dounce homogenized in a douncer for 20 strokes with a tight pestle and filtered through a 70µM pluriStrainer (pluriSelect, 43-50070). The samples were centrifuged at 900g for 5min at 4 °C and supernatant was discarded. Pellet was resuspended in 10mL PBS with 1mM phenylmethylsulfonyl fluoride (PMSF) (Sigma, 11359061001). The samples were centrifuged at 900g for 5min at 4 °C and supernatant was removed. The pellet was resuspended in 10mL cell wash buffer 1 (10mM Tris-HCl pH 8, 10mM EDTA, 0.5mM egtazic acid (EGTA), 0.25% Triton X-100) containing 1mM PMSF, centrifuged, and supernatant was discarded. Hereafter the pellet was resuspended in 10mL cell wash buffer 2 (10mM Tris-HCl pH 8, 1mM EDTA, 0.5mM EGTA, 200mM NaCl) containing 1mM PMSF. The samples were centrifuged, and supernatant was discarded. The pellet was resuspended in 1mL of ChIP buffer (50mM Trish-HCl pH 8, 1mM EDTA, 150mM NaCl, 1% Triton X-100, 0.1% deoxycholic Na, 0.1% SDS) with 1:200 PIC. 25µL Pierce ChIP-grade Protein A/G Magnetic Beads (Thermo Scientific, 26162) were prepared for each sample. The total volume of beads needed was aliquoted to a new tube and placed in a magnetic rack for 5 min. Supernatant was removed. Beads were washed twice by resuspending in 600µL ChIP buffer, placing the tube in a magnetic rack, turning 20x180°, leaving it for 2 min, and discarding supernatant. After the second wash the beads were resuspended in the initial volume of ChIP buffer (25µL per sample). 25µL of the prepared beads were added to each sample and they were incubated under rotation for 30 min at 4 °C. This preclearing gets rid of chromatin fragments that bind unspecifically to the beads, meaning that they bind to the beads without the presence of antibody. After incubation, the samples were placed in a magnetic rack, incubated for 5 min, and the supernatant was transferred to a new tube. 1% of the volume was transferred to a new tube and saved as an input. 5µL drosophila spike-in chromatin (active motif, 53083) and 2µL spike-in antibody (active motif, 61686) was added. The spike-in can be used to normalize for efficiency of immunoprecipitation after sequencing. However, this normalization method was not used in this report. 10µL of BRG1 antibody (Abcam, ab110641) was added to the samples, followed by ON incubation under rotation at 4 °C. 50µL of beads, prepared as described above, was added to each sample, and the samples were incubated for 1 hour under rotation at 4 °C. The samples were placed in a magnetic rack for 2 min and supernatant was discarded. Beads were subsequently washed by resuspending in different wash buffers, listed below, by placing the tube in a magnetic rack, turning 20x180°, incubating for 2 min, and discarding the supernatant. The washing buffers are: ChIP wash 1 (20mM Tris pH 8, 150mM NaCl, 2mM EDTA, 0.1% deoxycholic Na, 1% Triton X-100), ChIP wash 2 (20mM Tris pH 8, 500mM NaCl, 2mM EDTA, 0.1% deoxycholic Na, 1% Triton X-100), ChIP wash 3 (10mM Tris pH 8, 250mM LiCl, 1mM EDTA, 0.7% deoxycholic Na, 1% Igepal CA-630), and twice with TE buffer (10mM Tris-HCl pH 8, 1mM EDTA). After the final wash in TE buffer the supernatant was discarded and 150µL of elution buffer (1% SDS, 100mM NaHCO3) was added to the beads. The samples were incubated in a thermomixer at 65 °C at 1000 rpm for 30 min. The tubes were placed in a magnetic rack and incubated for 5 min. The supernatant was transferred to a new tube and the beads was resuspended with 150µL elution buffer and incubated in the thermomixer again. The tubes were transferred to a magnetic rack and the supernatant was pooled with supernatant from first elution and the beads were discarded. Elution buffer was added to the input samples for a final volume of 300µL. 12µL 5M NaCl was added to all samples, including inputs, and the samples were incubated at 65 °C ON to reverse DNA-protein crosslinking. 6µL 0.5M EDTA, 12µL 1M Tris-HCl pH 6.5, and 1µL 10mg/mL RNase A (Thermo Scientific, EN0531) was added to the samples. The samples were incubated at 37 °C for 30 min. 5µL 20 g/L proteinase K (Invitrogen, 25530049) was added and the samples was incubated at 56 °C for one hour. The DNA was purified using MinElute PCR purification kit. 7 volumes of buffer PB was added to the samples. 700µL of the sample was loaded onto the column, centrifuged at 16000g, and flowthrough was discarded. This process was repeated until the entire sample was passed through the column. The column was washed twice with buffer PE. Flowthrough was discarded and the column was centrifuged an additional time to get rid of excess ethanol, possible leftover ethanol around the O-ring was removed with a pipette. The column was transferred to a clean DNA LoBind tube (Eppendorf). 12µL of buffer EB was loaded to the center of the membrane, the samples were incubated for 1 min and centrifuged for 1 min. DNA concentrations of the samples was determined using Qubit. ChIP samples were generated using NEBNext® reagents and kits for Illumina® Sequencing following manufacturer's instrcutions. Samples were sequenced on an Illumina NovaSeq 6000 instrument.