List of tables
Chapter 3. DNA methylation changes in plasticity genes accompany the formation and maintenance of memory
3.2.3 Chromatin immunoprecipitation and sequencing (ChIP-seq)
ChIP protocol was optimized for low chromatin (0.5 to 1ug) input samples as described in the publication (Halder et al., 2015). ChIP-grade antibodies were used for this purpose, which was previously validated according to the Antibody Validation Database (Egelhofer et al., 2011).
ChIP-seq experiments were only performed for 1-hour time point in CA1 (cellular consolidation) using detailed methods described in (Halder et al., 2015). In brief, FACS sorted nuclei were centrifuged at 3200g for 15 minutes, the supernatant was removed, and nuclei were resuspended in RIPA buffer. Nuclei were transferred into a fresh Diagenode tube and samples were sheared in a Diagenode Bioruptor plus apparatus. 4 times 5 cycles were run with 30 Sec ON/OFF setting with high power. Samples were centrifuged in between after every 5 cycles. The sheared chromatin was centrifuged, and the supernatant was aliquoted in the DNA low binding tubes (Eppendorf). Chromatin was stored in -80oC after liquid nitrogen snap freezing.
DNA was isolated from an aliquot of chromatin using the SureClean method. In brief, 10 ul of chromatin was added in 20 ul of EB buffer. In the tube, 1 ul of RNase A (50 ng/ul) was added, and the solution was incubated for 30 minutes at 37oC. After the incubation, 1 ul of proteinase K (20 mg/ml) was added and incubated at 65oC for 2 hours with 800 RPM shaking. 3 ul of the co-precipitant (LPA 5 mg/ml) and 1 volume of SureClean was added. The solution was vortexed and incubated at room temperature for 10 minutes. Tubes were centrifuged at 15000g for 20 minutes
at room temperature, and the supernatant was removed. The pellet was dried, resuspended in 15 ul EB buffer and DNA content was measured using Qubit dsDNA HS assay kit. The size of the DNA fragments was also checked on an Agilent 2100 bioanalyzer using a DNA high sensitivity bioanalyzer chip. This shearing process should produce an average size of 300bp. If shearing was not good, samples were sonicated for more cycles and process was repeated.
The chromatin samples were diluted in IP buffer (10 times) including Roche Complete protease inhibitors, and chromatin was pre-cleared using BSA blocked protein A magnetic beads (Dynabeads from Invitrogen). The solution was incubated for 1 hour at 4oC. After incubation, tubes were kept on a magnetic rack and supernatant was taken. Chromatin was split into aliquots for using them in different immunoprecipitation (IP) assay. One tube with 10% Input (10% of one IP) was also aliquoted. To prepare BSA blocked protein-A magnetic beads, required volume of beads were calculated (based on samples), and taken into a 1.5 ml Vial and kept on a magnetic rack. The supernatant was removed, and beads were washed with 1 ml IP buffer. After that, beads were incubated with a solution containing 1 ml IP buffer and 0.5% BSA for 2 hours at 4oC. Beads were rewashed two times with the IP buffer and resuspended in the same volume of IP buffer as the starting volume. BSA blocked beads could be stored at 4oC for 2-3 days.
The amount of chromatin was optimized for the immunoprecipitation assay with the different ChIP-grade antibodies as described in (Table 3.2) below and incubated on a rotating wheel at 4oC for overnight.
Ab specificity Ab ID Input
chromatin/DNA [µg]
Ab [µg] per IP DNA recovery [ng]
H3 Abcam ab1791 0.1 0.5 6 – 15
H3K4me1 Abcam ab8895 0.5 0.5 3 – 10
H3K4me3 Abcam ab8580 1 1 1 – 5
H3K27ac Abcam ab4729 0.5 0.5 2 – 8
H3K9ac Millipore 07-352 0.5 1 (µL) 1.5 – 3
H3K79me3 Abcam ab2621 0.5 0.5 2 – 5
H3K27me3 Millipore CS200603
0.5 0,25 (µL) 2 – 5
Table 3.1 Antibody concentrations.
Details of the amount of chromatin and antibodies required for ChIP assays, adapted from (Halder et al., 2015).
After the overnight incubation, 15 ul of BSA blocked protein-A magnetic beads were added to each sample, and the tubes were incubated on a wheel for 2 hours at 4oC. The beads were washed two times with 200 ul cold IP buffer with 0.1% SDS and three times with 200 ul cold wash buffer. For the last two washes, samples were incubated 10 minutes on a rotator at 4oC. Beads were again washed twice with 200 ul cold IP buffer and twice with 200 ul cold TE (without any protease inhibitors). After the last washing step, the supernatant was removed, and the beads were incubated in EB buffer (1mM Tris pH 8) with the RNase A solution (0.1 ug/ul) for 30 minutes at 37oC. The RNase A treatment was also done on the Input sample that was saved in the previous step. To perform the de-crosslinking, the beads and the input samples were added with 2X Weinmann buffer (WB) without inhibitors and tubes were incubated in the presence of 1 ul proteinase K (0.5 ug/ul) and 1% SDS at 65oC for overnight shaking (800 RPM). Tubes were kept on the magnetic rack, and the supernatant was transferred to a fresh DNA low binding tube (Eppendorf). To increase the yield, the beads were washed one more time using EB and tubes were incubated for 10 minutes at 65oC with 800 RPM rotation. The supernatant was added again to the previous DNA low binding tube. The DNA was isolated using SureClean precipitation method as described earlier in the presence of LPA (linear acrylamide). The DNA pellet was washed two times using 70% ethanol. All the ethanol was removed by drying and using speedvac for 3 minutes at room temperature. The DNA was resuspended in EB (Tris 10 mM, pH 8) and the concentration was determined using Qubit dsDNA HS assay. The immunoprecipitation efficiency was also validated using qPCR with positive and negative regions.
Library preparation condition was optimized for a low amount (0.5 ng) of input materials to generate reliable and quantifiable libraries, as published in (Halder et al., 2015). The Diagenode MicroPlex kit or NEBNext Ultra DNA library preparation kit for Illumina (NEB) was used for this purpose. After template preparation and adapter ligation, the number of amplification cycles
was determined using a qPCR on a small amount of IP material to avoid over-amplification.
After the library amplification PCR, libraries were purified using the SureClean method as described earlier. After ethanol wash and drying, the pellet was resuspended in 10 mM Tris pH 8.
ChIP libraries were validated by qPCR assay and concentration was measured by a Qubit dsDNA HS assay kit. The library size was determined by a DNA High sensitivity bioanalyzer assay using the manufacturer’s protocol, and the library was adjusted to 2 nM concentration for the sequencing on a Hiseq 2000 (Illumina) according to the manufacturer’s protocol.