GSM1709164: 30 min Control 1 H3 3 IP Repeat ATCACG L008 R1 001.fastq rmdup; Mus musculus; ChIP-Seq
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
Histone
Antigen
H3.3
Cell type
Cell type Class
Neural
Cell type
Cortical neuron
NA
NA
Attributes by original data submitter
Sample
source_name
Primary Cortical Neurons (E16.5 – DIV8)
strain
C57BL/6
developemental stage
E16.5 – DIV8
treatment
Control (no stimulation) 30 min (repeat of #1)
tissue/cell type
Primary Cortical Neurons
chip antibody
H3.3
chip antibody vendor
Millipore
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Crosslinking and Sonicated ChIPs (H3.1/2, H3.3 and HA): Crosslinking ChIP assays were performed with approximately 2x107 neurons per sample (all in vitro experiments analyzed in biological triplicates/group, and in vivo experiments were analyzed in biological duplicates/group). Primary cortical neurons +/- isotonic KCl stimulation (30 min, 2 hrs or 5 hrs) or FACS sorted hippocampal NeuN+ nuclei from adult mice +/- EE (4 wks) were crosslinked with 1% paraformaldehyde (PFA) for 12 minutes at room temperature and then quenched for five minutes with 0.125 M glycine. Pelleted cells/nuclei were washed 5X with 1X PBS containing protease inhibitors before being subjected to lysis and sonication as previously described (Lee et al., 2006). Briefly, crosslinked cell/nuclear pellets were re-suspended and rotated for 10 minutes in 1 mL LB1 buffer [50 mM HEPES-KOH, pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton-X 100, 10 mM sodium butyrate, plus EDTA-free protease inhibitors and PhosSTOP phosphatase inhibitors] at 4°C. Following pelleting, cells were then re-suspended in 1 mL LB2 buffer [10 mM Tris-HCl, pH 8.0, 20 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 10 mM sodium butyrate, plus EDTA-free protease inhibitors and PhosSTOP phosphatase inhibitors] and rocked at room temperature for 10 minutes. Pelleted cells/nuclei were then re-suspended in 600 µl LB3 buffer [10 mM Tris-HCl, pH 8.0, 10 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 10 mM sodium butyrate, plus EDTA-free protease inhibitors and PhosSTOP phosphatase inhibitors], passed through a 27-gauge needle 3X and split into 300 µl aliquots for sonication using a Bioruptor (Diagenode) for 50 minutes (30 seconds on/30 seconds off; setting = high) at 4°C. All samples were sonicated to approximately 150-300 bp prior to further processing. Following sonication, chromatin was pooled/sample, 1/10 volume of 10% Triton-X 100 was added and samples were spun at 20,000 x g for 10 minutes at 4°C to crack nuclei. Supernatants were then collected and incubated with antibody (7.5 µg/sample) bound sheep anti-rabbit IgG M-280 Dynabeads (Invitrogen, 11203D) on a rotator at 4°C overnight. The following day, immunoprecipitates were washed 8X with 1 mL RIPA buffer [50 mM HEPES-KOH, pH 7.6, 500 mM LiCl, 1 mM EDTA, 1% NP-40, 0.7% Na-Deoxycholate, 10 mM sodium butyrate, plus EDTA-free protease inhibitors and PhosSTOP phosphatase inhibitors], rinsed 1X with 1 mL TE + 50 mM NaCl buffer and then eluted at 65°C for 30 minutes (shaking) in 210 µl elution buffer [50 mM Tris-HCl, pH 8.0, 10 mM EDTA and 1% SDS]. After centrifugation, samples, and respective inputs, were reversed crosslinked in elution buffer 65°C overnight. Next, RNA and protein were digested with RNAse (one hour at 37°C; Roche, 11119915001) and proteinase K (two hours at 55°C; Roche, 03115887001), respectively, prior to DNA fragment purification using a Qiagen PCR purification kit. Covalent Attachment of Tags to Capture Histones and Identify Turnover (CATCH-IT) For CATCH-IT analyses, primary cortical neurons were cultured as described above for 7 DIV before being switched to conditioned specialty media lacking methionine (AthenaES) but substituted with 4 mM L-Azidohomoalanine-HCl (Aha; Medchem Source) for 24 hours. On DIV 8, neurons (~35x106/sample, analyzed in biological triplicates/condition) were treated +/- KCl (five hours), pelleted and washed 2X with 1X PBS. CATCH-IT processing was then performed and validated as previously described (Deal et al., 2010). Briefly, cells were re-suspended and lysed in 1 mL of ice-cold TM2 buffer [10 mM Tris-HCl, pH 7.0, 2 mM MgCl2, 1% NP-40, 10 mM sodium butyrate, plus EDTA-free protease inhibitors and PhosSTOP phosphatase inhibitors] with vortexing (5X) for five seconds. Following collection of neuronal nuclei by centrifugation at 1,700 x g for five minutes at 4°C, samples were washed 2X with TM2 buffer minus NP-40 and then re-suspended in 200 µl HB125 buffer [15 mM Tris-HCl, pH 7.5, 125 mM sucrose, 15 mM NaCl, 40 mM KCl, 0.5 mM Spermidine, 0.15 mM Spermine, 10 mM sodium butyrate, plus EDTA-free protease inhibitors and PhosSTOP phosphatase inhibitors]. Next, cycloaddition reagents were added as follows: biotin alkyne (final concentration of 0.5 mM), a freshly prepared mixture of CuSO4 (final concentration of 1 mM) and ascorbic acid (final concentration of 5 mM). Nuclei were then placed on a rocking platform at 4°C for 30 minutes and pelleted at 17,000 x g for five minutes at 4°C. Re-suspension in HB125 plus cycloaddition reagents (all fresh) was repeated 1X, after which nuclei were re-suspended in 250 µl HB125 with 1 mM EDTA and 2 mM CaCl2 and warmed to 37°C. Next, 15 U MNase was added and samples were further incubated at 37°C for 3.5 minutes with occasional gentle mixing. Digestions were stopped by adding EDTA to a final concentration of 2 mM and nuclei were pelleted at 100 x g for five minutes at 4°C. Nulcei were then re-suspended in 300 µl CSB 350 buffer [1X PBS, 350 mM NaCl, 2 mM EDTA, 0.1% Triton-X 100, 10 mM sodium butyrate, plus EDTA-free protease inhibitors and PhosSTOP phosphatase inhibitors] and rocked at 4°C for ~16 hours to extract chromatin. Following centrifugation and removal of supernatants (including inputs), 25 µl of packed M280 streptavidin Dynabeads (Invitrogen, 11205D) were added to the solubilized chromatin and samples were rotated at 4°C for 1.5 hours. Magnetic beads were further collected and re-suspended in 1 mL of urea wash buffer [20 mM Tris-HCl, pH 8.0, 4 M urea, 300 mM NaCl, 1 mM EDTA, 10 mM sodium butyrate, plus EDTA-free protease inhibitors and PhosSTOP phosphatase inhibitors] for five minutes at 4°C with gentle rocking. Following urea wash buffer removal, beads were re-suspended in 1 mL of CSB 350 buffer with rocking for five minutes at 4°C. Finally, beads were re-suspended in 100 µl CSB 350 buffer plus 1% SDS, treated with RNAse A (10 minutes at 37°C) and proteinase K (10 minutes at 70°C). DNA fragments associating with newly made H3/H4 proteins were then purified with Qiagen MinElute DNA purification columns and processed for ChIP-seq as described below. Following DNA purification, ChIP-seq libraries were prepared according to the Illumina protocol and sequenced with the HiSeq 2000. Reads were mapped to the mouse genome (build 37, mm9) using the Bowtie alignment software (Langmead et al., 2009).