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| Status |
Public on Oct 04, 2020 |
| Title |
CUTRUN_CA1_CaMK2a_KA2_IgG |
| Sample type |
SRA |
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| Source name |
CA1_CaMK2a_KA2_IgG
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| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
age: 3-4 weeks
genotype: CaMK2a-Cre/+; LSL-Sun1-GFP/+
treatment: 2-3-hour kainic acid
antibody: Rabbit IgG (Cell Signaling Technologies 2729)
tissue: CA1 region of hippocampus
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Hippocampal tissue from mice was rapidly dissected and dounce homogenized. Dounce homogenization was performed in Buffer HB (0.25M sucrose, 25mM KCl, 5mM MgCl2, 20mM Tricine-KOH, pH 7.8 supplemented with protease inhibitors, 1mM DTT, 0.15mM spermine and 0.5mM spermidine) with tight pestle for 20 strokes in a total of 1.5mL volume. Tissue was then supplemented with 96uL 5% IGEPAL CA-630 and dounced an additional 5 strokes with tight pestle. Homogenate was then filtered through a 40-micron strainer to remove large debris and collected in a 15mL conical. 3.5mL of Buffer HB and 5mL of working solution (50% iodixanol with 25mM KCl, 5mM MgCl2, 20mM Tricine-KOH pH 7.8 supplemented with protease inhibitors, DTT, spermine and spermidine) was added. Homogenized tissue was underlaid with 1mL of 30% iodixanol and 1mL of 40% iodixanol (diluted from working solution) solutions. Samples were centrifuged at 10,000xg for 18 minutes. Nuclei were collected in a 250uL volume at the 30/40% iodixanol interface. Isolated nuclei were diluted two-fold with CUT&RUN wash buffer supplemented with 4mM EDTA and stained with DRAQ5 (Abcam ab108410) at a 1:500 dilution. CaMK2a+ (GFP+) nuclei, resulting from CaMK2a-Cre-mediated expression of Sun1-sfGFP-Myc, were isolated using flow cytometry using a Sony SH800Z Cell Sorter. Sorted nuclei were resuspended in 1 mL cold CUT&RUN wash buffer (20mM HEPES pH 7.5, 150mM NaCl, 0.2% Tween-20, 1mg/mL BSA, 10mM sodium butyrate, and 0.5mM spermidine supplemented with protease inhibitors), using 50,000 nuclei for each reaction. Nuclei were bound to magnetic Concanavalin-A (ConA) beads (Bangs Laboratories) that had been washed with CUT&RUN binding buffer (20mM HEPES-KOH pH 7.9, 10mM KCl, 1mM CaCl2, 1mM MnCl2). ConA-bead-bound nuclei were then incubated overnight in cold CUT&RUN antibody buffer (CUT&RUN wash buffer supplemented with 0.1% Triton X-100 and 2mM EDTA) and an in-house polyclonal Fos antibody (affinity eluted #1096) or rabbit IgG antibody (Cell Signaling Technologies # 2729). After antibody incubation, ConA-bead-bound nuclei were washed with CUT&RUN antibody buffer, resuspended in CUT&RUN Triton-wash buffer (CUT&RUN wash buffer supplemented with 0.1% Triton X-100), and Protein-A-MNase was added at a final concentration of 700ng/mL. Samples were incubated at 4°C for 1h. The ConA-bead-bound nuclei were then washed twice with CUT&RUN Triton-wash buffer and ultimately resuspended in 100uL of CUT&RUN Triton-wash buffer. 3uL of 100mM CaCl2 was added to each sample and samples were incubated on ice for 30 min. The reaction was stopped by adding 100uL of 2x STOP buffer (340mM NaCl, 20mM EDTA, 4mM EGTA, 0.04% Triton X-100, 20pg/mL yeast spike-in DNA, and 0.1ug/mL RNase A) and incubation at 37°C for 20 min. After incubation, ConA beads were captured using a magnet and supernatants containing DNA fragments released by Protein-A-MNase were collected. Supernatants were then treated with 2uL of 10% SDS and 2uL of 20mg/mL Proteinase-K and incubated at 65°C with gentle shaking for 1h. DNA was then purified using standard phenol/chloroform extraction with ethanol precipitation. DNA was resuspended in 30uL of 0.1x TE buffer.
CUT&RUN sequencing libraries were generated essentially as previously described (Hainer and Fazzio, Curr. Protoc. Mol. Biol., 2019), with the following modifications: Adapter ligation to end-repaired, A-tailed DNA was performed using Rapid T4 DNA ligase (Enzymatics). PCR-amplified libraries were purified from adapter dimers using a 1.1x ratio of AMPure XP beads, eluting in 20uL of 10mM Tris pH 8.0.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
Library strategy: CUT&RUN
Sequencing reads were trimmed for quality and remaining adapter sequence using Trimmomatic v0.36 (settings: LEADING:20 TRAILING:20 SLIDINGWINDOW:4:15 MINLEN:25) and kseq (default settings)
Trimmed reads were aligned to the mm10 genome using Bowtie2 v2.2.9 with the following parameters: --local --very-sensitive-local --no-unal --dovetail --no-mixed --no-discordant --phred33 -I 10 -X 700. Trimmed reads were also aligned to the sacCer3 genome with the same parameters to recover reads corresponding to yeast spike-in DNA used in CUT&RUN.
Genome-wide coverage of CUT&RUN fragments was generated using Bedtools v2.27.1 genomecov, normalizing to the number of yeast spike-in reads obtained for each sample.
BigWigs representing the average coverage across the three replicates were generated by concatenating BAM alignments for each individual replicate for a given condition aligned to either the mm10 or sacCer3 genome (for spike-in reads), and calculating normalized genome-wide coverage as above.
Peaks were identified for Fos CUT&RUN using SEACR v.1.1 using the following parameters: norm, relaxed. CUT&RUN performed using rabbit IgG was used as the negative control sample for peak calling. Peaks were subsequently filtered to identify peaks found in all three biological replicates for each condition using Bedtools v2.27.1 intersect, creating a conservative set of Fos-bound sites. Peaks within 150 bp of each other were then merged using Bedtools v2.27.1 merge.
Genome_build: mm10
Supplementary_files_format_and_content: BigWigs represent spike-in normalized genome-wide CUT&RUN coverage for each individual replicate, as well as a "MergedReplicates" sample that represents the average signal across three replicates. BED files represent peaks identified in Fos CUT&RUN experiments that are enriched relative to IgG in the KA condition. The peaks are filtered to preserve only those peaks observed in all three biological replicates.
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| Submission date |
Sep 30, 2020 |
| Last update date |
Oct 05, 2020 |
| Contact name |
Michael E. Greenberg |
| Organization name |
Harvard Medical School
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| Department |
Neurobiology
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| Lab |
Greenberg
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| Street address |
220 Longwood Ave
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| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02115 |
| Country |
USA |
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| Platform ID |
GPL19057 |
| Series (2) |
| GSE158838 |
Bidirectional perisomatic inhibitory plasticity of a Fos neuronal network [CUT&RUN] |
| GSE158843 |
Bidirectional perisomatic inhibitory plasticity of a Fos neuronal network |
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| Relations |
| BioSample |
SAMN16319989 |
| SRA |
SRX9221340 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4811929_CUTRUN_CA1_CaMK2a_KA2_IgG_spikein_norm_coverage.bigwig |
34.4 Mb |
(ftp)(http) |
BIGWIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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