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| Status |
Public on Mar 02, 2014 |
| Title |
BV2-ChIPseq-notx-CEBPb-Nmu |
| Sample type |
SRA |
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| Source name |
BV2 cells
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| Organism |
Mus musculus |
| Characteristics |
cell line: BV2
cell type: microglial
htt status: mutant Human Htt overexpression
chip antibody: CEBPb (sc-150)
expression construct: Nmu (HTT mutant)
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| Treatment protocol |
pCDH, Nwt, and Nmu Constructs: N-terminus human wild-type and N-terminus mutant huntingtin have been cloned from pCAG-Htt19550-15Q and pCAG-Htt1955-128Q respectively into MCS of pCDH-CMV-MCS-EF1-Puro (System Bioscience) using EcoRI and NotI. Lentiviral production and BV2 cells transduction were performed according to the manufacturer's protocol. Control cell line was generated by transducing BV2 cells with Lentivirus obtained from pCDH-CMV-MCS-EF1-Puro (empty vector). Validation of plasmids used in this study was performed by Western blotting.
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| Growth protocol |
Murine microglial BV2 cells, primary mouse microglia, mouse astrocyte maintained with DMEM (Cellgro) supplemented with 10% FBS (low endotoxin, Hyclone) and penicillin/streptomycin (Invitrogen). Adult microglia Purification: Cortex, striatum, and hippocampus were extracted and gently homogenized in staining buffer (HBSS (Life Technologies, 14175-095), 1% BSA, 1mM EDTA) on ice using a 2 ml polytetrafluoroethylene pestle (Wheaton), first in a 14 ml round-bottom tube (BD Falcon, 352059) and then in a 2 ml grinder chamber (Wheaton, 358029). Homogenates were filtered onto a 70 ?m cell strainer (BD Falcon, 352350) and centrifuged for 10 min at 400g. Cell pellets were resuspended in 6 ml of 37% isotonic Percoll (Sigma, P4937) and then underlayed with 5 ml of 70% isotonic Percoll in a 15ml centrifuge tube (Corning, 430790). Tubes were then centrifuged at 600g for 40 min at 18°C, with no acceleration or decelaration. Cells at the 37?70% Percoll interface were recovered and washed once in 15 ml HBSS. Cell were then incubated in staining buffer on ice with CD16/CD32 (eBioscience, clone 93) antibody for 25 min, and then with CD11b-PE (BioLegend 101208, clone M1/70) and CD45- Alexa488 (BioLegend 103122, clone 3-F11) antibodies for 30 min. Cells were then washed once and filtered onto a 40 ?m cell strainer (BD Falcon, 352340). Sorting was performed on a BD Influx cell sorter. Microglia were defined as singlets, CD11b+CD45Low events, and emcompassed 90-95% of all CD11b+ events. BMDM Isolation: Bone marrow was harvested and washed once with PBS, seeded/cultured in DMEM containing 20 % FCS, 30 % L-cell conditioned media (as source of macrophage colony stimulating factor, M-CSF), and 100 U/ml penicillin/streptomycin in non-tissue culture-treated petri dishes. After 6-8 days of culture, non-adherent cells were washed off with room temperature PBS and macrophages were obtained as a homogeneous population of adherent cells which were scraped and subsequently seeded onto tissue culture-treated petri dishes overnight in RPMI 1640 containing 10% FCS, 100 U/ml penicillin/streptomycin, and 10ng/ml M-CSF.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
20x10^6 cells were crosslinked in Formaldehyde/PBS 1% for 10 min at RT. After quenching the reaction by adding 125mM glycine, cells were washed 2X with PBS and were centrifuged (8 min, 800g, 4°C). Cells were resuspended in swelling buffer (10mM HEPES/KOH pH 7.9, 85mM KCl, 1mM EDTA, 0.5% IGEPAL CA-630, 1X protease inhibitor cocktail (Roche), 1mM PMSF) for 5 min. Cells were spun down and resuspended in 500μl lysis buffer (50mM Tris- HCl pH 7.4, 1% SDS, 0.5% EmpigenBB, 10mM EDTA, 1X protease inhibitor cocktail (Roche), 1mM PMSF) and chromatin was sheared to an average DNA size of 300–400bp by administering 5 pulses of 10 sec duration at 10 W power output with 30 sec pause on ice using a Misonix 3000 sonicator. The lysate was cleared by centrifugation (5 min, 16000g, 4°C), and supernatant was diluted 2.5-fold with 750μl dilution buffer (20mM Tris-HCl pH 7.4, 100mM NaCl, 0.5% Triton X-100, 2mM EDTA, 1X protease inhibitor cocktail (Roche)). The diluted lysate was pre-cleared by rotating for 2h at 4°C with 120μl 50% rProtein A sepharose Fast Flow (GE Healthcare). The beads were discarded, and 1% of the supernatant were kept as ChIP input. The protein of interest was immunoprecipitated by rotating the supernatant with 2.5 μg antibody overnight at 4°C, then adding 50μl blocked rProtein A sepharose and rotating the sample for an additional 1h at 4°C. The beads were pelleted (2 min, 1000g, 4°C), the supernatant discarded, and the beads were transferred in 400μl wash buffer I (WBI) (20mM Tris-HCl pH 7.4, 150mM NaCl, 0.1% SDS, 1% Triton X-100, 2mM EDTA) into 0.45 μm filter cartridges (Ultrafree MC, Millipore), spun dry (1 minute, 2200g, 4°C), washed one more time with WBI, and twice each with WBII (20mM Tris-HCl pH 7.4, 500mM NaCl, 1% Triton X-100, 2mM EDTA), WBIII (10mM Tris-HCl pH 7.4, 250mM LiCl, 1% IGEPAL CA-630, 1% Na-deoxycholate, 1mM EDTA), and TE. Immunoprecipitated chromatin was eluted twice with 100 μl elution buffer each (100mM NaHCO3, 1% SDS) into fresh tubes for 20 min. Eluates were pooled, the Na+ concentration was adjusted to 300mM with 5M NaCl and crosslinks were reversed overnight at 65°C in a hybridization oven. The samples were sequentially incubated at 37°C for 2h each with 0.33mg/ml RNase A and 0.5mg/ml proteinase K. The DNA was isolated using the QiaQuick PCR purification kit (Qiagen).
DNA from chromatin immunoprecipitation (10–50ng) was adapter-ligated and PCR amplified according to the manufacturer’s protocol (Illumina).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer IIx |
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| Description |
no treatment, 24h
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| Data processing |
ChIP-Seq: Reads were aligned to the mouse mm9 genome assembly (NCBI Build 37) using Bowtie allowing up to 2 mismatches. Only tags that mapped uniquely to the genome were considered for further analysis. ChIP-Seq experiments were normalized and visualized by using HOMER (http://biowhat.ucsd.edu/homer/) to generate custom tracks for the UCSC Genome Browser (http://genome.ucsc.edu/). Peak finding, motif finding, and peak annotation were performed using HOMER.
Genome_build: mm9
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| Submission date |
Jan 27, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Christopher Benner |
| E-mail(s) |
[email protected]
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| Organization name |
University of California, San Diego (UCSD)
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| Department |
Medicine
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| Street address |
9500 Gilman Dr. MC 0640
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| City |
La Jolla |
| State/province |
California |
| ZIP/Postal code |
92093-0640 |
| Country |
USA |
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| Platform ID |
GPL11002 |
| Series (1) |
| GSE54443 |
Mutant Huntingtin promotes neuronal death through cell autonomous microglial activation via myeloid lineage- determining factors |
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| Relations |
| BioSample |
SAMN02598189 |
| SRA |
SRX451627 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1315485_Sample18.CEBPb-Nmu.peaks.bed.gz |
598.0 Kb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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