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Status |
Public on Jan 22, 2024 |
Title |
CnR_IgG2: Satb2 flx/flx DIV14 primary cortical cultures, 1h bic, IgG, rep2 |
Sample type |
SRA |
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Source name |
Cortex
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Organism |
Mus musculus |
Characteristics |
tissue: Cortex cell type: DIV14 primary cortical cultures genotype: Satb2 flx/flx treatment: 1h bicucculine cut&run antibody: Rabbit IgG Negative Control Antibody (EpiCypher, 13-0042)
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Treatment protocol |
Cottical neurons were treated with 20 µM NBQX for 16 h on DIV13. After silencing, neurons were stimulated with 50 µM biccuculine for 1h on DIV14 and then collected.
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Growth protocol |
Corical tissue was dissected at P0-P1, dissociated with Papain and titurated. Cells were plated in serum-containing medium (MEM, 1% horse serum, 0.1% Pen/Strep, 0.1 % sodium piruvate). After 2h, MEM was excahnged with serum-free feeding medium (Neurobasal-A Medium, containing B-27, 0.1% Pen/Strep and 2 mM Glutamine. Cells were treated with 5µM cytosine arabinoside on DIV3 to inhibit glial cell proliferation. At DIV4, one third of the medium was replaced by fresh serum-free feeding medium. Neurons were cultured for 14 days in 5% CO2 at 37°C.
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Extracted molecule |
genomic DNA |
Extraction protocol |
For CUT&RUN, nuclei of 106 cells were isolated by addition of nuclei extraction buffer (NEB, 20 mM Hepes (KOH) pH 7.9, 10 mM KCl, 0.5 mM Spermidine, 0.1 % Triton X-100, 20 % Glycerol, 1 tablet cOmplete EDTA-free Protease inhibitor). Cells were incubated at 4 °C for 15 min, lifted from the dish using a cell lifter and gently resuspended. Nuclei suspension was spun down at 600 g at 4 °C for 3 min. Pellet was resuspended in NEB and intact nuclei were counted in a Neubauer chamber. 100 000 nuclei were used for each CUT&RUN reaction. After Concavalin-A conjugated beads activation (21-1401-EPC Epicypher), nuclei were demobilized and blocked in blocking buffer (20 mM Hepes pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, 0.1 % BSA, 2 mM EDTA, 1 tablet cOmplete EDTA-free Protease inhibitor) at RT for 5 min. After washing in washing buffer (20 mM Hepes pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, 0.1 % BSA, 1 tablet cOmplete EDTA-free Protease inhibitor) primary antibody was added and incubated at 4 °C for 2 h. Reaction was washed twice before adding pAG-MNAse (15-1016-EPC Epicypher) and incubated for 1h at 4°C. Next, beads were washed twice before addition of 100 mM CaCl2 for 30 min at 0 °C in an ice water bath. 2X STOP buffer (200 mM NaCl, 20 mM EDTA, 4 mM EGTA, 50 µg/ml RNAseA, 40 µg/ml Glycogen, 10 pg/ml E.Coli spike-in DNA) was added and beads were incubated for 20 min at 37 °C to release DNA fragments and to digest RNA. DNA was purified by Phenol-Chloroform extraction and precipitated in 95 % EtOH at -80 °C. Pellet was dissolved in TE buffer pH 8.0 (12090015, ThermoFisher) and stored at -20 °C. Libraries were prepared using the NEBNext Ultra Library prep Kit (E7645S, New England Biolabs) according to the manufacturer’s protocol. Adapters were used according to the DNA input at a working concentration of 1.5 µM. Library was PCR amplified with unique indexes (E7335S, New England Biolabs) for 14 cycles. After clean-up fragment size and concentration was analyzed before sending for sequencing.
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Library strategy |
OTHER |
Library source |
genomic |
Library selection |
other |
Instrument model |
Illumina NovaSeq 6000 |
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Data processing |
Illumina Universal adapters were trimmed with Trim Galore! with default parameters (--paired --illumina). The trimmed reads were aligned to mouse genome (mm10) with Bowtie2 (v2.4.5) with the following settings: bowtie2 --dovetail --local --very-sensitive-local --no-unal --no-mixed --no-discordant -q --phred33 -I 10 -X 700 -p 48 -x mm10. Reads with a mapping quality score (MAPQ) < 2 as well as optical and PCR duplicates are excluded. Replicate BAM files were merged and handled as single file for the next steps. Genome coverage bedgraph files were computed with bamCoverage (bin size 50 bp) and normalized to reads per kilobase per million (RPKM) to minimize bias arising from different sequencing depths and batches. Unspecific noise was filtered out by subtracting the mean signal from each value of the corresponding bedgraph files. Peaks were called with SEACR v1.4 under relaxed conditions and normalized to the IgG control bedgraph file. Assembly: mm10 Supplementary files format and content: bed Library strategy: CUT&RUN
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Submission date |
Jan 11, 2023 |
Last update date |
Jan 22, 2024 |
Contact name |
Nico Wahl |
E-mail(s) |
nico.wahl@i-med.ac.at
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Phone |
0043512900371255
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Organization name |
Medical University Innsbruck
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Department |
Institute for Neuroscience
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Street address |
Innrain 66
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City |
Innsbruck |
ZIP/Postal code |
6020 |
Country |
Austria |
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Platform ID |
GPL24247 |
Series (2) |
GSE222608 |
SATB2 organizes the 3D genome architecture of cognition in cortical neurons [CUT&RUN] |
GSE222609 |
SATB2 organizes the 3D genome architecture of cognition in cortical neurons |
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Relations |
BioSample |
SAMN32675334 |
SRA |
SRX19001678 |
Supplementary data files not provided |
SRA Run Selector |
Raw data are available in SRA |
Processed data are available on Series record |
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