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| Status |
Public on Jun 02, 2022 |
| Title |
IPS_GABA_H3K27ac_t15_Rep2 |
| Sample type |
SRA |
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| Source name |
NSB3188 (iPSC)
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| Organism |
Homo sapiens |
| Characteristics |
cell type: induced GABAergic neurons
treatment: depolarization
timepoint (minutes): 15
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| Treatment protocol |
Prior to depolarization, medium was replaced with serum free Neurobasal A/B27/Glutamax for 3 days. The neuronal culture was then silenced using 1μM of Tetrodotoxin citrate (TTX) (Labome 1069/1) and 100μM of DL-2-amino-5-phosphopentnoic acid (DL-AP5) (Abcam ab120271) for 12-16h. The neurons were then depolarized by adding depolarization buffer (170 mM KCl, 2 mM CaCl2, 1 mM MgCl and 10 mM HEPES directly into the media to a final concentration of 30% of the total culture media. Neurons were collected at different time points after depolarization for experiments.
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| Growth protocol |
IPS_GABA_H3K27ac_t30_Rep1
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
CUT&RUN was performed as previously described. Nuclei (50,000) were extracted from neurons using NE1 buffer (20 mM HEPES-KOH pH 7.9, 10 mM KCl, 0.5 805 mM Spermidine, 0.1% Triton X-100, 20% Glycerol and Roche Complete Protease Inhibitor EDTA-Free and used as the input source. Nuclei bound to Concanavalin-coated magnetic beads (Bangs Laboratories BP531) were incubated with the relevant antibody: H3K27ac (Abcam AB4729), FOS (Abcam AB208942), rabbit IgG (Abcam AB46540) or guinea pig IgG (Novus NBP1-2763). The next day, the beads were placed on a magnet to remove supernatant, washed, and incubated with Protein AG-MNase. To fragment the DNA, samples were first chilled to 0º C, the digestion was then activated by the addition of 2 mM of CaCl2. Reactions were stopped by 2×Stop buffer (340mM NaCl, 20mM EDTA, 4mM EGTA (Alfa Aesar J60767), 0.05% Digitonin, 100ug/mL RNAse A (Thermo Fisher Scientific EN0531), 50ug/mL Glycogen).
DNA fragments released from the nuclei were purified by extraction with phenol-chloroform and ethanol precipitated. DNA was quantified with a fluorescence-based method using Qubit 4 Fluorometer dsDNA HS assay. CUT&RUN DNA (10ng) was processed using NEBNext Ultra II DNA Library Prep Kit (New England Biolabs 816 E7645) for library preparation according to manufacturer’s instructions. Briefly, purified DNA was repaired, ligated with 1.5μM NEBNext Adaptors and amplified for 14 cycles by PCR. DNA was enriched for fragments of size 150-350bp for transcription factor and 150-800 bp for histone markers using Ampure XP beads. Final libraries were quantified with Qubit dsDNA HS assay and the size distribution was determined by Agilent 4200 TapeStation. Libraries were mixed at an equal ratio to achieve a final concentration as recommended by the manufacturer and sequenced on an Illumina HiSeq 4000 system.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 4000 |
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| Description |
DNA released from nuclei
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| Data processing |
Library strategy: CUT&RUN
Illumina adapters were trimmed from reads using trimmomatic: trimmomatic PE -threads 8 -phred33 R1_001.fastq.gz R2_001.fastq.gz ILLUMINACLIP:$adapterpath/TruSeqAdapters.fa:2:15:4:4:true LEADING:20 TRAILING:20 SLIDINGWINDOW:4:15 MINLEN:25
Trimmed reads were mapped to the human genome (hg38) using bowtie2: bowtie2 -p 8 --dovetail --phred33 --local --very-sensitive-local --no-unal --no-mixed --no-discordant -I 10 -X 700 -x $bt2idx/GRCh38_noalt_as
Aligned reads were sorted and indexed using samtools
Bigwig files were gneerated for visualizing sample using bamCoverage: bamCoverage -b aligned_sorted.bam -o aligned.bigwig -p 4 --normalizeUsing RPKM --extendReads 160
Broad peaks were called using MACS2: macs2 callpeak -t sorted_sample.bam -c sorted_control.bam-f BAMPE -g hs --nomodel --keep-dup all --broad
Genome_build: hg38 (GRCh38_noalt_as)
Supplementary_files_format_and_content: Bigwig files (.bigwig) can be used to visualize each sample on a gennome browser
Supplementary_files_format_and_content: Peak files (.broadPeak) contain the called peaks from MACS2
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| Submission date |
Feb 06, 2022 |
| Last update date |
Jun 02, 2022 |
| Contact name |
Matthew A Lalli |
| Organization name |
Icahn School of Medicine at Mount Sinai
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| Department |
Psychiatry
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| Street address |
1 Gustave L. Levy Pl
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| City |
New York |
| State/province |
NY |
| ZIP/Postal code |
10029 |
| Country |
USA |
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| Platform ID |
GPL20301 |
| Series (1) |
| GSE196207 |
Mapping cis-regulatory elements in human excitatory and inhibitory neurons links psychiatric disease heritability and activity-regulated transcriptional programs |
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| Relations |
| BioSample |
SAMN25689670 |
| SRA |
SRX14059555 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5862765_AD_15_H3K27ac_2.bigwig |
43.4 Mb |
(ftp)(http) |
BIGWIG |
| GSM5862765_AD_15_H3K27ac_2.broadPeak.gz |
1.7 Mb |
(ftp)(http) |
BROADPEAK |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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