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Status |
Public on Oct 16, 2023 |
Title |
ATAC-Pool2_C7_noStim |
Sample type |
SRA |
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Source name |
in vitro iPSC
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Organism |
Homo sapiens |
Characteristics |
tissue: in vitro iPSC cell type: iPSC derived neuron genotype: C7 treatment: baseline
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Treatment protocol |
iPSC derived neurons were stimulated by KCl-solution (170 mM KCl, 150 mM HEPES, 1 mM MgCl2, 2 mM CaCl2) for 5h on day 49 or untreated.
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Growth protocol |
iPSC derived neurons were generated via overexpression of pro-neural transcription factor Ngn2 plus small molecules and differentiated for 49 days on murine astrocytes.
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Extracted molecule |
genomic DNA |
Extraction protocol |
ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) was performed on bulk cryopreserved cell pellets of one 12-well according to previously published protocols8. Neuronal networks were gently thawed in DMEM/F12, pelleted (400 x g, 5 min, 4°C) and washed with PBS. Detergents for the lysis buffer were added freshly to the basic RSB buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2) and complete lysis buffer RSBI (RSB supplemented with 100 µg/ml digitonin, 1:100 Tween™20, 1:100 IGEPAL) and RSBII (1:100 Tween™20) were cooled down on ice. Cell pellets were resuspended in 50 µl RSBI with 4 mixes with the 200 µl pipette tip and then lysed on ice for 3 min. Afterwards, 1 ml of RSBII was added and the cell solution was gently mixed 4 times with a 1000 µl pipette tip. The nuclei were pelleted at 500 x g for 10 min at 4°C and counted (Luna-II). 50,000 nuclei were used for the transposition reaction at 37°C while shaking on a thermomixer (1000 rpm) for 30 min: 25 µl TD buffer (15027866, Illumina), 2.5 µl TDE1 tagment DNA enzyme (15027865, Illumina), 16.5 µl PBS, 0.5 µl Tween20, 0.5 µl digitonin [1% stock], and 5 µl water. Fragmented DNA was immediately column-purified and eluted in 22 µl EB provided with the kit. For the preparation of the library, 20 µl of the purified DNA was mixed with 25 µl NEBNext High-Fidelity master mix (M0541L, New England Biolabs), 2.5 µl FW (5’ AATGATACGGCGACCACCGAGATCTACACTCGTCGGCAGCGTCAGATGTG 3’) and 2.5 µl barcoding (for barcodes see Table S7) RV primer (5’ CAAGCAGAAGACGGCATACGAGATNNNNNNNNGTCTCGTGGGCTCGGAGATGT 3’)9. After the amplification, a double-sided bead cleanup with 0.55x of the volume and then 1x of the volume was performed. Before the pooling, libraries were quantified and verified for a successful transposition with the Bioanalyzer on a high sensitivity DNA chip. 10 libraries containing 10 different barcodes could therefore then be pooled equimolar for sequencing on a NovaSeq (50bp paired-end sequencing). Final pool was repurified with a right-left-cleanup of 0.5x and then 0.45x the volume.
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Library strategy |
ATAC-seq |
Library source |
genomic |
Library selection |
other |
Instrument model |
Illumina NovaSeq 6000 |
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Description |
Sample_ATACSeq5 ATACSeq_UnionPeakSet_rawCounts.txt
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Data processing |
Paired-end raw ATAC-Seq reads were aligned against hg19 using bowtie 2 (Langmead and Salzberg, 2012) version 2.3.5. Subsequently, duplicates were removed using picard tools and peak calling was performed using macs2 and standard human genome size (hs parameter). Resulting peak files of all samples were then merged using diffbind, only retaining those peaks that were present in at least 6 samples, giving rise to the union peak set employed in all further analyses. Next, bedtools multicov function was used to count fragments overlapping with peaks by at least one base. Assembly: hg19 Supplementary files format and content: raw count matrix for each peak and peak files from MACS2
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Submission date |
Feb 22, 2023 |
Last update date |
Oct 16, 2023 |
Contact name |
Michael J Ziller |
E-mail(s) |
ziller@uni-muenster.de
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Organization name |
University of Muenster
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Street address |
Busso-Peus-Str. 10
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City |
Muenster |
ZIP/Postal code |
48149 |
Country |
Germany |
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Platform ID |
GPL24676 |
Series (2) |
GSE225816 |
Massively parallel functional dissection of schizophrenia associated non-coding genetic variants [ATAC-seq] |
GSE225817 |
Massively parallel functional dissection of schizophrenia associated non-coding genetic variants |
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Supplementary file |
Size |
Download |
File type/resource |
GSM7056997_ATAC-Pool2_C7_noStim.bed.gz |
3.1 Mb |
(ftp)(http) |
BED |
Raw data not provided for this record |
Processed data provided as supplementary file |
Processed data are available on Series record |
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