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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Jan 21, 2015 |
| Title |
hNSC_CHD8_KD_G_3 |
| Sample type |
SRA |
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| Source name |
human Neural Stem Cells
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| Organism |
Homo sapiens |
| Characteristics |
cell type: human Neural Stem Cells (hNSCs)
transfected with: shCHD8 #G (CloneID: V3LHS_311510)
time point: 48 hours after transfection
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| Treatment protocol |
RNA-seq: All shRNAs were transfected using Amaxa Nucleofector (Laza) according to the manufacturer’s instructions. The following shRNAs were used: shCHD8 #C (CloneID: V2LHS_201084, Thermo), mature antisense sequence TAAAGACTCCAATGAGCAG); shCHD8#G (Clone ID: V3LHS_311510, Thermo), mature antisense sequence ACTGTTGAATCATCTGCCT). After transfection, cells were grown for 48 hours in KnockOut™ DMEM/F-12 medium. Cells were then trypsinized by Accutase (Life Science Technology) and sorted on FCS Aria II machine. Both western blotting and RT-qPCR were used to determine the downregulation of CHD8 expression.
ChIP-seq: Human brain tissue or mouse cortex tissue were briefly homogenized and crosslinked with 1% formaldehyde at room temperature with rotation for 15 minutes. Crosslinking was quenched with 150 mM glycine. hNSC cells were crosslinked on culture plates, quenched with glycine, scraped off plates, and transferred to 15 mL conical tubes. Cultured cells or tissue were harvested by centrifugation at 1500g for 5 min at 4C. Pelleted tissue was washed twice with cold PBS, flash frozen and stored at -80°C.
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| Growth protocol |
RNA-Seq: H9 derived human neural stem cells (hNSCs) were purchased from Life Science Technology (cat no. N7800100). Cells were maintained in KnockOu™DMEM/F12 medium (12660-012, Life Science Technology) supplemented with StemPro® supplement (A1050801, Life Science), 20ng/ml basic FGF recombinant protein (GF003, EMD Millipore Corporation), and 20ng/ml EGF recombinant protein (GF144, EMD Millipore Corporation). These cells were cultured as a attached monolayer in tissue culture flasks precoated with the BD Metrigel ™ hESC-qualified Matrix (354277, BD) in a 37C incubator with 5% CO2.
ChIP-seq: H9 derived human neural stem cells (hNSCs) were purchased from Life Science Technology (cat no. N7800100). Cells were maintained in KnockOu™DMEM/F12 medium (12660-012, Life Science Technology) supplemented with StemPro® supplement (A1050801, Life Science), 20ng/ml basic FGF recombinant protein (GF003, EMD Millipore Corporation), and 20ng/ml EGF recombinant protein (GF144, EMD Millipore Corporation). These cells were cultured as a attached monolayer in tissue culture flasks precoated with the BD Metrigel ™ hESC-qualified Matrix (354277, BD) in a 37oC incubator with 5% CO2. Human brain tissue was excised from frozen brain slabs with a rotary cutting instrument. Mouse embryonic tissue was dissected from E17.5 mouse embryos harvested from timed pregnant mothers.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
RNA-seq: Total RNA was extracted using the miRNeasy Micro Kit (217084, Qiagen), as described in the manufacturer’s instructions. The step of DNase I digestion was included to avoid potential DNA contamination.
Standard Illumina Multiplexed Paired-End Library Prep performed by the Yale Center for Genome Analysis (YCGA).
ChIP-seq: Nuclei were extracted in six pellet volumes of lysis buffer I (50 mM Tris pH 8.0, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100, 1x Roche Complete Protease inhibitor, 5 mM sodium butyrate) and incubating on ice for 15 minutes. Tissue was homogenized with a dounce homogenizer and nuclei were harvested by centrifugation at 2000g for 10 minutes at 4°C. Nuclei were resuspended in five pellet volumes of nuclear lysis buffer (10 mM Tris pH 8.0, 1 mM EDTA, 0.5 mM EGTA, 0.2% SDS, 1x Roche Complete Protease inhibitor, 5 mM sodium butyrate) and incubated on ice for 20 minutes. 300 mL nuclear lysates in 1.5 mL tubes were sonicated using a Misonix S4000 with 431A cup horn (10 second pulses, 10 second rest, 20 minutes total, 2°C maintained by circulating chiller). Following sonication insoluble material was pelleted by centrifugation at 16000g for 10 minutes at 4°C. A small sample of soluble crosslinked chromatin was purified, checked for correct sonication size range and DNA concentration was measured. 100 ug of chromatin was diluted to 450 mL with dilution buffer (16.7 mM Tris pH 8, 167 mM NaCl, 1.2 mM EDTA, 0.01% SDS, 1.1% Triton X-100, 1x Roche Complete Protease inhibitor, 5 mM sodium butyrate) and added to 50 uL of Protein G Dynabeads (Invitrogen) prebound with 10 mg of CHD8 antibody. Samples were incubated overnight at 4°C with rotation. Beads were precipitated on magnet stand and washed five times with 1 mL of wash buffer (100 mM Tris pH8.0, 500 mM LiCl, 1% NP-40, 1% deoxycholic acid, 1x Roche Complete protease inhibitor, 5mM sodium butyrate) and once with TE. Immunoprecipiated chromatin was eluted from beads with 50 mL of elution buffer (TE + 1% SDS) at 65°C for 10 minutes. Crosslinks were reversed overnight at 65°C. Chromatin was treated with RNAseA and proteinase K then purified with Qiagen PCR cleanup kit. Concentration of eluted chromatin was measured with PicoGreen (Invitrogen).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
Bowtie (v0.12.9)
zcat /sequencers/sequencerO/runs/131021_SN7001144_0397_AH7LYYADXX/Data/Intensities/BaseCalls/Unaligned/Project_Jc798/Sample_JCOT_146_004/JCOT_146_004_TGACCA_L001_R1_010.fastq.gz | bowtie -B 1 -p 8 --chunkmbs 256 -m 1 -x 1000 /home/jc798/GENOME/contam/contam - --al=131021_SN7001144_0397_AH7LYYADXX_Sample_JCOT_146_004/JCOT_146_004_TGACCA_L001_R1_010.contam --un=131021_SN7001144_0397_AH7LYYADXX_Sample_JCOT_146_004/JCOT_146_004_TGACCA_L001_R1_010.unaligned 131021_SN7001144_0397_AH7LYYADXX_Sample_JCOT_146_004/JCOT_146_004_TGACCA_L001_R1_010.multi ; bowtie -B 1 -p 8 --chunkmbs 256 -m 1 -x 1000 /home/jl56/GENOME/hg19/dna/hg19_nh 131021_SN7001144_0397_AH7LYYADXX_Sample_JCOT_146_004/JCOT_146_004_TGACCA_L001_R1_010.unaligned 131021_SN7001144_0397_AH7LYYADXX_Sample_JCOT_146_004/JCOT_146_004_TGACCA_L001_R1_010.bowtie
Custom perl script
Cotney et al, Cell 2013.
RSEQTools (0.6.0)
cat Sample_JCOT_166_141_DFC_Chd8.bowtie | bowtie2mrf genomic > ../mrf/Sample_JCOT_166_141_DFC_Chd8.mrf
RSEQTools (0.6.0) and wigToBigWig
cat Sample_JCOT_148_hNSC_H3K27ac_1.mrf | mrf2wig Sample_JCOT_148_hNSC_H3K27ac_1; cat Sample_JCOT_148_hNSC_H3K27ac_1_chr*.wig | grep -v track > Sample_JCOT_148_hNSC_H3K27ac_1.wig; rm Sample_JCOT_148_hNSC_H3K27ac_1_chr*.wig; wigToBigWig -clip Sample_JCOT_148_hNSC_H3K27ac_1.wig /home/jl56/GENOME/hg19/dna/hg19_nh.chrom.sizes Sample_JCOT_148_hNSC_H3K27ac_1.bw
Tophat (v2.0.9)
tophat -p 8 -G /data/ss64/ajw54/Tophat/transcriptome_data/Homo_sapiens/UCSC/hg19/Annotation/Genes/genes.gtf -o 4_Sample_JCOT_214_004_ --no-novel-juncs --no-novel-indels --read-mismatches 1 --read-gap-length 0 --read-edit-dist 1 --read-realign-edit-dist 0 --b2-N 1 -g 1 -m 0 -M --library-type=fr-firststrand /data/ss64/ajw54/Tophat/transcriptome_data/Homo_sapiens/UCSC/hg19/Sequence/Bowtie2Index/genome ../Sample_JCOT_214_004_R1.fastq.gz ../Sample_JCOT_214_004_R2.fastq.gz
Htseq (v0.5.4)
samtools view ../4_Sample_JCOT_214_004_/accepted_hits.sorted.bam | htseq-count -q -s reverse - /data/ss64/ajw54/Tophat/transcriptome_data/Homo_sapiens/UCSC/hg19/Annotation/Genes/genes.gtf > 4_Sample_JCOT_214_004_counts.txt
Genome_build: hg19 for human samples
Genome_build: mm9 for mouse samples
Genome_build: UCSC KnownGene annotation (05/13/2013)
Supplementary_files_format_and_content: bam (anonymized aligned read cooridinates)
Supplementary_files_format_and_content: bed (peak calls in UCSC bed format)
Supplementary_files_format_and_content: bigWig (signal tracks)
Supplementary_files_format_and_content: counts.txt (abundance measurements per gene reported by HTSeq)
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| Submission date |
May 07, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Justin Cotney |
| E-mail(s) |
cotney@uchc.edu
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| Organization name |
UConn Health
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| Department |
Genetics and Genome Sciences
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| Lab |
Cotney Lab
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| Street address |
400 Farmington Ave.
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| City |
Farmington |
| State/province |
CT |
| ZIP/Postal code |
06030-6403 |
| Country |
USA |
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| Platform ID |
GPL16791 |
| Series (1) |
| GSE57369 |
The chromatin modifier CHD8 targets autism risk genes during human neurodevelopment |
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| Relations |
| BioSample |
SAMN02763022 |
| SRA |
SRX534861 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1381239_hNSC_Chd8_KD_G_3.counts.txt.gz |
98.4 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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