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| Status |
Public on Jul 01, 2024 |
| Title |
GIC-3_H3K27me3_shMETTL7B |
| Sample type |
SRA |
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| Source name |
GIC19
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| Organism |
Homo sapiens |
| Characteristics |
cell line: GIC19
cell type: Glioblastoma initiating cells
genotype: METTL7B knockdown
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| Growth protocol |
GIC were maintained in NeuroCult NS-A Proliferation kit medium, 1% Pen/Strep, 2µg/ml Heparin, 20ng/ml murine EGF and 10ng/ml human FGF. iNSC were cultured in 0.5X Advanced DMEM/F-12 , 0.5X Neurobasal medium, 1X Neurobasal Induction supplement and 1X pen/strep
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
H3K27me3 ChIP was performed using ChIP-IT High Sensitivity kit (active Motif) as per manufacturer’s instructions. Briefly, cells were fixed with the formaldehyde-based fixing solution for 15 min at room temperature and lysed with provided lysis solution supplemented with proteases inhibitors. Next, nuclei pellets were lysed and chromatin sonicated with Bioruptor Plus sonication device (Diagenode) to obtain fragments within the recommended 200-1200 bp range. 5 µg of sheared chromatin was then incubated with 4 µg of antibody against H3K27me3 (Diagenode) overnight at 4 °C with rotation. Following incubation with Protein G agarose beads, bound chromatin was washed, eluted and purified following the manufacturer’s protocols. Validation by qPCR-ChIP on target genes was done before proceeding to sequencing.
ChIPed DNA was end-repaired, A-tailed and adapter-ligated before size selection and amplification. The obtained libraries were QC’ed and multiplexed before 150-bp paired-end sequencing on Illumina NovaSeq 6000
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
Quality and adapter trimming was performed using Trimgalore v0.6.5
Alignment to hg19 Genome was performed using Bowtie2 v2.4.5 with default parameters
samtools v1.9 was used to obtain bam from sam files that . Bam files were then sorted by cooirdinate and indexed
Bams were filtered to include only uniquely mapping reads (ie unmapped reads and multimapping were removed) using sambamba
Peak calling was performed with MACS2, BAM mode was used (-f BAM and also -g 'hs’) and with the broad peak calling (--broad and --broad-cutoff 0.05) and 300bp fragment (shift) size ( –extsize 300)
Further quality assessment of peak calling was performed using R package ChIPQC
Differential peak analysis was performed with R package with DiffBind
Assembly: hg19
Supplementary files format and content: MACs2 output bedGraph files
Supplementary files format and content: bigWig files gnerated from bedGraphs
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| Submission date |
Sep 05, 2023 |
| Last update date |
Jul 01, 2024 |
| Contact name |
James Nicholson |
| E-mail(s) |
[email protected]
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| Organization name |
Queen Mary University of London
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| Department |
Blizard Institute
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| Street address |
4 Newark St
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| City |
London |
| ZIP/Postal code |
E1 2AT |
| Country |
United Kingdom |
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| Platform ID |
GPL24676 |
| Series (2) |
| GSE242326 |
METTL7B is an essential epigenetic regulator of lineage specification in glioblastoma [ChIPseq] |
| GSE243132 |
METTL7B is an essential epigenetic regulator of lineage specification in glioblastoma |
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| Relations |
| BioSample |
SAMN37286344 |
| SRA |
SRX21638655 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7758132_GIC-3_H3K27me3_shMETTL7B_treat_pileup.bedgraph.gz |
107.1 Mb |
(ftp)(http) |
BEDGRAPH |
| GSM7758132_GIC3_H3K27me3_shMETTL7B.SeqDepthNorm.bw |
189.8 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
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