|
| Status |
Public on May 04, 2023 |
| Title |
EZH1, WT, H3K27me3, R2 |
| Sample type |
SRA |
| |
|
| Source name |
cells derived from ventral mesencephalon region of fetal brain
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: cells derived from ventral mesencephalon region of fetal brain
cell line: ReNcells VM
cell type: immortalized human neural progenitor line
genotype: EZH1 WT
treatment: anti-H3K27me3 (Cell Signaling (D5A7))
|
| Treatment protocol |
ReNcells were transduced to generate stable lines expressing WT or mutant EZH1 with EGFP. Cells were incubated with lentiviruses for 48 hrs, after which media was replaced with fresh ReNcell media and cells cultured for 5 days before sorting with BD FACS Aria Fusion flow cytometer
|
| Growth protocol |
ReNcells VM were cultured in a 1:1 ratio of N2 and B27 media supplemented with 20 ng/ml Fibroblast Growth Factor (bFGF) and 20 ng/ml Epidermal Growth Factor (bEGF). N2 media contains DMEM/F12 (Gibco #11330032), 1X N-2 neural supplement (Gibco #17502048), 5 g/ml Insulin (MilliPore Sigma #I9278), 1X GlutaMAX™ (Gibco # 35050061), 100 M MEM Non-Essential Amino Acids Solution (Gibco #11140050), 100 M -Mercaptoethanol (Gibco #21985023), 1 mM Sodium Piruvate (Gibco #11360070) and 1% Penicillin-Streptomycin (Gibco #15140122). B27 media contains Gibco Neurobasal™ medium (Gibco #21103049) supplemented with 1X B-27 neural supplement (Gibco #17504044), 1X GlutaMAX™and and 1% Penicillin-Streptomycin.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were fixed with 1% formaldehyde for 10 min at room temperature. Fixation was quenched with glycine (0.125 M). Cross-linked chromatin was fragmented by sonication carried out using a Bioruptor to generate an average fragment size of 200-500 bp.
DNA libraries were constructed with the TAKARA ThruPLEX® DNA-Seq Kit and the Illumina-compatible TAKARA DNA Single Index Kit
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Description |
WT_K27_merged_CPM.bw
|
| Data processing |
ChIP-seq reads were aligned to the hg19 reference genome using bowtie2 with parameters: -q --local --no-mixed --no-unal –dovetail
Uniquely aligned reads (mapping quality score >= 20) and concordant alignments were kept for downstream analysis using command: samtools -q 20 -f 0x2.
H3K27me3 peaks were called using MACS2 with parameters: --broad --keep-dup all -p 1e-5 --broad-cutoff 1e-5
ChIP-seq signal was normalized to sequencing depth using deepTools: bamCoverage --normalizeUsing CPM
ChIP-seq signal around peaks was computed and visualized using deepTools computeMatrix and plotHeatmap functions, respectively
Assembly: hg19
Supplementary files format and content: bigWig
Supplementary files format and content: broadPeak
|
| |
|
| Submission date |
Aug 03, 2022 |
| Last update date |
May 06, 2023 |
| Contact name |
Carolina C Gracia-Diaz |
| E-mail(s) |
ccgracia@pennmedicine.upenn.edu
|
| Organization name |
The Children's Hospital of Philadelphia
|
| Department |
Raymond G. Perelman Center for Cellular and Molecular Therapeutics
|
| Lab |
Akizu Lab
|
| Street address |
3501 Civic Center Boulevard
|
| City |
Philadelphia |
| State/province |
PA |
| ZIP/Postal code |
19104 |
| Country |
USA |
| |
|
| Platform ID |
GPL24676 |
| Series (2) |
| GSE210465 |
Gain and loss of function variants in EZH1 disrupt neurogenesis and cause dominant and recessive neurodevelopmental disorders [ChIP-seq] |
| GSE227016 |
Gain and loss of function variants in EZH1 disrupt neurogenesis and cause overlapping neurodevelopmental disorders. |
|
| Relations |
| BioSample |
SAMN30127277 |
| SRA |
SRX16819836 |
