|
| Status |
Public on Jun 29, 2020 |
| Title |
Line 17 H3.3 rep1 |
| Sample type |
SRA |
| |
|
| Source name |
Line XVII DIPG
|
| Organism |
Homo sapiens |
| Characteristics |
histone h3.3 status: mutant (K27M)
antibody: H3.3
|
| Treatment protocol |
Single-stranded donor oligonucleotide (ssODN) templates were designed with the desired mutations to facilitate homology directed repair (HDR) after cleavage of H3F3A by Cas9n. cells were co-transfected with a combination of pSpCas9n(BB)-2A-GFP-guideA (0.5μg/well), pSpCas9n(BB)-2A-Puro-guideB (0.5μg/well), and ssODN HDR (1μg/well) template using XtremeGene-HP (Sigma).
|
| Growth protocol |
All DIPG cells were cultured in Tumor Stem Medium (Nagaraja et al, 2017), which contains DMEM/F12 1:1 (Invitrogen), Neurobasal-A (Invitrogen), 10mM HEPES (Invitrogen), 1X MEM sodium pyruvate (Invitrogen), 1X MEM nonessential amino acids (Invitrogen), 1% GlutaMax (Invitrogen), human-bFGF (20 ng/ml) (Shenandoah), human-EGF (20 ng/ml) (Shenandoah), human PDGF-A and PDGF-B (20ng/ml) (Shenandoah), heparin (10 ng/ml) (StemCell Technologies) and B27 without Vitamin A (Invitrogen).
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Lysates were clarified from sonicated nuclei and protein-DNA complexes were isolated with antibodies to H3K27me3 (Cell Signaling C36B11) or H3.3 (Millipore #09-838).
Libraries were prepared with the Nextera library prep kit
Libraries were sequenced on the NovaSeq machine with 150 base paired-end sequencing
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Description |
Line 17 H3.3 rep1
|
| Data processing |
Reads were called with illumina bclfastq
Reads were aligned to the hg19 genome with BWA (version 0.7.13-r1126 )
Peaks were called with MACS2 (version 2.1.0.20151222)
Differential peak occupancy analysis was performed with DiffBind (R version 3.5.2)
Downstream analysis was performed on the Galaxy webserver
Genome_build: hg19
Supplementary_files_format_and_content: bed files of peaks
|
| |
|
| Submission date |
Apr 12, 2019 |
| Last update date |
Jul 01, 2020 |
| Contact name |
Paul Knoepfler |
| E-mail(s) |
knoepfler@ucdavis.edu
|
| Organization name |
University of California Davis
|
| Department |
Cell Biology and Human Anatomy
|
| Street address |
2425 Stockton Blvd, Room 633
|
| City |
Sacramento |
| State/province |
CA |
| ZIP/Postal code |
95817 |
| Country |
USA |
| |
|
| Platform ID |
GPL24676 |
| Series (1) |
| GSE129765 |
Reciprocal gene editing defines targetable mutant H3.3 oncohistone effectors in pediatric glioma |
|
| Relations |
| BioSample |
SAMN11411054 |
| SRA |
SRX5678410 |
