|
| Status |
Public on May 09, 2024 |
| Title |
SU-DIPG-XXXVI_sgCHD2_RNAseq_rep2 |
| Sample type |
SRA |
| |
|
| Source name |
patient-derived cell culture
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: Diffuse intrinsic pontine glioma (DIPG) cells
genotype: CHD2 KO
|
| Growth protocol |
Patient-derived DMG cells (SU-DIPG-IV, SU-DIPG-VI, SU-DIPG-XVII, SU-DIPG-XXXVI) were obtained from Dr. Michelle Monje at Stanford University and maintained as neurospheres in knockout DMEM/F12 media (Gibco) supplemented with StemPro Neural Supplement (Gibco), 20 ng/ml human FGF-basic (bFGF) (Gibco), 20 ng/ml human EGF (Gibco), 2 mM L-Glutamine (Gioco), 1% penicillin/streptomycin (Invitrogen) and plasmocin (InvivoGen). Alternatively, the DMG cells were maintained as adherent culture using 24 μg/ml fibronectin-coated dishes. Mouse DMG cells with H3.3K27M mutation were obtained from Dr. Oren Becher at Mount Sinai Hospital and maintained as neurospheres in NeuroCult™ Basal Medium (STEMCELL Technologies) containing Neurocult proliferation supplement (STEMCELL Technologies), 20 ng/ml human bFGF (Gibco), 10 ng/ml human EGF (Gibco), 2 μg/mL Heparin (STEMCELL Technologies) and 1% penicillin/streptomycin (Invitrogen). All cells were cultured at 37℃ with 5% CO2, and were periodically tested mycoplasma negative by PCR.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
CUT&RUN was performed as previously described (Skene and Henikoff, 2017) with minor modifications. Total RNA samples were purified the same as for RT-PCR.
RNA-seq libraries were prepared and deep sequencing was performed by Columbia University Genome Center. CUT&RUN libraries were prepared using ACCEL-NGSTM 1S plus DNA library kit and library PCR was performed using standard Illumina Nextera Dual Indexing primers. Samples were pooled and sequenced using paired-end sequencing on Illumina NextSeq 500 platforms at Columbia University Genome Center.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
DIPG36_CHD2KO_RNAseq_rep2
|
| Data processing |
Raw reads were trimmed to remove sequencing adaptors and low-quality reads using Trim Galore (version 0.6.7) with default parameters.
Sequence reads were mapped back to the human (hg19) reference genome using Bowtie2 software. Only consistent pair-end reads were chosen for further analysis. RNA-Seq reads were aligned to hg19 reference using STAR.
Coverage for each base pair of the human genome was computed using genomeCoverageBed from BEDTools (version 2.29.2) and normalized to library size (reads-per-million; RPM).
Sparse Enrichment Analysis for CUT&RUN (SEACR version 1.3) was used for peak calling (Meers et al., 2019). Overlapped peaks between replicates were retained and then merged to obtain a consensus peak set for each Cut&Run target using BEDTools (version 2.29.2) (Quinlan and Hall, 2010).
Read count on gene body or promoters were calculated by FeatureCount. Differential genes and log fold change were calculated by edgeR. The P value of each gene was adjusted using the Benjamini–Hochberg (BH) procedure. Genes with a BH-adjusted P value less than 0.05 and a fold change greater than 1.5 (upregulated) or less than 0.67 (downregulated) were considered as differentially expressed genes (DGEs).
Assembly: hg19
Supplementary files format and content: bigwig files were generated using UCSC bedGraphToBigWig (version 359), scores represent the normalization reads density.
|
| |
|
| Submission date |
Aug 11, 2022 |
| Last update date |
May 09, 2024 |
| Contact name |
Zhiguo Zhang |
| E-mail(s) |
zz2401@cumc.columbia.edu
|
| Phone |
212-851-4936
|
| Organization name |
Columbia University
|
| Department |
Pediatric and Genetics and Development
|
| Lab |
Irving Cancer Research Center
|
| Street address |
1130 St. Nicholas Avenue
|
| City |
New York |
| State/province |
NY |
| ZIP/Postal code |
10032 |
| Country |
USA |
| |
|
| Platform ID |
GPL18573 |
| Series (1) |
| GSE211074 |
CHD2 Regulates Neuron-glioma Interactions in Pediatric Glioma |
|
| Relations |
| BioSample |
SAMN30273512 |
| SRA |
SRX17035835 |
