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| Status |
Public on Jul 01, 2017 |
| Title |
SH-EP H3K27ac, ChIP-seq |
| Sample type |
SRA |
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| Source name |
SH-EP
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| Organism |
Homo sapiens |
| Characteristics |
cell type: Neuroblastoma cell line
chip antibody: ab4729 (rabbit polyclonal, Abcam)
treatment: none
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| Treatment protocol |
RNA-seq: Cells were not treated unless it is specified
RNA-seq: Treatment of cell lines with chemotherapy: SK-N-SH cells were plated in 6-well plates and then treated with cisplatin (7.5 µM) or doxorubicin (100 nM) for 7 days, medium and drugs were changed every 2 days. RNAs were extracted using NucleoSpin RNA kit (Macherey-Nagel).
RNA-seq: Doxycycline-inducible shRNA systems: PHOX2B-specific short hairpin RNAs sh1783 (5’-CCGGTGGAAGGCAGAAACCATTAAA- CTCGAGTTTAATGGTTTCTGCCTTCCATTTTTG-3’) and sh1437 (5’-CCGGAGTAATCG- CGCTAAGAATAAACTCGAGTTTATTCTTAGCGCGATTACTTTTTTG-3’) were selected from Sigma Mission shRNA library and cloned into the pLKO-Tet-On all-in-one system46 (Addgene). Lentiviral particles were produced in HEK293T cells and CLB-GA cells were infected as previously described4. SH-SY5Y cells were incubated with viral particules for 48 hours without polybrene. Selection with puromycin (Invitrogen) at 400 ng/ml or 1 µg/ml, respectively, was performed 24 h after infection and maintained during all culture experiments, for CLB-GA and SH-SY5Y cells, respectively. PHOX2B knockdown efficacy was assessed by Western blot 24 h/48 h/96 h after the addition of doxycycline (100 ng/ml or 1 μg/ml). For colony formation assays, 6x104 transduced cells were plated at day 0 in 6-well dishes and stained with crystal violet at day 11.
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| Growth protocol |
ChIP-seq: Neuroblastoma cell lines were cultured at 37°C with 5% CO2 in a humidified atmosphere in RPMI (GE Healthcare, for CLB cell lines, SH-EP, GICAN and NB69), in IMDM (Gibco) for NB-EBc1 (according to the provided conditions) or DMEM (GE Healthcare, for other cell lines), with 10%, 15% or 20% FCS (Eurobio) and 100 μg/ml penicillin/streptomycin (Gibco). Primary hNCC lines were grown as previously described under bioethical approval PFS14-011 from the French Biomedical Agency for the use of human embryonic material to S. Zaffran. Briefly, cells were grown in Glutamax DMEM:F12 [Gibco] supplemented with 12% FCS (Eurobio), 100 μg/ml penicillin/streptomycin, 10 mM HEPES, 100 ng/ml hydrocortisone, 10 μg/ml transferrin, 400 pg/ml 3,3,5-thio-iodo-thyronine, 10 pg/ml glucagon, 100 pg/ml epidermal growth factor, 1 ng/ml insulin and 200 pg/ml fibroblast growth factor 2 (all products supplied by Sigma-Aldrich except EGF and FGF2 from Gibco).
RNA-seq: Neuroblastoma cell lines were cultured at 37°C with 5% CO2 in a humidified atmosphere in RPMI (GE Healthcare, for CLB cell lines, SH-EP, GICAN and NB69), in IMDM (Gibco) for NB-EBc1 (according to the provided conditions) or DMEM (GE Healthcare, for other cell lines), with 10%, 15% or 20% FCS (Eurobio) and 100 μg/ml penicillin/streptomycin (Gibco). Primary hNCC lines were grown as previously described under bioethical approval PFS14-011 from the French Biomedical Agency for the use of human embryonic material to S. Zaffran. Briefly, cells were grown in Glutamax DMEM:F12 [Gibco] supplemented with 12% FCS (Eurobio), 100 μg/ml penicillin/streptomycin, 10 mM HEPES, 100 ng/ml hydrocortisone, 10 μg/ml transferrin, 400 pg/ml 3,3,5-thio-iodo-thyronine, 10 pg/ml glucagon, 100 pg/ml epidermal growth factor, 1 ng/ml insulin and 200 pg/ml fibroblast growth factor 2 (all products supplied by Sigma-Aldrich except EGF and FGF2 from Gibco).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIP-seq: Chromatin preparation and ChIP were performed with Ideal ChIP-seq kit for histones or transcription factors according to the supplier's protocol (Diagenode )
ChIP-seq: llumina sequencing libraries were prepared from ChIP and input DNAs using the TruSeq ChIP library preparation kit according to the manufacturer’s protocol. Briefly, DNA were subjected to consecutive steps of end-repair, dA-tailing and ligation to TruSeq indexed Illumina adapters. Size-selection was performed only for the H3K27ac ChIP (100 – 600 bp). After a final amplification step, the resulting DNA libraries were quantified using a qPCR method (KAPA library quantification kit) and sequenced on the Illumina HiSeq2500 instrument (rapid run mode; single reads 100 nts).
RNA-seq: Total RNA was extracted from fresh cells or frozen tumors using TRIzol® Reagent (Invitrogen), or AllPrep DNA/RNA Mini Kit (Qiagen) or NucleoSpin RNA kit (Macherey-Nagel; for the SK-N-SH cell line treated with chemotherapy). All samples were subjected to quality control on a Bioanalyzer instrument and only RNA with RIN (RNA Integrity Number) > 6 were used for sequencing.
RNA-seq: RNA sequencing libraries were prepared from 1 µg of total RNA using the Illumina TruSeq Stranded mRNA Library preparation kit which allows performing a strand-specific sequencing. A first step of polyA selection using magnetic beads is done to focus sequencing on polyadenylated transcripts. After fragmentation, cDNA synthesis was performed and resulting fragments were used for dA-tailing and then ligated to the TruSeq indexed adapters. PCR amplification is finally achieved to create the final cDNA library. After qPCR quantification, sequencing was carried out using 2 x 50 cycles (paired-end reads 50 nts) for all samples (except SH-EP, 2 x 100; Pair1-Relapse and Pair3-Relapse, 2 x 75; Pair2-Relapse, 2 x 150). Sequencing was performed with the Illumina HiSeq2500 instrument (high output mode).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
ChIP-seq: Alignment with Bowtie2, default options
ChIP-seq: Quality filtering: Q > 20; duplicates kept
ChIP-seq: Peak calling with HMCan v1.30
ChIP-seq: Renormalization of wig profiles among all H3K27ac samples with an in-house R script
Genome_build: hg19
Supplementary_files_format_and_content: bigWig files correspond to renormalized signal; score in .bed files correspond to the likelihood of a region to be enriched in H3K27ac or to be a binding site of a TF
RNA-seq:TopHat2 v2.0.643 with the following parameters: global alignment, no mismatch in the 22 bp seed, up to three mismatches in the read, library type fr-firststrand
Genome_build: hg19/GRCh37
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| Submission date |
Jun 12, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Valentina Boeva |
| Organization name |
Institut Cochin
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| Department |
Inserm U1016
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| Street address |
24 rue du Faubourg Saint-Jacques
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| City |
Paris |
| ZIP/Postal code |
75014 |
| Country |
France |
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| Platform ID |
GPL16791 |
| Series (1) |
| GSE90683 |
Heterogeneity of neuroblastoma cell identity revealed by transcriptional circuitries |
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| Relations |
| BioSample |
SAMN07222650 |
| SRA |
SRX2911540 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2664333_SHEP.K27ac.rep3.wig.bw |
64.9 Mb |
(ftp)(http) |
BW |
| GSM2664333_SHEP.K27ac.rep3_peaks.narrowPeak.gz |
3.8 Mb |
(ftp)(http) |
NARROWPEAK |
| GSM2664333_SHEP.K27ac.rep3_regions.bed.gz |
3.1 Mb |
(ftp)(http) |
BED |
SRA Run Selector |
| Processed data provided as supplementary file |
| Raw data are available in SRA |
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