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Sample GSM1521758

Query DataSets for GSM1521758

Status

Public on Nov 02, 2014

Title

MRG_rep2_H3K4me3

Sample type

SRA

Source name

neural precursor cell: mid radial glial

Organism

Homo sapiens

Characteristics

cell type: ES-derived neural progenitor cells
genetic background: HES5::eGFP BAC transgenic human ES cells (H9; WA-09; Wicell) expressing GFP under the HES5 promoter
chip-antibody: Abcam,ab8580,GR68224-1

Growth protocol

Culturing undifferentiated human ES cells: HES5::eGFP BAC transgenic human ES cells (H9; WA-09; Wicell) expressing GFP under the HES5 promoter were cultured on mitotically inactivated mouse embryonic fibroblasts (MEFs) (Globalstem). Undifferentiated ES cells were maintained as described previously1 in medium containing DMEM/F12, 20% KSR, 1mM Glutamine, 1% Penicillin/Streptomycin, non essential amino acids and beta-mercaptoethanol. Undifferentiated ES cells were purified with pluripotency markers Tra-1-60 and PE-conjugated SSEA-3 (BD Pharmingen). Neural induction and long-term propagation of NPCs: Neural differentiation of ES cells was performed as described previously1-3. Briefly, neuroectodermal cells were generated either by monolayer induction – with dissociated ES cells plated on Matrigel (BD biosciences), or by co-culture on MS5 stromal cells. In both cases neural fate was directed by dual SMAD inhibition protocol2. Neural rosettes were harvested mechanically beginning on day 8-10 of differentiation. Rosettes were replated on culture dishes pre-coated with 15 μg/mL polyornithine (Sigma), 1 μg/mL Laminin (BD Biosciences) and 1ug/ml Fibronectin (BD Biosciences) (Po/Lam/FN) in N2 medium composed of DMEM/F12 and N2 supplement (Invitrogen). N2 supplement contained Insulin, Apo-transferin, Sodium Selenite, Putrecine and Progesterone. This medium was supplemented with SHH (200 ng/mL), FGF8 (100 ng/mL) and BDNF (20 ng/mL) (all from R&D Systems) to maintain early anterior regionalization of the neural plate. These factors were gradually replaced by FGF2 (20 ng/mL) and EGF (20 ng/mL) in the following two weeks of differentiation in order to maintain a proliferative (FGF and EGF responsive) NPC state. NPCs from all stages were collected at indicated days and FACS purified for HES5::GFP (NE to L-RG) or EGFR for LNPs to purify for the highest NPC state for each stage. Neuroectodermal cells were collected at day 12 of differentiation, Neuroepithelial/early radial glial cells were collected at day 14, mid neurogenesis radial glial cells were collected at day 35, late gliogenic radial glial cells were collected at day 80, and long term NPCs were collected at day 220. At each stage cells were either split for the next passage or subjected to FACS purification for HES5::GFP as described. Cells were replated onto Po/Lam/FN culture dishes. For neuronal, astroglial or oligodendroglial differentiation, NPCs were seeded at high density and subjected to differentiated for 17 days in the presence of AA/BDNF, 5% Fetal Bovine Serum (FBS) (Invitrogen), or AA/BDNF/SHH/FGF8, respectively. For more details, please see Edri et al.3.

Extracted molecule

genomic DNA

Extraction protocol

ChIP_Seq:To extract DNA and create the Illumina library we used AMPure XP beads (Agencourt) Solid-phase reversible immobilization (SPRI). SPRI beads were added to the samples, mixed 15 times, incubated at room temperature for 2 minutes. Supernatant was extracted from the beads from the beads 4 minutes on a magnet. We used 70% ethanol to wash the beads and then dried for 4 minutes. 40 ul EB buffer (10 mM Tris-HCl pH 8.0) was used to elute the DNA.
ChIP_Seq:The following steps of Illumina library construction (end-repair, addition of A-base, ligation of barcoded adaptors and PCR enrichment) we used a general SPRI cleanup procedure: addition of PEG buffer (20% PEG and 2.5 M NaCl), and extracted and washed as above. The enzymatic reactions were carried as follows: 1. DNA end-repair: T4 PNK and T4 polymerase (New England Biolabs) incubated 12C for 15 min, 25C for 15 min; 2. A-base addition: Klenow (3’->5’ exonuclease; New England Biolabs) incubated at 37C for 30 min. 3. Adaptor ligation: DNA ligase (New England Biolabs) and indexed oligo adaptors and incubated 25C for 15 min, followed by 0.7X SPRI/reaction to remove non-ligated adaptors. 4. PCR enrichment: PCR mastermix (primer set, dNTP mix, Pfu Ultra Buffer (Agilent), Pfu Ultra-II Fusion (Agilent), water), for 20 cycles. The PCR amplified libraries we cleaned up using 0.7X SPRI/reaction (size selection mode) to remove excessive primers. Roughly 5 picomoles of DNA library was then applied to each lane of the flow cell and sequenced on Illumina HiSeq 2000 sequencers according to standard Illumina protocols.

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina HiSeq 2500

Description

Processed data file: FinalIDR_MRG_H3K4me3_macs2.regionPeak.narrowpeak

Data processing

ChIP-Seq data was aligned to the hg19/GRCh37 reference genome using MAQ14 version 0.7.1 with default parameter settings or Bowtie 2 version 2.05 (ref 15). Reads were filtered for duplicates and extended by 200 bp at the end of the read. Visualization of read count data was performed by converting raw bam files to .tdf files using IGV tools16 and normalizing to 1 million reads. Fragment length extended, duplicate and quality-filtered reads were used for subsequent analysis.
For H3K27ac and H3K4me3 histone marks, the Irreproducible Discovery Rate (IDR) framework20 with a cutoff of 0.1 in combination with the MACS221 peak caller version 2.1 was used to identify peaks taking advantage of both replicates for each condition. For MACS2 peak calling, we used an initial p-value cutoff of 0.01 and the corresponding whole cell extract (WCE) control library as background.
Genome_build: hg19
Supplementary_files_format_and_content: All coordinates are based on hg19. ENCODE narrowPeak Format: chromosome,chromstart,chromend,peak name,macs2 integer score, strand, fold change over background,-log10pvalue,-log10qvalue,relative summit position to peak star WGBS/RRBS bed format, each row describes one CpG: chrom,chromstart,chromend,number of reads where CpG was methylated/total number of reads coverging CpG,numeric score for UCSC display,strand-ignore RNA-Seq txt format: cuffdiff output file: cufflinks ChIP-Seq bed format containing 1kb tiles above a minimal enrichmend level: chrom,chromstart,chromend

Submission date

Oct 08, 2014

Last update date

May 15, 2019

Contact name

Michael Johannes Ziller

Organization name

Max Planck Institute of Psychiatry

Department

Translational Psychiatry

Lab

Ziller lab