Comment[GEAAccession]	E-GEAD-349
MAGE-TAB Version	1.1
Investigation Title	ChIP-analysis of neural stem cells from wild type and Fbl mutant mice
Experiment Description	Cells were either FACS-sorted from E11, E12 and E14 Hes1-d2-EGFPTG/+ mice or from E14 cortices of control (P53-/- Emx1Cre/+, Fblflox/+P53-/- and Fblflox/floxP53-/-), heterozygotes (Fblflox/+P53-/- Emx1Cre/+) and DKO (Fblflox/floxP53-/-Emx1Cre/+). Cells were fixed with 0.25% PFA in PBS for 10 min at RT, washed with 0.1M glycine in PBS for three times. ChIP-seq was performed using H3K4me3 or H3K27me3 antibody.
Experimental Design	genetic modification design	time series design	genotyping design	replicate design
Experimental Factor Name	genotype
Experimental Factor Type	genotype
Person Last Name	wu
Person First Name	quan
Person Affiliation	RIKEN Center for Biosystems Dynamics Research
Person Roles	submitter
Public Release Date	2021-12-15
Protocol Name	P-GEAD-395	P-GEAD-396	P-GEAD-397	P-GEAD-398	P-GEAD-399
Protocol Type	sample collection protocol	nucleic acid extraction protocol	nucleic acid library construction protocol	nucleic acid sequencing protocol	normalization data transformation protocol
Protocol Description	Cells were either FACS-sorted from E11, E12 and E14 Hes1-d2-EGFPTG/+ mice or from E14 cortices of control (P53-/- Emx1Cre/+, Fblflox/+P53-/- and Fblflox/floxP53-/-), heterozygotes (Fblflox/+P53-/- Emx1Cre/+) and DKO (Fblflox/floxP53-/-Emx1Cre/+). Cells were fixed with 0.25% PFA in PBS for 10 min at RT, washed with 0.1M glycine in PBS for three times.Cells were collected after centrifugation at 1500 g for 5 min.	After remove of supernatant, cells were re-suspended with ChIP buffer (10 mM Tris-HCl pH 8.0, 200 mM KCl, 1mM CaCl2, 0.5% NP40) at the concentration of 1X106 cells/ml. After a brief sonication (TOMY HandySonic, 10s level 10), micrococcal nuclease (Worthington) was added at the concentration of 50 U/ml. The mixer was incubated at 37degree_C for 20 min. EDTA was added to stop MNase reaction at the concentration of 10 mM. Cells were collected after centrifugation at 15000 g for 5 min and were re-suspended with RIPA buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 2mM EDTA, 1% NP40, 0.5% Sodium Deoxycholate, 0.1% SDS). After sonication for three times (10s at level 10), lysate was centrifuged at 15,000g for 5 min and supernatant was collected. For each ChIP experiment, 100 microL lysate was used. 25microL Dynabeads-Anti Rabbit or Mouse IgG (Invitrogen) were washed with 500 microL ChIP buffer. Beads were incubated with blocking buffer (5mg/ml BSA, 0.5% NP40, 0.1% Tween20 in PBS) including primary antibody anti-rabbit H3K27me3 (CST, #9733) and anti-mouse H3K4m3 (monocle, 307-34813) at 4degree_C with gentle rotation for one night. After wash with ChIP buffer three times, the beads were mixed with blocking buffer and 100microL ChIP lysate and incubated 1h at 4degree_C. Beads were washed five time with low salt wash buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 2mM EDTA, 1% Triton-X100 and 0.1% SDS) and high salt wash buffer (20 mM Tris-HCl pH 8.0, 500 mM NaCl, 2 mM EDTA, 1% Triton-X100 and 0.1% SDS), respectively, followed by the release of chromatin by incubation of the beads with 200 microL elution buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA and 1% SDS) for 30 min at 65degree_C. The supernatant was removed into a new tube and was incubated at 65 for 4 h. RNase A was added and incubate at 37degree_C for 10m to degrade RNA. The supernatant was incubated at 55degree_C overnight with 5 microL proteinase K. Genomic DNA was extracted with phenol:chloroform extraction.	Library preparation for ChIP-sequencing (ChIP-seq) was using the KAPA LTP Library Preparation Kit (KAPA Biosystems) and 2 ng of input DNA, or the entire amount of the ChIP DNA obtained. KAPA Real-Time Library Amplification Kit (KAPA Biosystems) was used in conjunction with the library preparation kits described above to minimize the number of PCR cycles for library amplification.	Sequencing was performed in HiSeq1500 (Illumina) with the HiSeq SR Rapid Cluster Kit v2 (Illumina).	Sequenced reads with poor quality were trimmed with Trim Galore! (--phred33 -q 30 --length 35). Reads were mapped onto mouse genome mm10 using bowtie with the parameter --m1 --best --strata. Mapped sam files were transferred into bam files and were sorted with samtools. Duplicates were marked and removed with Picard. Peaks of ChIP-seq were called using MACS2 (2.1.1). Q-value to cutoff H3K4me3 peaks was set at 0.01. For call peaks of H3K27me3 --broad function was used and q-value was set at 0.01 and 0.05 to cutoff narrow/strong regions and broad/weak regions, respectively. For each sample, specified input was used as control.
SDRF File	E-GEAD-349.sdrf.txt
Comment[AEExperimentType]	ChIP-seq
Comment[BioProject]	PRJDB9278
Comment[Last Update Date]	2021-12-15