GEO Logo
   NCBI > GEO > Accession DisplayHelp Not logged in | LoginHelp
GEO help: Mouse over screen elements for information.
Sample GSM4665702 Query DataSets for GSM4665702
Status Public on Nov 10, 2020
Title E12_PFL_wt_ChIP_H3K27ac
Sample type SRA
Source name Proximal forelimb
Organism Mus musculus
Characteristics genotype: wild-type
strain: B6CBAF2
embryonic day: E12.5
chip antibody: H3K27ac
Extracted molecule genomic DNA
Extraction protocol For E9.5 ChIP samples, 15 to 17 pairs of forelimb buds were used for each experiment. For the E12.5 H3K27ac ChIP sample, proximal forelimbs were dissected from wild-type mice. For E12.5 CTCF ChIP samples, the four whole limbs were micro-dissected from one mouse embryo either from wild type or mutant lines (del CTCF(CS38), delCTCF(CS38;CS40), inv(CS38-40)). All samples were taken from homozygous crosses.Tissues were dissected, fixed in 1% formaldehyde (in PBS) for 10 min at room temperature and the reaction was quenched with Stop Solution from the ChIP-IT High sensitive kit (Active Motif). Samples were then washed 3 times with working Washing Solution (ChIP-IT, Active Motif) and then snap-frozen in liquid nitrogen and stored at -80ºC until further processing. Limb tissues were disrupted with a polytron device, lysed in RIPA buffer or Prep Buffer (ChIP-IT, Active Motif) and sonicated in Diagenode Bioruptor Pico. All experiments were processed following the ChIPmentation protocol (Schmidl et al., 2015) as in (Rodríguez-Carballo et al., 2019). Around 0.6 million cells were used for each IP on a final volume of 800 to 1000ul of RIPA-LS buffer (10mM Tris-HCl pH8, 140mM NaCl, 1mM EDTA pH8, 0.1% SDS, 0.1% sodium deoxycholate, 1% Triton x-100 and proteinase inhibitors). They were incubated overnight with the respective antibodies (CTCF Active Motif 61311; H3K27ac Diagenode C15410196; RAD21 abcam ab992) and precipitated after a 2h long incubation with magnetic beads (Dynabeads Protein A, Invitrogen 10001D) rotating at 4ºC. Washes were performed as follows: two times RIPA-LS, two times RIPA-HS (10mM Tris-HCl pH8, 500mM NaCl, 1mM EDTA pH8, 0.1% SDS, 0.1% sodium deoxycholate, 1% Triton x-100 and proteinase inhibitors), two times RIPA-LiCl (10mM Tris-HCl pH8, 250mM LiCl, 1mM EDTA pH8, 0.5% NP-40, 0.5% sodium deoxycholate and proteinase inhibitors) and once with 10mM Tris-HCl pH8. Beads were resuspended in 24ul of tagmentation buffer (10mM Tris pH8, 5mM MgCl2, 10% dimethylformamide). They were incubated at 37ºC for either 2 minutes (CTCF, RAD21), or 10 minutes (H3K27ac) with 1 µl of Tn5 transposase (Illumina 15027865, from Nextera DNA Library Prep Kit 15028212). Samples were then resuspended and washed twice in 1ml of RIPA-LS and twice in 1ml TE buffer (10mM Tris-Hcl pH8, 1mM EDTA pH8). Beads were magnetised, DNA was eluted in ChIP elution buffer (10mM Tris-HCl pH8, 5mM EDTA pH8, 300mM NaCl, 0.4% SDS) with 2ul of proteinase K (20mg/ml stock) and then incubated for 1 hour at 55ºC and 6 hours to overnight at 65ºC. After de-crosslinking, the supernatant was recovered and beads were resuspended again in 19ul ChIP elution buffer with 1ul of proteinase K and left 1 hour at 55ºC. The two supernatants were combined and purified with MinElute kit (Qiagen) in 22ul of EB buffer. Relative quantitation was performed using SYBR-green as in (Schmidl et al, 2015; PMID:26280331) using 2ul of DNA. Libraries were amplified according to the Cq values obtained in the previous step, purified and size selected with CleanNGS magnetic beads (CleanNA) and eluted in 15ul of water. DNA was sequenced in a HiSeq 4000 machine as 50 bp or 100 bp reads.
Libraries of ChIP experiments were generated following the ChIPmentation protocol (Schmidl et al, 2015; PMID:26280331).
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model Illumina HiSeq 4000
Description Proximal forelimb mouse E12_wt_ChIP_H3K27ac
Data processing Analyses were performed using the facilities of the Scientific IT and Application Support Center of EPFL. Sequences were filtered and adapters were removed using cutadapt (v1.16) with parameters -m 15 -q 30 -a CTGTCTCTTATACACATCTCCGAGCCCACGAGAC. Reads were mapped on mm10 using bowtie2 (v2.3.4.1) using default parameters. Only reads with a mapping quality above 30 were kept. A profile was obtained with macs2 (version option –extsize 300). Bedgraphs were normalized to their number of million tags used in the profile (bedtools v2.27).
Genome_build: mm10
Supplementary_files_format_and_content: bedgraph
Submission date Jul 10, 2020
Last update date Apr 12, 2021
Contact name Eddie Rodríguez-Carballo
Organization name Université de Genève
Department Department of Genetics and Evolution
Street address 4, Boulevard d'Yvoy
City Geneva
ZIP/Postal code 1205
Country Switzerland
Platform ID GPL21103
Series (2)
GSE154187 Chromatin topology and the timing of enhancer function at the HoxD locus [ChIP-seq]
GSE154189 Chromatin topology and the timing of enhancer function at the HoxD locus
BioSample SAMN15502758
SRA SRX8706924

Supplementary file Size Download File type/resource
GSM4665702_E12_PFL_wt_ChIP_H3K27ac.bedGraph.gz 80.1 Mb (ftp)(http) BEDGRAPH
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

| NLM | NIH | GEO Help | Disclaimer | Accessibility |
NCBI Home NCBI Search NCBI SiteMap