|
|
GEO help: Mouse over screen elements for information. |
|
Status |
Public on Sep 29, 2022 |
Title |
ATAC-seq__limb_E115_wt_mouse_Rep-2 |
Sample type |
SRA |
|
|
Source name |
forelimb
|
Organism |
Mus musculus |
Characteristics |
Stage: E115 strain: C57Bl.6/J genotype: wt
|
Treatment protocol |
ATAC-seq was performed as described previously (Buenrostro et al., 2015). Briefly, 1x105 isolated limb cells were employed per biological replicate. Cells were washed in cold PBS, lysed in fresh lysis buffer (10mM TrisCl pH7.4, 10mM NaCl, 3mM MgCl2, 0.1% (v/v) Igepal CA-630) for 2 min on ice, and finally pelleted for 10 min at 500 x g and 4°C. Following supernatant aspiration, nuclei-containing pellets were subjected to transposition using Tn5 Transposase for 30 min at 37° C.
|
Growth protocol |
Chicken, opossum and mouse limb buds were microdissected from embryos in cold PBS. Isolated limbs were then trypsinised 5 minutes at 37°C with continuous agitation with a P1000 pipette until no visible clumps remained. Limb cell suspensions were then passed through a 40 µm filter, centrifuged at 250 g for 5 min. Supernatants were then removed from isolated ESCs or limb cells which could then be used for downstream applications.
|
Extracted molecule |
genomic DNA |
Extraction protocol |
Resulting DNA was then purified using MinElute Reaction Clean up kit, eluted in 11 µl of elution buffer and stored in -20° C, if not immediately processed further. Barcoded adapters were added to the transposed fragments by PCR. To avoid saturation in our PCR, we initially performed 5 cycles and extracted a 5 µl aliquot for qPCR to identify the number of cycles required without overamplification. Nextera qPCR primers were used for the amplification. The remaining 45 µl of the PCR reaction were then amplified for the desired number of cycles which never exceeded 12. Finally, samples were purified on AMPure XP beads and eluted in 20 µl. Concentration was measured with Qubit and the quality of the samples was estimated by Bioanalyzer analysis. ATAC-seq samples were sequenced yielding for 50 million 75 bp paired-end reads and each experiment was performed in duplicate.
|
|
|
Library strategy |
ATAC-seq |
Library source |
genomic |
Library selection |
other |
Instrument model |
Illumina HiSeq 2500 |
|
|
Data processing |
Raw sequencing fastq files were processed using cutadapt (Martin, 2011) for adapter trimming, Bowtie2 (Langmead and Salzberg, 2012) for mapping, SAMtools (Li et al., 2009) for filtering, sorting and removing duplicates, and deepTools (Ramirez et al., 2016) for generating coverage tracks. Genome_build: mm10 galgal6 and mondom5 Supplementary_files_format_and_content: bigwig files
|
|
|
Submission date |
Oct 12, 2021 |
Last update date |
Oct 01, 2022 |
Contact name |
Michael I Robson |
E-mail(s) |
robson@molgen.mpg.de
|
Organization name |
Max Planck Institute for Molecular Genetics
|
Street address |
Max Planck Institut für molekulare Genet, Ihnestrasse 63-73
|
City |
Berlin |
ZIP/Postal code |
14195 |
Country |
Germany |
|
|
Platform ID |
GPL17021 |
Series (2) |
GSE185774 |
Promoter repression and 3D-restructuring resolves divergent developmental gene expression in TADs [ATAC-Seq] |
GSE185775 |
Repression and 3D-restructuring resolves regulatory conflicts in evolutionarily rearranged genomes |
|
Relations |
BioSample |
SAMN22232714 |
SRA |
SRX12581259 |
Supplementary file |
Size |
Download |
File type/resource |
GSM5623638_ATAC-seq_forelimb_E115_mm10_wt_Rep2_L9152.cpm.bw |
477.6 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
Raw data are available in SRA |
Processed data provided as supplementary file |
|
|
|
|
|