|
| Status |
Public on Jul 23, 2024 |
| Title |
D0_WT_H3K27ac_HiChIP_rep2 |
| Sample type |
SRA |
| |
|
| Source name |
mouse embryonic stem cells
|
| Organism |
Mus musculus |
| Characteristics |
cell type: mouse embryonic stem cells
strain: E14
growth media: 2i/vitaminC/LIF
genotype: DnmtWT
|
| Growth protocol |
Day 0 post-FGF2/Activin A treatment
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Hi-ChIP experiments were performed using the Arima Hi-ChIP kit (Arima Genomics) according to the manufacturer’s instructions. 15g of chromatin were used per sample and experiments were performed in duplicates. Briefly, cells were cross-linked with 2% formaldehyde for 10 min at room temperature, lysed and chromatin was digested with two different restriction enzymes included in the kit. Overhangs were filled-in in the presence of biotinylated nucleotides, followed by ligation. Ligated DNA was sonicated using the Covaris M220 to an average fragment size of 500 bp with the following parameters (Peak incident power: 50; Duty factor: 10%; Cycles per burst: 200; Treatment time: 250s). DNA was then immunoprecipitated overnight using 2.5µg of H3K27Ac (Active Motif 91193) or CTCF antibody (Active Motif 91285). After a double-size selection to retain DNA fragments between 200 and 600 bp using Ampure XP beads (Beckman Coulter) the biotin-ligated DNA was precipitated with streptavidin-coupled magnetic beads (included in the kit). The Hi-ChIP libraries were prepared on beads using the Accel-NGS 2D library Kit (Swift bioscience) following instructions from the Arima Hi-ChIP kit. Final libraries were analyzed using 4200 TapeStation system (Agilent) and sequenced. The Hi-ChIP libraries were prepared on beads using the Accel-NGS 2D library Kit (Swift bioscience) following instructions from the Arima Hi-ChIP kit. Final libraries were analyzed using 4200 TapeStation system (Agilent) and sequenced.
The Hi-ChIP libraries were prepared on beads using the Accel-NGS 2D library Kit (Swift bioscience) following instructions from the Arima Hi-ChIP kit. Final libraries were analyzed using 4200 TapeStation system (Agilent) and sequenced. The Hi-ChIP libraries were prepared on beads using the Accel-NGS 2D library Kit (Swift bioscience) following instructions from the Arima Hi-ChIP kit. Final libraries were analyzed using 4200 TapeStation system (Agilent) and sequenced.
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Data processing |
CUT&Run data: PCR duplicate removal using BBmap clumpify (Bushnell et al., 2014)(v38.18) and parameters: reorder dedupe=t k=19 passes=6 subs=(0.01 x readlength). Trimmomatic (Bolger et al., 2014) (v0.39) and parameters: CROP:36 ILLUMINACLIP:adapters.fa:2:30:10 SLIDINGWINDOW:4:20 MINLEN:24
Read pairs that survived trimming were aligned to the mouse reference genome (build mm10) using Bowtie2 (Langmead et al., 2019)(v2.4.5) for CUT&Run using parameters: --local –very-sensitive –no-mixed –dovetail –no-discordant –phred33 -I 10 -X 700
Processed bigwig files were generated using deeptools (Ramírez et al., 2016) bamCoverage (v3.5.1) and parameters: --binSize 1 –smoothLength 0 –minMappingQuality 10 –normalizeUsing CPM –outFileFormat bigwig –blackListFileName ENCFF547MET.bed –ignoreForNormalization chrX chrM chrY
HiChIP data: the HiC-Pro pipeline (v3.1.0) (Servant et al. 2015) was used to digest the mm10 genome (^GATC G^ANTC), align reads and generate contact map matrices. Single-end alignment bam files were used for peak calling using macs2 (v2.2.7.1) (Zhang et al. 2008) and generating bigwigs using bamCompare (as described above). Loops were called using hichipper (v0.7.7) (Lareau and Aryee 2018b) and parameters “-mu -pp 1000” and differential loops were calculated using diffloop (Lareau and Aryee 2018a) and quickAssoc normalization. bigInteract tracks of differential looping were colour coded based on statistical significance (FDR<0.05).
Assembly: mm10
Supplementary files format and content: Bigwig format. CUT&Run: CPM-normalized counts. HiChIP: CPM-normalized counts and bigBedInteract loop files (considering 1kb padding around achors)
Library strategy: HiChIP
|
| |
|
| Submission date |
Nov 03, 2023 |
| Last update date |
Jul 23, 2024 |
| Contact name |
Julien Richard Albert |
| E-mail(s) |
julien.richardalbert@ijm.fr, jrichardalbert@gmail.com
|
| Organization name |
Institut Jacques Monod, CNRS, Université de Paris
|
| Department |
UMR 7592
|
| Lab |
Chromatin Dynamics in Mammalian Development
|
| Street address |
15 Rue Hélène Brion
|
| City |
Paris |
| ZIP/Postal code |
75013 |
| Country |
France |
| |
|
| Platform ID |
GPL24247 |
| Series (1) |
| GSE246984 |
The impact of the embryonic DNA methylation program on CTCF-mediated genome regulation |
|
| Relations |
| BioSample |
SAMN38091991 |
| SRA |
SRX22366300 |
