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Sample GSM7881741

Query DataSets for GSM7881741

Status

Public on Jul 23, 2024

Title

D0_TKO_CTCF_CUTnRUN_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: DnmtTKO

Growth protocol

Day 0 post-FGF2/Activin A treatment

Extracted molecule

genomic DNA

Extraction protocol

We performed CUT&RUN according to the original protocol (Skene and Henikoff 2017) with the following modifications: 500,000 cells were used for each sample, and the primary antibody was incubated overnight at 4ºC on a rotator. After incubation with pAG-MNase and performing the MNAse reaction, the samples were placed on a magnetic rack and the supernatant containing the DNA samples was recovered. Following addition of 0.1% SDS and 0.17mg/ml Proteinase K, samples were incubated at 50°C for 1h. Purified DNA was obtained by phenol/chloroform extraction and precipitated with 100% Ethanol by centrifugation. The DNA pellet was washed in 80% ethanol, spun down and air-dried before being resuspended in 25µl of 1mM Tris-HCl pH8.0. We used a primary CTCF antibody (Cell D31H2) or IgG control (SigmaAldrich I5006) and no secondary antibody was used for these experiments. The pAG-MNase plasmid was obtained from Addgene (#123461), and the protein was purified by the Curiecoretech Recombinant Protein Platform.
Sequencing library preparation was made using the NEBNext® UltraTM II DNA Library Prep Kit for Illumina (NEB) following the procedure described in “Library Prep for CUT&RUN with NEBNext® UltraTM II DNA Library Prep Kit for Illumina® (E7645) V.1” available in protocols.io. Quality control for the finalized libraries was performed using a TapeStation 420 system (Agilent). Libraries were sequenced by Novogene Co on a NovaSeq using paired-end 150 base pair parameters, requesting 4 gigabytes of data per sample, or approximately 13 million reads.

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: CUT&Run

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