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Sample GSM7360749

Query DataSets for GSM7360749

Status

Public on May 16, 2024

Title

12552_ID_Cfa6.66_noins_BioRep1

Sample type

SRA

Source name

Brainstem

Organism

Canis lupus familiaris

Characteristics

genotype: noins
cell type: Brainstem
chip antibody: input

Extracted molecule

genomic DNA

Extraction protocol

We extracted nuclei from flash frozen brainstem tissue using the Active Motif High sensitivity kit (Active Motif). Briefly, we re-suspended 50mg of flash-frozen tissue in Tissue fixation solution (4.35ml Molecular grade water, 0.5ml Active Motif 10x PBS, and 140ul 37% (v/v) formaldehyde). We rotated the tube for 8 minutes, added 51.5ul of Active Motif Stop Solution, and followed by 5 minutes of rotation.
ChIP-seq libraries were prepared using the Active Motif High sensitivity kit complemented with the Active Motif Next Gen DNA library Prep kit and Active Motif Next Gen Indexing kit with 15 PCR cycles. 1 ng of DNA was used as starting material for input and ip samples. Post amplification libraries were size selected at 250-450bp in length using Agencourt AMPure XP beads from Beckman Coulter. Libraries were validated using the Agilent High Sensitivity DNA Kit.

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina NovaSeq 6000

Data processing

Basecalls were performed using bcl2fastq v2.17 for Novaseq output.
ChIP-seq reads were trimmed to remove barcodes and reads with a qulaity cutoff score of greater than 20 (q>20) were reatined using Trim Galore v0.0.6
ChIP-seq reads were aligned to the canfam3 genome using Bowtie version 1.1.2. Only uniquely mapping reads with distinctive "unqiue molecular identifier" sequences were retained.
To create ChIP-seq coverage plots, we used deeptools v3.5.1 and normalized all libraries to the largest library size. ChIP-seq reads were extended 100bp.
Peaks were called uing MACS v2.2.0 with the significance cut-off q-value <=0.05 and 0.1 for E2F1 and H3K27ac Chip libraries, respectively.
Bigwig files were generated in deeptools v3.5.1. Score represents the normalized coverage of DNA fragments at a given genomic coordinate. narrowPeak files were generated using MACS v2.2.0. broadPeak files were generated with MACS v2.2.0, with the q cutoff as 0.1 and --broad-cutoff as 0.1.
Assembly: canFam3.1
Supplementary files format and content: bigWig, narrowPeak/broadPeak (except for Input sample)

Submission date

May 16, 2023

Last update date

May 16, 2024

Contact name

Dhriti Tandon

E-mail(s)

dtandon@princeton.edu

Organization name

Princeton University

Department

Ecology and Evolutionary Biology