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Sample GSM4819888

Query DataSets for GSM4819888

Status

Public on Dec 08, 2020

Title

LncMyoD-KD H3K27ac Rep 1 Input

Sample type

SRA

Source name

Skeletal muscle

Organism

Mus musculus

Characteristics

strain: C57BL/6
cell type: Muscle stem cells
genotype: LncMyoD-KD
chip antibody: none

Treatment protocol

Cultured satellite cells were treated with siRNA targeting LncMyod for 48h before harvest

Growth protocol

Satellite cells were isolated from mouse hindlimb and cultured in F10 supplemented with 10% HS and P/S

Extracted molecule

genomic DNA

Extraction protocol

Lysates were clarified from sonicated nuclei and histone-DNA complexes were isolated with antibody.
Library was constructed using ChIPmentation protocol from Schmidl et al., (2015). Briefly, antibody-bound DNA was tagmented using TruePrep DNA Library Prep Kit V2 for Illumina (Vazyme) and amplified to construct ChIP-seq library. Library was then sequenced using DNBSEQ-G400 sequencer (BGI) (2 x 100).

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

DNBSEQ-G400

Data processing

Reads were mapped to mm10 using bowtie2 v2.3.2 (Langmead et al., 2009) with default parameters.
Duplicates reads were marked using the MarkDuplicates module of Picard v2.9.2 with default settings. Unmapped and duplicate reads as well as read alignments with a mapping quality below 10 were removed using samtools v1.3 (Li et al., 2009) with parameters ‘-F 1540 -q 10 -b’.
Reads from sample replicates were pooled together into a single file using samtools v1.3 (Li et al., 2009).
For each individual sample replicate as well as pooled replicates, the predominant fragment length was estimated for use during peak calling using phantompeakqualtools v1.2 (Kharchenko, Tolstorukov & Park, 2008).
Relaxed narrow and broad peak calling to determine enriched regions was performed on individual sample replicates as well as pooled replicates using MACS2 v2.1.1 (Zhang et al., 2008) with treatment and control as input files and parameters ‘-g mm -p 1e-2 --nomodel --shift 0 --extsize {estimated fragment length from last step} -B --SPMR’.
Peaks called in the pooled replicates were kept and considered a consensus peak if they had at least a 50% overlap with peaks called in the individual replicates. This was done using intersect in bedtools v2.26.0 (Quinlan, Hall, 2010).
Fold-change signals were generated with the bdgcmp command in MACS2 v2.1.1 (Zhang et al., 2008) and then converted to BigWig format using the UCSC bedGraphToBigWig v332 tool.
Genome_build: mm10
Supplementary_files_format_and_content: narrowPeak files containing consensus peak calls of pooled samples; BigWig treatment over control fold change tracks

Submission date

Oct 06, 2020

Last update date

Dec 08, 2020

Contact name

Tom Cheung

Organization name

Hong Kong University of Science and Technology

Department

Division of Life Science

Street address

Clear Water Bay, Kowloon