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Sample GSM5710319

Query DataSets for GSM5710319

Status

Public on Jul 01, 2022

Title

H1 Day 18-20 (Time Point)- Input (Experiment#2)

Sample type

SRA

Source name

Day 18-20 cardiomyocytes

Organism

Homo sapiens

Characteristics

cell type: WT cardiomyocytes Day 18-20
chip antibody: none
name in the manuscript: H1 day 18-20 (Time point)

Treatment protocol

hESC-derived cardiomyocytes were cultered on Vitronectin (VTN-N) coated plates in presence of differentiation medium and RNA extraction was performed around 15-18 of differentiation when the monolayer started beating

Growth protocol

hESCs H1 were grown on Vitronectin-coated plates in presence of E8 medium, dissociated using Accutase and differentiated into cardiomyocytes using the The PSC Cardiomyocyte Differentiation Kit (Thermo Fisher Scientific) according to the Manufacturer ‘s instruction

Extracted molecule

genomic DNA

Extraction protocol

Briefly, about 8×106 hESCs and 5×106 hESC-derived cardiomyocytes were used for each immunoprecipitation. Cells were detached using Accutase, fixed in PBS containing 1% formaldehyde and quenched with 125 mM glycine. Cells were lysed in ChIP lysis buffer (1% SDS, 10 mM EDTA and 50 mM Tris-HCl at pH 8.1) and sonicated (E220evolution Focused-ultrasonicator, Covaris) to generate 250-bp DNA fragments. Soluble chromatin was diluted eightfold in ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl at pH 8.1, and 167 mM NaCl). Chromatin was incubated overnight at 4°C with KDM1A or T7-tag antibodies and recovered using Dynabeads Protein G (Thermo Fisher Scientific). Beads were washed sequentially with buffers at different salt concentrations: low-salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl (pH 8.1) and 150 mM NaCl), high-salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl (pH 8.1) and 500 mM NaCl), lithium chloride wash buffer (0.25 M lithium chloride, 1% Nonidet P-40, 1% deoxycholate, 1 mM EDTA and 10 mM Tris-HCl (pH 8.1)) and TE buffer. Immunocomplexes were eluted in ChIP elution buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl (pH 8.1) and 500 mM NaCl), at 30°C for 20 minutes and cross-linked overnight at 65°C. DNA was purified using QIAquick PCR columns (Qiagen) according to the manufacturer's instructions.
DNA libraries were generated using the MicroPlex Library Preparation Kit v2 (Diagenode) according to the manufacturer's instructions
ChIP-seq sequencing has been performed on Illunima Hiseq 4000 or Novaseq 6000 SP1 with 150 bp paired-edn method

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina NovaSeq 6000

Description

H1 Day 18-20 (Time Point)- Input (Experiment#2)
Input HiSeq 4000 Replicate #2

Data processing

Raw data was quality-checked and trimmed with BBDuk (version December 10, 2015) [Bushnell et al. 2017a] with a minimum read length of 35 bp and a minimum Phred quality score of 20. High-quality reads were then mapped against the human genome (GRCh38) using bowtie2 (version 2.3.4.1) [Langmead B et al 2019]. Read duplicates were removed using Picard MarkDuplicates (version 2.8.1) [http://broadinstitute.github]. Peak calling was performed using MACS2 (version 2.2.4) [Zhang Y et al 2008] using default ‘sharp’ settings, setting the format of the tag file to “BAMPE.” Peak annotation was carried out using the R package ChIPseeker (version 1.26.2) [Yu G et al 2015] with the UCSC knownGene annotation derived from the R package TxDb.Hsapiens.UCSC.hg38.knownGene. Peak annotation was further enriched for active or poised enhancers using H3K4m1 (ENCFF238YJA, ENCFF558IKG, ENCFF694ENI) and H3K27ac (ENCFF045CUG, ENCFF162HPV) ChIP-seq public data (https://www.encodeproject.org/). Thus, distal intergenic peaks overlapping with both H3K27ac and H3K4me1 peaks were annotated as active enhancers, while distal intergenic peaks overlapping only with H3K4m1 peaks were annotated as poised enhancers.
Excel files including the annotated peak for each ChIP and the narrowPeak files containing the peak locations together with peak summit, p-value, and q-value.
Genome_build: GrCh38 release 91

Submission date

Dec 01, 2021

Last update date

Jul 01, 2022

Contact name

Antonio Adamo

E-mail(s)

antonio.adamo@kaust.edu.sa

Organization name

King Abdullah University of Science and Technology, KAUST

Department

Biological and Environmental Science and Engineering Division, BESE