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Sample GSM3754960

Query DataSets for GSM3754960

Status

Public on May 31, 2021

Title

7mG_E24RO_R1

Sample type

SRA

Source name

H9 cells

Organism

Homo sapiens

Characteristics

type: embryonic stem cells
treatment: Mesendoderm 24h + RO3306
biological replicate: Replicate 1

Treatment protocol

H9 cells were synchronized in their cell cycle by incubating them with nocodazole (100 ng/ml) for 16h. Then, nocodazole was withdrawn and cells induced to differentiated into mesendoderm for 24h (In CDM-PVA medium supplemented with Insulin (7ug/ml), Activin A (100 ng/ml), BMP (10 ng/ml), LY294002 (10 mM ), and CHIR99021 (3 mM). To block cells in their S phase during differentiation, we added Hydroxyurea (200 mM) at the start of the differentiation. To block the cells in their G2/M phases during differentiation, we added nocodazole 8h after the induction of the differentiation. In this way, we made sure that the cells were able to re-initiate the cell cycle and get blocked in the following G2/M phase. After 24h of differentiation, cells were collected and processed for ChIP-seq library preparation.

Growth protocol

H9 cells were grown in E8 medium containing penicillin (50 units/ml) and streptomycin (100 mg/ml)mycin, on plates coated with Vitronectin-XF (Stem Cells Technologies). Cells were incubated at 37ºC in atmosphere of 5% CO2

Extracted molecule

total RNA

Extraction protocol

Libraries were prepared for sequencing using NEBNext® Ultra™ II DNA Library Prep Kit for Illumina; Ref N: E7645S

Library strategy

ChIP-Seq

Library source

transcriptomic

Library selection

ChIP

Instrument model

Illumina HiSeq 4000

Description

RNA-IP-seq
Mesendoderm 24h + RO3306

Data processing

Base calling was done using illumina package RTA v2.7.7, and instrument control package called HCS v3.4.0.38.
Adaptors were clipped before alignment and then re-inserted
Alignment files were converted into BAM files by the sequencing facility. To calculate differential enrichments we used the exomePeak r package from bioconductor. Because exomepeak package only accepts single-end files, pair-end bam files were converted back to fasq files and then only one of the ends was re-mapped using hisat2. This new bam files were used for the calculation of differential enrichments.
Genome_build: GRCh38.84
Supplementary_files_format_and_content: All bigWig files were generated using the function bamCompare from the Deeptools software for Histone marks vs Input, with a bin size = 10, normalized using RPKM method, and an effective genome size of 2913022398

Submission date

May 07, 2019

Last update date

May 31, 2021

Contact name

Rodrigo Andres Grandy

Organization name

University of Cambridge

Department

Surgery

Lab

Vallier Lab