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| Status |
Public on Jul 20, 2022 |
| Title |
ChIP DE Input, replicate B |
| Sample type |
SRA |
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| Source name |
Definitive Endoderm (DE)
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| Organism |
Homo sapiens |
| Characteristics |
cell type: Definitive Endoderm (DE)
chip antibody: none
time: Day 2
sequencing run: 2
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| Growth protocol |
H9 hESCs were routinely propagated feeder-free in mTeSR1 on cell culture plates previously coated with Matrigel following the manufacturer instructions. Undifferentiated hESCs were propagated and passed at least 3 times after thawing and plated for the differentiation when at 80% of confluence. Cells were maintained in culture and expanded at high quality with particular care to avoid any spontaneous differentiation, which would confound downstream differentiation. The day before the induction of the differentiation, hESCs were washed twice in PBS, dissociated with Accutase, plated in mTeSR1 + Rock Inhibitor (Y27632, 5uM) to promote cell survival and incubated for 12 hours at 37 °C, 5% CO2 for 12 hours. The day after, 30% confluent hESCs were induced to differentiate into definitive endoderm, by incubation with Medium A (Gibco cat. n. A30621-01) for 24 hours and with Medium B (Gibco cat. n. 30624-01) for the following 24 hours at 37 °C, 5% CO2. The third day DE was patterned into AFG by the addition of CDM2 medium with A-83-01, 1uM and DM3189, 250nM. The composition of CDM2 basal medium was as follows: 50% IMDM (+GlutaMAX, +HEPES, +Sodium Bicarbonate; Gibco, 31980-097) + 50% F12 (+GlutaMAX; Gibco, 31765-092) + 1 mg/mL polyvinyl alcohol (Sigma, P8136-250G) + 1% v/v concentrated lipids (Gibco, 11905-031) + 450 µM monothioglycerol (Sigma, M6145) + 0.7 µg/mL insulin (Roche, 1376497) + 15 µg/mL transferrin (Roche, 652202) and incubated at 37 °C, 5% CO2. From day four to day 5, Retinoic Acid 200 nM was added at the CDM2 medium with A-83-01, 1 uM and DM3189, 250 nM and medium was changed every 24 hours.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIP experiments were performed on chromatin extracts according to the manufacturer's protocol (MAGnify ChIP, Life Technologies Cat. n. 492024). For each immunoprecipitation reaction, 10ug of sheared chromatin from DE and PE differentiated cells was used (two biological replicates per ChIP experiment). Sheared chromatin was incubated O.N. with 5μg of anti- H3K27me3 (Abcam Cat. n. ab6002), H3K4me3 (Active Motif Cat. n. 39159), H3K27ac (Active Motif Cat. n. 39133), or H3K4me1 (Abcam ab8895) antibodies.
Immunoprecipitated (IP) and input DNA samples were quantified by Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA) and the DNA integrity was checked with 4200 TapeStation (Agilent Technologies, Palo Alto, CA, USA). NEBNext Ultra DNA Library Preparation kit was used following the manufacturer’s recommendations (Illumina, San Diego, CA, USA). Briefly, the ChIP DNA was end-repaired and adapters were ligated after adenylation of the 3’ ends. Adapter-ligated DNA was size selected, followed by clean up, and limited cycle PCR enrichment. The ChIP library was validated using Agilent TapeStation and quantified using Qubit 2.0 Fluorometer as well as real time PCR (Applied Biosystems, Carlsbad, CA, USA).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 4000 |
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| Data processing |
Image analysis and base calling were conducted by the HiSeq Control Software (HCS). Raw sequence data (.bcl files) generated from Illumina HiSeq was converted into fastq files and de-multiplexed using Illumina's bcl2fastq v2.17 software.
Sequencing adapters and low-quality bases were trimmed using the Trimmomatic v0.39 software with parameters ILLUMINACLIP:/path/to/adapter:2:30:10:1:true SLIDINGWINDOW:20:15 MINLEN:36.
Preprocessed reads were then aligned to hg38 genome using Bowtie2 v2.3.4.1 with parameters --wrapper basic-0 --fr -X 2000.
Aligned reads were filtered using SAMtools v1.11 to keep only concordant primary alignments having a minimum mapping quality of 30. PCR or optical duplicates were marked using Picard v2.25.1 tool and removed. Reads mapping to mitochondrial DNA and to unplaced contigs were filtered out. Reads falling in ENCODE Blacklist regions were removed using BEDTools v2.30.0 pairToBed tool.
deepTools v3.5.1 bamCoverage tool was employed to convert BAM files to bigWig files with RPGC normalization and the genomic bin size set to 10 bp.
Chromatin state discovery was performed using the ChromHMM v1.22 software. As a first step, all the BAM files were binarized with the BinarizedBam module, using a bin size of 200 bp. Concatenated model learning was conducted with the LearnModel module, using the input samples as control data to adjust the binarization threshold locally. This module was employed to build models with a number of states ranging from 6 to 16. We decided to focus on a model with 10 states for the subsequent analyses, since it delivered a compact and meaningful representation of the main chromatin states that can be produced with the 4 histone marks under analysis. The LearnModel module produced 200 bp chromatin state calls for DE and PE cell types, that were renamed based on the function that is commonly associated to known HM combinations and on their overlap with annotated functional regions. The two quiescent states were collapsed into a single Quies state.
Assembly: hg38
Supplementary files format and content: Sample-specific read coverage tracks (bigWig format).
Supplementary files format and content: Expanded ChromHMM state calls for DE and PE (BED format).
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| Submission date |
Jun 21, 2022 |
| Last update date |
Jul 20, 2022 |
| Contact name |
Alessio Colantoni |
| E-mail(s) |
alessio.colantoni@uniroma1.it
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| Organization name |
"Sapienza" University of Rome
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| Department |
Dipartimento di Biologia e Biotecnologie "Charles Darwin"
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| Lab |
Tartaglia Lab
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| Street address |
Piazzale Aldo Moro, 5
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| City |
Roma |
| State/province |
RM |
| ZIP/Postal code |
00185 |
| Country |
Italy |
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| Platform ID |
GPL20301 |
| Series (2) |
| GSE206566 |
Multi-omics characterization of human Embryonic Stem Cells-derived Pharyngeal Endoderm cells (Histone Modification ChIP-Seq) |
| GSE208319 |
Multi-omics characterization of human Embryonic Stem Cells-derived Pharyngeal Endoderm cells |
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| Relations |
| BioSample |
SAMN29222423 |
| SRA |
SRX15808712 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6256952_ChIP_DE-Input-B.nobl.bw |
454.9 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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