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Sample GSM3333907

Query DataSets for GSM3333907

Status

Public on Jan 08, 2019

Title

EndoC_BH1_ChIP_seq_H3K9me3

Sample type

SRA

Source name

Pancreas

Organism

Homo sapiens

Characteristics

cell line: EndoC-BH1 human pancreatic beta cell line
biomaterial provider: Univercell_Bioslutions, EndoCells, INSERM
age: Not applicable
Sex: Not applicable
unos id: Not applicable
ethnicity: Not applicable
chip antibody: H3K9me3

Growth protocol

EndoC-ßH1 cells provided by EndoCells/INSERM were cultured and passaged as previously described (Ravassard et al., 2011). Briefly, cells were seeded at a density of approximately 600,000 cells/cm2 on tissue culture-treated plates pre-coated overnight with extracellular matrix (Sigma) and fibronectin (Sigma) in EndoC-ßH1 complete medium. Cells were passaged approximately every 7 days. Cells were harvested at various passages and distinct sites (e.g., NHGRI, JAX-GM) for karyotyping, genotyping, ATAC-seq, ChIP-seq, RNA-seq, Hi-C, and Pol2 ChIA-PET analyses.

Extracted molecule

genomic DNA

Extraction protocol

CTCF, H3K27ac, H3K27me3, H3K36me3, H3K4me1, H3K79me2, H3K4me3, H3K9me3 ChIP-seq was performed as previously described (Stitzel et al., 2010)

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina HiSeq 2500

Description

EndoC_BH1_ChromHMM_annotations.txt

Data processing

EndoC-ßH1 RNA Pol2 ChIA-PET libraries were generated and sequenced reads were processed and analyzed according to the protocol in (Li et al., 2017b). ChIA-PET interactions were identified using ChIA-PET2 (Li et al., 2017a) using the “bridge linker mode” option. ChIA-PET and Hi-C loops were further filtered using the Bioconductor package InteractionSet_1.8.0 (Lun et al., 2016) to retain only those in which both interacting sites (anchors) overlapped OCRs. ChIA-PET anchors were annotated to the nearest gene.
Hi-C libraries were generated as described in (Rao et al., 2014) and analyzed using the Juicer Tools version 1.75 pipeline (Durand et al., 2016a). We sequenced 6,065,763,792 Hi-C read pairs in EndoC-ßH1 cells, yielding 1,909,699,446 Hi-C contacts; we also sequenced 6,009,242,588 Hi-C read pairs in islet cells, yielding 1,516,995,339 Hi-C contacts. Loci were assigned to A and B compartments at 500 kB resolution. Loops were annotated using HiCCUPS at 5kB and 10kB resolutions with default Juicer parameters. This yielded a list of 9,100 loops in EndoC-ßH1 cells and 2,580 loops in Islet cells.
EndoC-ßH1 ATAC-seq libraries were prepared as previously described (Varshney et al., 2017) and sequenced on an Illumina NextSeq 500 with 2 x 125 bp cycles. Paired-end ATAC-seq reads were quality trimmed using Trimmomatic version 0.33 (Bolger et al., 2014) and parameters “TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36”. Trimmed reads were aligned to human genome (hg19) using BWA version 0.7.12 (Li, 2013), specifically using the bwa mem –M option. Duplicate reads were removed using “MarkDuplicates” from Picard-tools version 1.95 (The Broad Institute, 2013). After preprocessing and quality filtering, peaks were called on alignments with MACS version 2.1.0 (Zhang et al., 2008) using the parameters “-g 'hs' --nomodel --keep-dup all --broad --broad-cutoff 0.05 -f BAMPE”. Peaks located in blacklisted regions of the genome were removed.
Total RNA was extracted and purified from EndoC using Trizol as previously described (Varshney et al., 2017). All sequencing was performed on an Illumina NextSeq 500 with 2 x 101 bp cycles. Paired-end RNA-seq reads were trimmed using Trimmomatic with the same parameters as used for ATAC-seq reads. Trimmed reads were aligned to human genome (hg19) using STAR version 2.53 (Dobin et al., 2012) with default parameters and expression levels of all genes were determined using QoRTs version 1.2.42 (Hartley and Mullikin, 2015) with default parameters and Gencode v19 transcript annotations.
Total RNA was extracted and purified from 2 x 106 EndoC-ßH1 cells using Trizol (Life Technologies). RNA quality was confirmed with Bioanalyzer 2100 (Agilent); EndoC- ßH1 cells RNA RIN scores were > 9.0. miRNA libraries were prepared at the NIH Intramural Sequencing Core (NISC) from 1 µg total RNA using Illumina’s TruSeq Small RNA Library Kit according to the manufacturer’s guidelines, except a 10% acrylamide gel was used for better separation of library from adapters. Libraries were pooled in groups of about 8 for gel purification. Single-end 51 base sequencing was performed on Illumina HiSeq 2500 sequencers in Rapid Mode using version 2 chemistry. Data was processed using RTA version 1.18.64 and CASAVA 1.8.2. All resulting data was processed with miRquant 2.0 (Kanke et al., 2016).
CTCF, H3K27ac, H3K27me3, H3K36me3, H3K4me1, H3K79me2, H3K4me3, H3K9me3 ChIP-seq was performed as previously described (Stitzel et al., 2010) and sequenced on an Illumina HiSeq 2500 using 2 x 100 bp cycles. Harmonized ChromHMM states for EndoC-ßH1 and NIH Roadmap cells/tissues were determined as previously described (Varshney et al., 2017).
Genome_build: Human genome GRch37/hg19, and Gencode v19 reference gene annotations
Supplementary_files_format_and_content: Tab delimited text or BED files. HiC files contain HiCCUPS loop locations. ChIAPET file contains RNA Pol2 loop locations. ChromHMM file contains chromatin position and chromatin state annotation information as determined by ChromHMM algorithm (this file is meant to be a "summary" processed data file representing the amalgamation of all 13 ChIP-seq samples). ATACseq file contains broad peak positions as determined by MACS2 software. RNAseq file contains raw counts and Fragments Per Kilobase of transcript per Million mapped reads (FPKM) values for each gene. miRNA file contains reads per million mapped miRNA (RPMMM) values for each miRNA.

Submission date

Aug 15, 2018

Last update date

Jan 08, 2019

Contact name

Michael Stitzel

E-mail(s)

Michael.Stitzel@jax.org

Organization name

The Jackson Laboratory

Street address

10 Discovery Drive