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Sample GSM2829006

Query DataSets for GSM2829006

Status

Public on Aug 03, 2018

Title

Fast ATAC-Seq Rep1

Sample type

SRA

Source name

H9 hESCs

Organism

Homo sapiens

Characteristics

cell type: H9 human Embryonic Stem Cell Line

Extracted molecule

genomic DNA

Extraction protocol

For Hi-C, cells were crosslinked with 1% formaldehyde for 10 min at room temperature and quenched with glycine, nuclei were isolated and chromatin digested by HindIII, filled in with biotin-dCTP, and ligated. After ligation, nuclei were digested by ProteinaseK and chromatin were reverse-crosslinked overnight, followed by ethanol precipitation to get purified DNA. DNA were sonicated to 200-500 bp fragment and enriched with streptavidin dynal beads, followed by illumina library preparation. For ChIP-seq, nuclear lysates were sonicated to generate 200-500 bp fragments . Chromatin was precleared with Protein A or G Dynabeads at 4°C for 2 hours and incubated with antibody overnight at 4°C. Immunoprecipitated chromatin was eluted, reverse crosslinked and purified by standard methods, followed by illumina library preparation. For HiChIP nuclei were isolated and chromatin digested by DpnII, filled in with biotin-dCTP, and ligated. After ligation, chromatin was sonicated and precleared with Protein A and G Dynabeads at 4°C for 2 hours, then precipitated using Protein A or G dynalbeads pre-incubated with proper antibodies overnight. Immunoprecipitated chromatin was eluted, reverse crosslinked and purified by standard methods, followed by streptavidin beads pull down and illumina library preparation. For ATAC-seq, H9 cells grown to exponential stage in 96-well plates cultured at 37°C or heat stressed at 42°C were collected and used for Fast-ATAC and OMNI-ATAC protocol. For fast-ATAC, cells on 96-well plate were washed with PBS and incubated in 50 µl transposes Tn5 mixture (0.01% digitonin for permeabilizing cell membrane, 2.5 µl Tn5, 25 µl TD buffer) at 37°C for 20 min with occasional shaking. For OMNI-ATAC, cells on 96-well plate were washed with PBS and nuclear membrane was disrupted by soaking in lysis buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3mM MgCl2, 0.1% NP40, 0.1% Tween-20, and 0.01% Digitonin) on ice, nuclei was washed once in cold lysis buffer without NP40 or digitonin, and then incubated in Tn5 transposes mixture (25 ul 2x TD buffer, 2.5 ul transposase (100nM final), 16.5 ul PBS, 0.5 ul 1% digitonin, 0.5 ul 10% Tween-20, 5 ul H2O) at 37°C for 20 min with occasional shaking. After the reaction is over, DNA was directly extracted by Minelute Kit (Qiagen). Purified tagmented DNA were used to make illumina Nextera libraries. For eU-Seq, Ctrl and HS cells were labeled with EU in growth medium for 30 min at 37°C and 42°C respectively before RNA extraction with TRIZOL reagent. Purified RNAs were converted by click-chemistry into Biotin-U labeled RNAs and pulled down with streptavidin beads, followed by fragmentation and reverse transcription into cDNA. Illumina libraries were made from dsDNAs after second strand synthesis on cDNA and amplified with barcoded primers for sequencing.
Libraries were constructed using the standard protocol. Genomic fragments were end repaired (NEBNext End Repair Module), A-tailed by adding adenosine to the 3’ ends of fragment using Klenow fragment (3’ to 5’ exo minus, New England Biolabs), and adaptors were ligatedat room temperature for 1 hr with T4 DNA ligase (New England Biolabs). Libraries were amplified with Illumina primers using the KAPA SYBR FAST qPCR Master Mix. Nextera libraries were amplified with Nextera barcoded primers using the KAPA SYBR FAST qPCR Master Mix.

Library strategy

ATAC-seq

Library source

genomic

Library selection

other

Instrument model

Illumina HiSeq 2000

Description

fast ATAC-Seq
Fast ATAC-Seq under normal conditions
ATAC-Seq.bw

Data processing

All experiments results are mapped to human hg38 genome build. ChIP-Seq and ATAC-Seq were paired end mapped using bowtie and PCR duplicates were removed. Hi-C and HiChIP reads were processed using the Juicer pipeline. eU-Seq were mapped by TOPHAT using default parameters, and FPKM were calculated by Cufflinks.
Genome_build: hg38
Supplementary_files_format_and_content: bigWig containing coverage of ChIP-seq or ATAC-seq and .hic files containing Hi-C and contact matrices.

Submission date

Oct 24, 2017

Last update date

May 15, 2019

Contact name

Victor Corces

E-mail(s)

xiaowenlyu@gmail.com

Phone

4042687305

Organization name

Emory University